Reactive oxygen species (ROS), which constitute a representative group of substances associated with skin health, are promising indicators of oxidative stress. A local electrochemical system can measure ROS with minimal invasiveness. In this study, a microneedle-based amperometric sensor is developed for real-time monitoring of changes in the H2O2 concentration of the skin. Two electrodes can be mounted on a single open-tip porous microneedle (PMN), which allows local measurement without intradermal noise caused by the high resistance and non-uniform distribution of the potential difference. The PMN electrode shows a current response in accordance with the H2O2 concentration, and amperometric measurement of ex vivo skin samples is successfully performed. The results indicate that a compact system based on a single needle is a promising tool for intracutaneous sensing of biological signals.

<title>Abstract</title>A microneedle array is an attractive option for a minimally invasive means to break through the skin barrier for efficient transdermal drug delivery. Here, we report the applications of solid polymer-based ion-conductive porous microneedles (PMN) containing interconnected micropores for improving iontophoresis, which is a technique of enhancing transdermal molecular transport by a direct current through the skin. The PMN modified with a charged hydrogel brings three innovative advantages in iontophoresis at once: (1) lowering the transdermal resistance by low-invasive puncture of the highly resistive stratum corneum, (2) transporting of larger molecules through the interconnected micropores, and (3) generating electroosmotic flow (EOF). In particular, the PMN-generated EOF greatly enhances the transdermal molecular penetration or extraction, similarly to the flow induced by external pressure. The enhanced efficiencies of the EOF-assisted delivery of a model drug (dextran) and of the extraction of glucose are demonstrated using a pig skin sample. Furthermore, the powering of the PMN-based transdermal EOF system by a built-in enzymatic biobattery (fructose / O₂ battery) is also demonstrated as a possible totally organic iontophoresis patch.

A totally soft organic subdural electrode has been developed by embedding an array of poly(3,4-ethylenedioxythiophene)-modified carbon fabric (PEDOT-CF) into the polyvinyl alcohol (PVA) hydrogel substrate. The mesh structure of the stretchable PEDOT-CF allowed stable structural integration with the PVA substrate. The electrode performance for monitoring electrocorticography (ECoG) was evaluated in saline solution, on ex vivo brains, and in vivo animal experiments using rats and porcines. It was demonstrated that the large double-layer capacitance of the PEDOT-CF brings low impedance at the frequency of brain wave including epileptic seizures, and PVA hydrogel substrate minimized the contact impedance on the brain. The most important unique feature of the hydrogel-based ECoG electrode was its shape conformability to enable tight adhesion even to curved, grooved surface of brains by just being placed. In addition, since the hydrogel-based electrode is totally organic, the simultaneous ECoG-fMRI measurements could be conducted without image artifacts, avoiding problems induced by conventional metallic electrodes.

Contact lens with built-in electronics is a next-generation wearable product with potential applications such as biomedical sensing and wearable displays. However, fabricating a wireless-powered circuit on a moist, soft contact lens, via common dry lithography, makes producing smart contact lenses challenging. Here, electrochemically (EC) printing a wireless-powered circuit onto a moist, soft contact lens is demonstrated. EC printing involves adding a conductive polymer at the interface between a metal contact and a hydrogelbased contact lens, resulting in strong adhesion of the circuit to the lens without losing high power transfer efficiency (50%) from an eyeglass transmitter to the printed receiver lens. The energy transfer characteristics during eye movement are modeled using the Neumann equation and Kirchhoff's voltage law for wireless power transfer. The energy transfer efficiency between the eyeglass transmitter and the printed receiver lens is derived, and illumination of a wireless-powered single light-emitting diode display as a function of eye rotation angle is demonstrated. This work opens the door to integrating more complex circuits at soft contact lens interface to produce smart contact lens with increased functionality.

Successful treatment of age-related macular diseases requires an effective controlled drug release system with less invasive route of administration in the eye to reduce the burden of frequent intravitreal injections for patients. In this study, we developed an episcleral implantable device for sustained release of ranibizumab, and evaluated its efficacy on suppression of laser-induced choroidal neovascularization (CNV) in rats. We tested both biodegradable and non-biodegradable sheet-type devices consisting of crosslinked gelatin/chitosan (Gel/CS) and photopolymerized poly(ethyleneglycol) dimethacrylate that incorporated collagen microparticles (PEGDM/COL). In vitro release studies of FITC-labeled albumin showed a constant release from PEGDM/COL sheets compared to Gel/CS sheets. The Gel/CS sheets gradually biodegraded in the sclera during the 24-week implantation; however, the PEGDM/COL sheets did not degrade. FITC-albumin was detected in the retina during 18 weeks implantation in the PEGDM/COL sheet-treated group, and was detected in the Gel/CS sheet-treated group during 6 weeks implantation. CNV was suppressed 18 weeks after application of ranibizumab-loaded PEGDM/COL sheets compared to a placebo PEGDM/COL sheet-treated group, and to the intravitreal ranibizumab-injected group. In conclusion, the PEGDM/COL sheet device suppressed CNV via a transscleral administration route for 18 weeks, indicating that prolonged sustained ranibizumab release could reduce the burden of repeated intravitreal injections.

Rapid clearance and low ocular bioavailability are drawbacks of conventional ophthalmic eye drops. To increase the ocular drug resistance time and improve efficacy, an in situ forming and thermosensitive chitosan-gelatin hydrogel was developed. The feasibility of using this hydrogel as a topical eye drop formulation for sustained release of timolol maleate was evaluated. The flexible hydrogel that was co-crosslinked with β‑glycerophosphate disodium salt hydrate (β-GD) and genipin showed a fast gel formation at 37 °C. The swelling properties and in vitro biodegradation characteristics showed a strong relationship with the initial genipin concentration. In vitro release profiles demonstrated that crosslinking with genipin reduced the release rate of entrapped model drugs and timolol maleate. In vitro cytotoxicity tests showed that the hydrogel was non-toxic to Chinese hamster fibroblast V79 cells. The hydrogel was further applied as eye drop formulations for sustained release of timolol maleate to reduce intraocular pressure (IOP). A fast gel formation was observed after instilling the chitosan-gelatin solution into the lower conjunctival sac of the rabbit eyes, and the in situ formed hydrogels protected the drugs from clearance by tears, and released the drugs in a sustained manner. Furthermore, administration of timolol maleate containing chitosan-gelatin hydrogels showed a long-lasting and effective IOP lowering efficacy for up to 24 h compared with the conventional eye drops. These results suggested that β-GD and genipin co-crosslinked chitosan-gelatin hydrogels could be a useful ocular drug delivery platform with enhanced therapeutic effects and reduced side effects.

Herein, we report a sheet-type device capable of self-deployment and sustained release of protein type drugs. The device consisted of a thin photopolymerized polyethylene glycol dimethacrylate (PEGDM) sheet and collagen microparticles (COLs), which were embedded in the sheet as drug carriers and for increased drug permeation. When the density of the COLs in the sheet was increased to be sufficiently interconnected, the drug permeability was increased. In addition, since protein type drugs electrostatically interacted with the COLs, a prolonged sustained release was possible. The PEGDM/COLs device was flexible enough to be rolled up, and the device maintained its structure due to van der Waals attractive forces between the sheet surfaces. When the device was immersed in water, the attractive forces acting between the sheet surfaces were relieved by water. Subsequently, the device unfolded by bending-stress relaxation. Moreover, the rolled-up device could be injected through a conventional syringe needle into water to recover its original shape. The developed sheet-type device provides the possibility of minimally invasive transplantation into diseased tissues and organs, and could provide better therapeutic outcomes and reduce possible side effects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 780–786, 2018.

A flexible polymer film was coated with titanium oxide and a fluoroacrylate polymer to make the surface superhydrophobic and then patterned with superhydrophilic open microfluidic channels consisting of fractal branching structures. The lateral transport of liquid driven by the imbalance of the Laplace pressure in the flow channels with a width gradient allowed the collection of tiny aqueous droplets from the entire surface of the film at the converging point at the center within a second. The proposed fractal patterns were well-defined (i.e., mathematically determined in a unique manner) space-filling trees with only a few geometrical parameters. With the optimized geometrical parameters, the fluid volume collected to the film center (2.0 mm radius, 7.3% of total pattern area) reached 74% ± 9%, where the areal density of liquid was 12 times higher than that of an unpatterned surface.

Subcutaneous space is a potential site for the transplantation of cells such as islets for treatment of type 1 diabetes. To enhance engraftment, an optimal space for the growth of the transplanted cells is needed along with neovascularization. In this study, we developed a device using a photocurable resin, poly(ethyleneglycol) dimethacrylates (PEGDM), for controlled release of basic fibroblast growth factor (bFGF) to create a subcutaneous neovascular bed in rats. The device consists of a disk-shaped capsule with micropores and is composed of tri(ethyleneglycol) dimethacrylate (TEGDM) and a drug formulation of PEGDM. The release rate was tuned by changing the number of pores and the composition of water and PEGDM in the drug formulation. bFGF released from devices incubated in phosphate-buffered saline (PBS) enhanced the growth of fibroblasts, indicating bioactivity of bFGF after release. Histological evaluation showed a significant increase in the extent of vasculature that was dependent on the amount of bFGF loaded into the device. A perfusion study using fluorescein isothiocyanate dextran 2000 kDa showed linear and capillary staining patterns, indicating potent functional vasculature. In conclusion, the controlled bFGF releasing device could provide a neovascular bed with the required vascularization in the subcutaneous space. (c) 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3017-3024, 2017.

Wound healing on skin involves cell migration and proliferation in response to endogenous electric current. External electrical stimulation by electrical equipment is used to promote these biological processes for the treatment of chronic wounds and ulcers. Miniaturization of the electrical stimulation device for wound healing on skin will make this technology more widely available. Using flexible enzymatic electrodes and stretchable hydrogel, a stretchable bioelectric plaster is fabricated with a built-in enzymatic biofuel cell (EBFC) that fits to skin and generates ionic current along the surface of the skin by enzymatic electrochemical reactions for more than 12 h. To investigate the efficacy of the fabricated bioelectric plaster, an artificial wound is made on the back skin of a live mouse and the wound healing is observed for 7 d in the presence and absence of the ionic current of the bioelectric plaster. The time course of the wound size as well as the hematoxylin and eosin staining of the skin section reveals that the ionic current of the plaster leads to faster and smoother wound healing. The present work demonstrates a proof of concept for the electrical manipulation of biological functions by EBFCs.

Transscleral drug delivery is becoming increasingly popular to manage posterior eye diseases. To evaluate the clinical application of a transscleral, sustained, unoprostone (UNO)-release device (URD) constructed of photopolymerized tri(ethyleneglycol) dimethacrylate and poly (ethyleneglycol) dimethacrylate, we evaluated physicochemical and biological properties of this device. The URD consists of a drug-impermeable reservoir and a semipermeable cover. The in vitro release rate of UNO from the URD increased with increasing temperatures from 20 to 45 degrees C. Scanning electron microscopy and atomic-force microscopy showed that the border between the reservoir and drug formulation was sharply defined but that between the cover and drug was poorly determined, indicating that UNO could permeate only through the cover. For stability tests, the URDs were sterilized with ethylene oxide gas and stored at 40 degrees C/75% for 3 and 6 months and at 25 degrees C/60% for 3, 6, 9, 12, 18, and 24 months; UNO content and release rate at 37 degrees C were then evaluated. There was no significant decrease in either UNO content or release rate after the storage conditions. Cytotoxicity was evaluated by examining the colony formation of Chinese hamster fibroblast V79 cells in a media extract of the URD without UNO. This extract did not affect colony formation of V79 cells, indicating the cytocompatibility of the URD. In conclusion, the URD was physically stable for 24 months and is potentially useful for clinical application.

Angiogenesis plays a critical role in many diseases, including macular degeneration. At present, the pathological mechanisms remain unclear while appropriate models dissecting regulation of angiogenic processes are lacking. We propose an in vitro angiogenesis process and test it by examining the co-culture of human retinal pigmental epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVEC) inside a microfluidic device. From characterisation of the APRE-19 monoculture, the tight junction protein (ZO-1) was found on the cells cultured in the microfluidic device but changes in the medium conditions did not affect the integrity of monolayers found in the permeability tests. Vascular endothelial growth factor (VEGF) secretion was elevated under low glucose and hypoxia conditions compared to the control. After confirming the angiogenic ability of HUVEC, the cell-cell interactions were analyzed under lowered glucose medium and chemical hypoxia by exposing ARPE-19 cells to cobalt (II) chloride (CoCl2). Heterotypic interactions between ARPE-19 and HUVEC were observed, but proliferation of HUVEC was hindered once the monolayer of ARPE-19 started breaking down. The above characterisations showed that alterations in glucose concentration and/or oxygen level as induced by chemical hypoxia causes elevations in VEGF produced in ARPE-19 which in turn affected directional growth of HUVEC.

A stretchable, electrochromic film of a uniform composite of poly(3,4-ethylenedioxythiophene):p-toluene sulfonic acid (PEDOT:PTS) and polyurethane (PU) (PEDOT/PU) was fabricated, and its integration with a hydrogel as a free-standing, stretchable electrochromic (EC} display was demonstrated. The PEDOT/PU composite film was prepared by the spill coating of a solution containing an EDOT monomer and PU, followed by oxidative polymerization using iron(III) tosylate at elevated temperature. The fabricated film showed reversible electrochromism without an external conductive support. The color change of the film can be used to quantify the progress of the redox reactions by means of digital camera image analysis and a custom mobile: phone app.

We describe an electrochemical method of harvesting cells cultured on a biodegradable polymeric nanosheet (cell/nanosheet construct), which is stabilized on a self-assembled monolayer (SAM) of thiol molecules. A poly(lactic-co-glycolic acid) (PLGA) nanosheet was attached by hydrophobic interactions onto the surface of a SAM of L-cysteine coated onto a gold electrode. Retinal pigment epithelial cell lines (RPE-J cells) were cultured on the nanosheet to form a monolayer. An AA-size dry battery was used to apply a negative electrical potential, causing reductive desorption of the SAM from the gold surface. Within one minute of application of the voltage, the cell/nanosheet of several mm in diameter was successfully detached without the loss of cell viability in a gentle stream of the electrolyte solution. The use of a porous electrode shortened the detachment time due to the more efficient permeation of the electrolyte solution to the electrode surface. Cell transplantation following the harvesting process was demonstrated by the local delivery of RPE-J cell/nanosheet constructs into the subretinal space of rat eyes through a capillary needle. This nanosheet-based approach that allows the on-demand harvesting of cell/nanosheet constructs and their subsequent transplantation in a minimally-invasive manner could play an important role in cell transplantation therapy.

This paper presents an energy-autonomous bio-sensing system with the capability of wireless communication. The proposed system includes a biofuel cell as a power source and sensing frontend associated with the integrated supply-sensing sensor. The sensor consists of a digital-based gate leakage timer, supply-insensitive time-domain temperature sensor, and inductive-coupling transmitter. A test chip using 65-nm CMOS technology was operated with a supply of 0.19 V and consumed 53 mu W to successfully demonstrate wireless communication with an asynchronous receiver.

An Au film electrode supported by a conductive elastic film was tightly bonded on a stretchable double network hydrogel sheet by means of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) grown from the electrode surface into the hydrogel. This electrode/hydrogel composite showed stable resistance of 35 +/- 5 Omega sq(-1) even during successive 20% stretching because of the pre-formed, designed cracks in the Au film. The large interfacial electric double layer capacitance (9.5 +/- 0.3 mF cm(-2)) of the PEDOT adhesive layer at the interface of the layered composite was found to stabilize the electrode potential against external noises, and decrease the electric impedance at the frequency of 5-500 Hz, which is the typical range of electromyographic signals. The electrical robustness and shape-conformability of the composite electrode were demonstrated by monitoring electromyographic signals of the joint of a human forefinger. In addition, it was also demonstrated that an ionic liquid-containing gel (ionogel) serves as a substrate of the composite for longer-term monitoring over 3 days on air-exposed human skin. (C) 2016 Published by Elsevier B.V.

© 2016 IEEE.We developed all-organic patches and devices that fit to skin. Devices for power generation from biofuel as well as transport of biologically relevant molecules into and from the body through the skin are presented. Integration of these technologies may lead to self-powered healthcare electronics on the skin.

It has been suggested that unoprostone isopropyl (UNO) has potent neuroprotective activity in the retina. The effect of sustained transscleral UNO delivery to the posterior segment of the eye on photoreceptor degeneration was evaluated. UNO was loaded into a device made of poly(ethyleneglycol) dimethacrylate by polydimethylsiloxane mold-based UV-curing. The amount of UNO diffusing from these devices was measured using high-performance liquid chromatography. The polymeric devices that released UNO at 1.8 mu g/day were implanted on the sclerae of S334ter rats at postnatal 21 days, and electroretinograms (ERGs) were compared with those of topical application and placebo devices. Retinal thickness was evaluated by histological examination. Western blots of specimens 4 weeks after implantation were performed. ERGs showed that the UNO-loaded device prevented the reduction of ERG amplitudes 2 and 4 weeks after implantation, compared with results using a placebo device or topical application. Histological examination showed that the UNO-loaded device prevented the reduction of retinal thickness, and Western blots of specimens indicated that the UNO-loaded device decreased expression of ERK1/2, phosphorylated ERK1/2, and caspase-3. A device that provided sustained UNO administration protected against retinal degeneration in rhodopsin mutant rats, and thus, may have translational potential as a sustainable method to administer drugs to treat retinitis pigmentosa. (C) 2015 Wiley Periodicals, Inc.

A commercial painless microneedle was filled with physiological saline agar, and this needle-based salt bridge was inserted into the skin (a piece of porcine skin and a flank skin of a live mouse) to make an electrical contact with its subepidermal region. The transepidermal potential (TEP), the potential difference between the skin surface and the subepidermal region, was measured using this inner electrode and a conventional agar electrode on the surface of the skin. Control of penetration depth of the inner electrode with a spacer and hydrophilic pretreatment with ozone plasma were found to be necessary for stable measurement. The TEP was reduced upon damages on the skin surface by tape stripping and acetone defatting, which indicated the fabricated needle electrode is useful for the minimally-invasive measurement of TEP and evaluation of skin barrier functions. Furthermore, we showed that the device integrating two electrodes into a single compact probe was useful to evaluate the local barrier functions and their mapping on a skin. This device could be a personal diagnostic tool in the fields of medicine and cosmetics in future.

© 2016 IEEE.We have achieved development of a completely organic, totally disposable iontophoresis patch with a built-in enzymatic biofuel cells (BFCs) that utilizes enzymes as electrocatalysts, which can work under mild conditions, i.e., body temperature and at approximately neutral pH. We used fructose as the fuel because the enzyme for oxidation of fructose (fructose dehydrogenese, FDH) can work without the presence of electron mediators for electrocatalysis, resulting in a simple configuration of the patch. The patch, when mounted on the skin, generates a transdermal ionic current with the osmotic flow from anode to cathode, thereby administrating the chemical into the skin.

An energy-autonomous, disposable supply-sensing biosensor based on bio fuel cells and a 0.23-V 0.25-mu m zero-Vth all-digital CMOS supply-controlled ring oscillator with a current-driven pulse-interval-modulated inductive-coupling transmitter was demonstrated. All-digital and current-driven architecture using zero-Vth transistors enables low-voltage operation and small footprint in cost-competitive legacy CMOS. Measured results with 0.25-mu m CMOS testchip successfully demonstrated operation under a 0.23-V supply, which is the lowest supply voltage among reported proximity transmitters. An energy-autonomous biosensing operation using organic bio fuel cells was also demonstrated.

Age-related macular degeneration (AMD) is one of the major ophthalmic diseases that cause visual impairment and blindness. Although transplantation of autologous peripheral cells has been attempted by injecting cell suspensions, limited visual improvement resulted due to the low viability of the injected cells in the subretinal tissue. We developed micropatterned polymeric nanosheets toward local delivery of retinal pigment epithelial (RPE) cells. The micropatterned nanosheet directed growth and morphogenesis of the RPE cells, and allowed for the injection of an engineered RPE monolayer through syringe needles flexibly into the subretinal space of rat eyes. Such an ultra-thin flexible carrier has the promise of a minimally invasive delivery of organized cellular structures into narrow tissue spaces.

This paper presents an energy-autonomous biosensing system with the capability of proximity communication. The proposed biosensor includes a biofuel cell as a power source and sensing frontend associated with the integrated supply-sensing sensor. The sensor consists of a digital-based gate leakage timer, supply-insensitive time-domain temperature sensor, and current-driven inductive-coupling transmitter and achieves low-voltage operation. The timer converts the output supply-voltage from a biofuel cell to period output. The supply-insensitive temperature sensor provides PWM output without dependency of the supply voltage. The following inductive-coupling transmitter enables proximity communication. A test chip using 65-nm CMOS technology was operated with a supply of 0.19 V and consumed 53 mu W to successfully demonstrate proximity communication with an asynchronous receiver. The measurement results show the possibility of energy-autonomous operation using biofuel cells.

We evaluated the effects of a transscleral drug delivery device, consisting of a reservoir and controlled-release cover, which were made of photopolymerized polyethylene glycol dimethacrylate and triethylene glycol dimethacrylate, combined at different ratios. Geranylgeranylacetone (GGA), a heat-shock protein (HSP) inducer, was loaded into the device. The GGA was released from the device under zero-order kinetics. At both 1 week and 4 weeks after device implantation on rat sclera, HSP70 gene and protein expression were up-regulated in the sclerachoroid-retinal pigment epithelium fraction of rat eyes treated with the GGA-loaded device compared with rat eyes treated with saline-loaded devices or eyes of non-treated rats. Flash electroretinograms were recorded 4 days after white light exposure (8000 lx for 18 h). Electroretinographic amplitudes of the a- and b-waves were preserved significantly in rats treated with GGA-loaded devices compared with rats treated with saline-loaded devices. Histological examination showed that the outer nuclear layer thickness was preserved in rats that had the GGA-loaded device. These results may show that transscleral GGA delivery using our device may offer an alternative method to treat retinal diseases.

A porous polymer microneedle array with an average pore diameter of similar to 1 mu m was fabricated by photopolymerization of an acrylate monomer in the presence of a porogen within a mold. Porosity measurement and water absorption testing revealed that the pores are interconnected throughout the microneedle, which enabled rapid wetting of the microneedle by capillary action. The porosity of the polymer microneedles can be modulated by varying porogen content, and this allowed balancing fast water absorption speed and sufficient mechanical strength to penetrate a skin. The developed porous polymer microneedle array is a potentially versatile tool for rapid fluid transport from and into a body through the skin.

A sheet-type, stretchable biofuel cell was developed by laminating three components: a bioanode textile for fructose oxidation, a hydrogel sheet containing fructose as fuel, and a gas-diffusion biocathode textile for oxygen reduction. The anode and cathode textiles were prepared by modifying carbon nanotube (CNT)-decorated stretchable textiles with fructose dehydrogenase (FDH) and bilirubin oxidase (BOD), respectively. Enzymatic reaction currents of anode and cathode textiles were stable for 30 cycles of 50% stretching, with initial loss of 20-30% in the first few cycles due to the partial breaking of the CNT network at the junction of textile fibers. The assembled laminate biofuel cell showed power of similar to 0.2 mW/cm(2) with 1.2 k Omega load, which was stable even at stretched, twisted, and wrapped forms. (C) 2015 Elsevier B.V. All rights reserved.

Age-related macular degeneration is the leading cause of legal blindness among older individuals. Therefore, the development of new therapeutic agents and optimum drug delivery systems for its treatment are crucial. In this study, we investigate whether clotrimazole (CLT) is capable of protecting retinal cells against oxidative-induced injury and the possible inhibitory effect of a sustained CLT-release device against light-induced retinal damage in rats. In vitro results indicated pretreatment of immortalized retinal pigment epithelium cells (RPE-J cells) with 10-50 mu M CLT before exposure to oxygen/glucose deprivation conditions for 48 h decreased the extent of cell death, attenuated the percentage of reactive oxygen species-positive cells, and decreased the levels of cleaved caspase-3. The device consists of a separately fabricated reservoir, a CLT formulation, and a controlled release cover, which are made of poly(ethyleneglycol) dimethacrylate (PEGDM) and tri(ethyleneglycol) dimethacrylate (TEGDM). The release rate of CLT was successfully tuned by changing the ratio of PEGDM/TEGDM in the cover. In vivo results showed that use of a CLT-loaded device lessened the reduction of electroretinographic amplitudes after light exposure. These findings indicate that the application of a polymeric CLT-loaded device may be a promising method for the treatment of some retinal disorders.

A grid micropattern of neuronal cells was formed on a free-standing collagen film (35 mu m thickness) by directing migration and extension of neurons along a Matrigel pattern previously prepared on the film by the microcontact printing method. The neurons migrated to reach the nodes on the grid pattern and extended neurites to bridge cell bodies at the nodes. The resulting neuronal micropattern on the collagen film containing culture medium can be handled and deformed with tweezers with maintenance of physiological activity of the neurons, as examined by response of cytosolic Ca2+ concentration to a dose of bradykinin. This portability is the unique advantage of the present system that will open novel possibility of cellular engineering including the on-demand combination with analytical devices. The repetitive lamination of the film on a microelectrode chip was demonstrated for local electrical stimulation of a specific part of the grid micropattern of neurons, showing Ca2+ wave propagation along the neurites. The molecular permeability is the further advantage of the free-standing hydrogel substrate, which allows external supply of nutrients and dosing with chemical stimulants through the film even under rolled and laminated conditions.

This study demonstrates an energy-autonomous disposable supply-sensing biosensor platform for big-data-based healthcare application for the first time. The proposed supply-sensing biosensor platform is based on bio fuel cells and a 0.23-V 0.25-mu m zero-Vth all-digital CMOS supply-controlled ring oscillator with a current-driven pulse-interval-modulated inductive-coupling transmitter. The all-digital and current-driven architecture using zero-Vth transistors enables low-voltage operation and small footprint even in the cost-competitive legacy CMOS, which enables converterless energy-autonomous operation using bio fuel cell for disposable healthcare application. To verify its effectiveness, a test chip was fabricated using 0.25-mu m CMOS technology. The measured results successfully demonstrated operation under a 0.23-V supply, which is the lowest supply voltage among reported proximity transmitters. In addition, an energy-autonomous biosensing operation using organic bio fuel cells for transdermal patch was successfully demonstrated.

A free-standing cell-cultured hydrogel sheet with stretchability was prepared for an in vitro cellular assay with mechanical stimulation. The anti-fouling surface of the stretchable double network (DN) hydrogel composed of poly(2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt) (PNaAMPS) and poly(N, N-dimethylacrylamide) (PDMAAm) was modified as the cell adhesive by additional polymerization of PNaAMPS at the surface region of the DN hydrogel in the depth of around 170 mu m, while preserving the original stretchability and molecular permeability. The mechanically stable and flexible hydrogel sheet holding the culture medium has the advantage that it can be handled with tweezers in air, and cells adhered on the sheet maintain physiological activity during transportation of the film for in vitro assays. The stretchability of the free-standing culture sheet allows for the observation of the Ca2+ response of epidermal keratinocytes upon mechanical stimulation.

We here describe a thin, highly stretchable transparent and molecular permeable porous electrode with a well-defined honeycomb structure fabricated by a combination of a bottom-up breath figure process and top-down sputtering of metals. Furthermore, the honeycomb electrode-hydrogel hybrids show higher durability, optical transparency, and electrical conductivity. The optical and electrical properties of the electrode, and its potential for use as a stretchable transparent electrode, are discussed.

Hydrogel-based, molecular permeable electronic devices are considered to be promising for electrical stimulation and recording of living tissues, either in vivo or in vitro. This study reports the fabrication of the first hydrogel-based devices that remain highly electrically conductive under substantial stretch and bending. Using a simple technique involving a combination of chemical polymerization and electropolymerization of poly (3,4-ethylenedioxythiophene) (PEDOT), a tight bonding of a conductive composite of PEDOT and polyurethane (PU) to an elastic double-network hydrogel is achieved to make fully organic PEDOT/PU-hydrogel hybrids. Their response to repeated bending, mechanical stretching, hydration-dessication cycles, storage in aqueous condition for up to 6 months, and autoclaving is assessed, demonstrating excellent stability, without any mechanical or electrical damage. The hybrids exhibit a high electrical conductivity of up to 120 S cm(-1) at 100% elongation. The adhesion, proliferation, and differentiation of neural and muscle cells cultured on these hybrids are demonstrated, as well as the fabrication of 3D hybrids, advancing the field of tissue engineering with integrated electronics.

We developed micropatterned polymeric ultra-thin films (nanosheets) consisting of biodegradable poly(lactic-co-glycolic acid) (PLGA) and magnetic nanoparticles (MNPs), on which monolayer of retinal pigment epithelial (RPE) cells were formed. The viability of RPE cells on the micropatterned nanosheets were evaluated along the syringe manipulation equipped with a clinical needle. Finally, subretinal injectability of the macropatterned nanosheets was demonstrated by using ex vivo swine eyes in terms of handleability and physical stability. The micropatterned nanosheets injectable by clinical syringe hold a great promise to transplant functional RPE cells minimum invasively.

We show the fibrous protein fibrin can serve as biocompatible glue with which to bind synthetic cationic or anionic hydrogels together. Both the bonding to and detachment from the hydrogels by fibrin (gelation and degradation, respectively) proceeded enzymatically under physiological conditions. We built a hydrogel-based actuator to demonstrate the method. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.

Enzymatic fuel cells (EFCs) are power devices in which enzymes are used as electrocatalysts to convert biochemical energy directly into electricity, in contrast to metallic catalysts commonly used in fuel cells. This chapter describes the three types of MEFCs fabricated using a series of microelectromechanical system (MEMS)-related techniques. An insertion miniature enzymatic fuel cells (MEFCs) is a type of cell that generates electricity from sugars in living organisms. A microfluidic MEFC consists of microchannels for continuous fuel supply, in which the power generation and performance depend on several fluidic parameters including the flow velocity, electrode configuration, and channel dimensions. The series and parallel stacking of microfluidic MEFCs increase the level of output voltage and net lifetime, respectively. Finally, a sheet-shaped MEFC is described that could be combined with wearable electronics of the future. Engineering advances focused on miniaturization described to promote early practical applications and commercialization of EFCs.

We have developed a technique to fabricate Au microelectrodes on hydrogel substrates such as polyacrylamide. Electropolymerization of poly(3,4-ethylenedioxythiophene) (PEDOT) anchors the Au electrodes to the hydrogels. The resulting soft, molecularly permeable hydrogel-based microelectrodes were effective for the electroporation of adherent cells by direct lamination. The electroporation-assisted uptakes of dye molecules and genes were demonstrated for normal human dermal fibroblast cells and human induced pluripotent stem (iPS) cells.

The design of drug delivery systems that can deliver multiple drugs to the posterior segment of the eye is a challenging task in retinal disease treatments. We report a polymeric device for multi-drug transscleral delivery at independently controlled release rates. The device comprises a microfabricated reservoir, controlled-release cover and three different fluorescent formulations, which were made of photopolymeized tri(ethyleneglycol)dimethacrylate (TEGDM) and poly(ethyleneglycol)dimethacrylate (PEGDM). The release rate of each fluorescent is controlled by varying the PEGDM/TEGDM ratio in its formulation and the cover. The release kinetics appeared to be related to the swelling ratio of the PEGDM/TEGDM polymers. When the devices were implanted onto rat sclerae, fluorescence was observable in the ocular tissues during 4 weeks' implantation and distributed locally around the implantation site. Our polymeric system, which can administer multiple compounds with distinct kinetics, provides prolonged action and less invasive transscleral administration, and is expected to provide new tools for the treatment of posterior eye diseases with new therapeutic modalities. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

We constructed brain-derived neurotrophic factor (BDNF) expressing rat retinal pigment epithelial (RPE) cells by stable transfection of BDNF cDNA, and the RPE cells were cultured on a cross-linked collagen sheet (Coll-RPE-BDNF). BDNF expression of the Coll-RPE-BDNF was confirmed by western blot, and the Coll-RPE-BDNF was transplanted into the rabbit sclera. In vivo BDNF expression was confirmed by His expression that was linked to the expressing BDNF. The effect of the released BDNF was examined in a rabbit acute high intraocular pressure system by electroretinogram and histological examination. Statistically significant preservation of ERG b wave amplitude was observed in the rabbits treated by Coll-RPE-BDNF when compared to that of no treatment. Statistically significant preservation of the thickness of the inner nuclear layer at the transplanted area was observed in the rabbits treated by Coll-RPE-BDNF compared to that of no treatment. Intra-scleral Coll-RPE-BDNF transplantation may partially rescue retinal cells from acute high intraocular pressure.

Two types of hydrogel-based bioassay sheets were developed for in vitro evaluation of contraction-dependent metabolic regulation in skeletal muscle cells: one is an oxygen sensor sheet and the other is an immunocapture sheet for a myokine, interleukin-6 (IL-6). These soft, molecularly permeable hydrogel-based bioassay sheets were directly laminated to another hydrogel on which myotubes were micro-patterned, and displayed usefully measurable changes in local oxygen consumption and IL-6 secretion of myotubes upon electrically-induced contraction.

The effects of pre-treatment with surfactants on the electrocatalytic reaction of multi-copper oxidases were quantitatively evaluated using a well-structured carbon nanotube forest electrode. It was found that both the charge polarity of the head group and the aromatics in the tail part of the surfactants affect the efficiency of enzymatic electrocatalysis.

We established a sustained vasohibin-1 (a 42-kDa protein), delivery device by a novel method using photopolymerization of a mixture of polyethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and collagen microparticles. We evaluated its effects in a model of rat laser-induced choroidal neovascularization (CNV) using a transscleral approach. We used variable concentrations of vasohibin-1 in the devices, and used an enzyme-linked immunosorbent assay and Western blotting to measure the released vasohibin-1 (0.31 nM/day when using the 10 μM vasohibin-1 delivery device [10VDD]). The released vasohibin-1 showed suppression activity comparable to native effects when evaluated using endothelial tube formation. We also used pelletized vasohibin-1 and fluorescein isothiocyanate-labeled 40 kDa dextran as controls. Strong fluorescein staining was observed on the sclera when the device was used for drug delivery, whereas pellet use produced strong staining in the conjunctiva and surrounding tissue, but not on the sclera. Vasohibin-1 was found in the sclera, choroid, retinal pigment epithelium (RPE), and neural retina after device implantation. Stronger immunoreactivity at the RPE and ganglion cell layers was observed than in other retinal regions. Significantly lower fluorescein angiography (FA) scores and smaller CNV areas in the flat mounts of RPE-choroid-sclera were observed for the 10VDD, VDD (1 μM vasohibin-1 delivery device), and vasohibin-1 intravitreal direct injection (0.24 μM) groups when compared to the pellet, non-vasohibin-1 delivery device, and intravitreal vehicle injection groups. Choroidal neovascularization can be treated with transscleral sustained protein delivery using our novel device. We offer a safer sustained protein release for treatment of retinal disease using the transscleral approach. © 2013 Onami et al.

Similar to conventional electrolyte batteries, biofuel cells often need to be stacked in order to boost their single cell voltage (<1 V) up to a practical level. Here, we report a laminated stack of biofuel cells that is composed of bioanode fabrics for fructose oxidation, hydrogel sheets containing electrolyte and fuel (fructose), and O-2-diffusion biocathode fabrics. The anode and cathode fabrics were prepared by modifying fructose dehydrogenase and bilirubin oxidase, respectively, on carbon nanotubes-decorated carbon fiber fabrics. The total thickness of the single set of anode/gel/cathode sheets is just 1.1 mm. The laminated triple-layer stack produces an open-circuit voltage of 2.09 V. which is a 2.8-fold increase over that of a single set cell (0.74 V). The present layered cell (5 mm x 5 mm) produces a maximum power of 0.64 mW at 1.21 V. a level that is sufficient to drive light-emitting diodes. (C) 2012 Elsevier B.V. All rights reserved.

We report the stepwise modification of Os-complex mediator (polyvinylimidazole-[Os(bipyridine)2Cl] (PVI-[Os(bpy)2Cl]) and glucose oxidase (GOD) within the inner nano-space of a carbon nanotube forest (CNTF) film. Owing to the controlled alignment of enzyme/mediator/ electrode in the ensemble, the prepared film electrode has both a high-efficiency (turnover rate of ca. 650 s-1) and a large net oxidation current (ca. 15 mA cm-2). The previous GOD electrodes developed by monolayer-based and polymer-based approaches have either of the performances (efficiency or net activity). In addition, the present GOD electrode is a flexible film that could be used by winding on needle devices. © Published under licence by IOP Publishing Ltd.

We developed techniques for preparing a muscular cells-arrayed hydrogel sheet and a microelectrodes-printed hydrogel sheet. The combination of these two hydrogel sheets enables the metabolic assay for muscular tissue under a controlled contractile motion.

Molecularly ordered composites of polyvinylimidazole-[Os(bipyridine)2Cl] (PVI-[Os(bpy)2Cl]) and glucose oxidase (GOD) are assembled inside a film of aligned carbon nanotubes. The structure of the prepared GOD/PVI-[Os(bpy)2Cl]/CNT composite film is entirely uniform and stable; more than 90% bioelectrocatalytic activity could be maintained even after storage for 6 d. Owing to the ideal positional relationship achieved between enzyme, mediator, and electrode, the prepared film shows a high bioelectrocatalytic activity for glucose oxidation (ca. 15 mA cm-2 at 25 degrees C) with an extremely high electron-transfer turnover rate (ca. 650 s-1) comparable to the value for GOD solutions, indicating almost every enzyme molecule entrapped within the ensemble (ca. 3 x 1012 enzymes in a 1 mm x 1 mm film) can work to the fullest extent. This free-standing, flexible composite film can be used by winding on a needle device; as an example, a self-powered sugar monitor is demonstrated.

A strip of carbon fabric (CF) electrode modified with multiwalled carbon nanotubes and subsequently fructose dehydrogenase (FDH) showed an oxidation current density of similar to 11 mA cm(-2) in stirred 200 mM fructose solution. Obtaining a sufficient dispersion of the nanotubes during its modification was found to be critical to ensure such a performance of the FDH anode. For use with this anode, a CF strip modified with ketjenblack (KB) and bilirubin oxidase (BOD) served as a gas-diffusion cathode for the reduction of O-2 from air at a current density of similar to 2 mA cm(-2.) The FDH-modified CF strip and the BOD-modified CF strip were stacked with an agarose film that retained an electrolyte solution and fuel (fructose) to construct a totally flexible sheet-shaped biofuel cell. This assembly allowed bending of 44 degrees without affecting the maximum output power density, 550 mu W cm(-2) obtained at 0.4 V. (C) 2012 Published by Elsevier Ltd.

Cellular impedance biosensors offer an alternative to conventional analytical techniques with potential advantages over optical methods of high speed, accuracy, sensitivity, non-invasiveness, and ease of direct computer analysis. We first review the original examples of impedimetric sensing where there were several tens or hundreds of cells on the electrodes. Recent works have dealt with single cells. We will highlight impedance sensing for cells grown on electrodes, as this method allows the study of the motion of mammalian cells in real time and in conditions as close as possible to their in vivo environments. Working at alternating current of low frequencies, it is possible to probe intrinsic properties of the cells and their interaction with substrates. In some cases, electrical measurements have been shown to be sensitive to changes in cell properties that are not visible optically. Possible applications may be relevant to a wide range of subjects, such as wound healing, mitosis and pharmacological apoptosis. © Scrivener Publishing LLC.

A patch-type oxygen imaging sheet useful for in vitro cellular metabolic assays was developed. Oxygen-responsive fluorescent microbeads were embedded into a biocompatible polyacrylamide gel sheet, which can be directly attached onto target cells for fluorescent imaging of metabolic activity. The sensor beads were directed in a microfluidic device using AC and DC electric manipulation techniques, followed by encapsulation in a hydrogel. Fluorescent imaging of oxygen-consuming activity was demonstrated for glucose oxidase-modified microparticles as cellular models to show the applicability of the imaging sheet to bioassays. (C) The Electrochemical Society of Japan, All rights reserved.

We report the fabrication of totally organic hydrogel-based microelectrodes of poly(3,4-ethylenedioxythiophene) (PEDOT), which exhibit a lowered sheet resistivity of about 100 Omega/square. The preparation process starts with the electrodeposition of conductive PEDOT (ca. 20 S cm(-1)) on Pt microelectrodes. After laminating hydrogels onto the PEDOT-modified Pt electrode substrates, a second PEDOT (low conductivity) layer was electrodeposited to anchor the first PEDOT film to the hydrogel. Finally, the hydrogel sheet with PEDOT micropatterns was peeled off by taking advantage of the electroactuation property of PEDOT. The process proved to be versatile, allowing the use of most natural and synthetic hydrogels including agarose, collagen, polyacrylamide, and so on.

Nanostructured carbons have been widely used for fabricating enzyme-modified electrodes due to their large specific surface area. However, because they are random aggregates of particular or tubular nanocarbons, the post-modification of enzymes to their intra-nanospace is generally hard to control. Here, we describe a free-standing film of carbon nanotube forest (CNTF) that can form a hybrid ensemble with enzymes through liquid-induced shrinkage. This provides in-situ regulation of its intra-nanospace (inter CNT pitch) to the size of enzymes, and eventually serves as a highly active electrode. The CNTF ensemble with fructose dehydrogenase (FDH) showed the oxidation current density of 16 mA cm -2 in stirred 200 mM fructose solution. The power density of a biofuel cell using the FDH-CNTF anode and the Laccase-CNTF cathode reached 1.8 mW cm -2 (at 0.45 V) in the stirred oxygenic fructose solution, more than 80 % of which could be maintained after continuous operation for 24 h. Application of the free-standing, flexible character of the enzyme-CNTF ensemble electrodes is demonstrated via their use in the patch or wound form. © 2012 Materials Research Society.

Contractile C 2C 12 myotube line patterns supported by a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. Myotube line patterns cultured on dish were transferred with 100% efficiency to the surface of fibrin gel sheets. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (one week) than myotubes adhered on a conventional culture dish. The gel sheet-supported C 2C 12 myotube micropatterns were combined with a microelectrode array chip to fabricate a skeletal muscle cell-based bioassay system. The contractile behavior of each myotube line pattern on the gel was individually controlled by localized electrical stimulation using microelectrode arrays that had been previously modified with the electropolymerized conducting polymer. We successfully demonstrated fluorescent imaging of the contraction-induced translocation of the glucose transporter, GLUT4, from intracellular vesicles to the plasma membrane of the myotubes. This device is applicable for the bioassay of contraction-induced metabolic alterations in a skeletal muscle cell. © 2011 IEEE.

Enzymatic biofuel cells have attracted much attention for their potential to directly use biochemical energy sources in living organisms such as animals, fruits, etc. However, generally natural organisms have a skin, and the oxygen concentration in the organisms is lower than that of biofuels like sugars. Here, we fabricated a novel miniature assembly that consists of a needle bioanode for accessing biofuels in organisms through their skins and a gas-diffusion biocathode for utilizing the abundant oxygen in air. The performance of the biocathode was fourfold improved by optimizing its hydrophobicity. The assembled device with four needle anodes for fructose oxidation was inserted into a raw grape, producing a maximum power of 26.5 mu W (115 mu W cm(-2)) at 0.34 V. A light-emitting diode (LED) with the cell served as a self-powered indicator of the sugar level in the grape. Power generation from blood sugar was also investigated by inserting a needle anode for glucose oxidation into a blood vessel in a rabbit ear. Prior coating of the tip of the needle anode with an anti-biofouling agent was effective to stabilize the output power.

Nanostructured carbons have been widely used for fabricating enzyme-modified electrodes due to their large specific surface area. However, because they are random aggregates of particular or tubular nanocarbons, the postmodification of enzymes to their intrananospace is generally hard to control. Here, we describe a free-standing film of carbon nanotube forest (CNTF) that can form a hybrid ensemble with enzymes through liquid-induced shrinkage. This provides in situ regulation of its intrananospace (inter-CNT pitch) to the size of enzymes and eventually serves as a highly active electrode. The CNTF ensemble with fructose dehydrogenase (FDH) showed the oxidation current density of 16 mA cm(-2) in stirred 200 mM fructose solution. The power density of a biofuel cell using the FDH-CNTF anode and the Laccase CNTF cathode reached 1.8 mW cm(-2) (at 0.45 V) in the stirred oxygenic fructose solution, more than 80% of which could be maintained after continuous operation for 24 h. Application of the free-standing, flexible character of the enzyme CNTF ensemble electrodes is demonstrated via their use in the patch or wound form.

A transscleral drug-delivery device, designed for the administration of protein-type drugs, that consists of a drug reservoir covered with a controlled-release membrane was manufactured and tested. The controlled-release membrane is made of photopolymerized polyethylene glycol dimethacrylate (PEGDM) that contains interconnected collagen microparticles (COLs), which are the routes for drug permeation. The results showed that the release of 40-kDa FITC-dextran (FD40) was dependent on the COL concentration, which indicated that FD40 travelled through the membrane-embedded COLs. Additionally, the sustained-release drug formulations, FD40-loaded COLS and FD40-loaded COLs pelletized with PEGDM, fine-tuned the release of FD40. Capsules filled with COLs that contained recombinant human brain-derived neurotrophic factor (rhBDNF) released bioactive rhBDNF in a manner dependent on the membrane COL concentration, as was found for FD40 release. When capsules were sutured onto sclerae of rabbit eyes, FD40 was found to spread to the retinal pigment epithelium. Implantation of the device was easy, and it did not damage the eye tissues. In conclusion, our capsule is easily modified to accommodate different release rates for protein-type drugs by altering the membrane COL composition and/or drug formulation and can be implanted and removed with minor surgery. The device thus has great potential as a conduit for continuous, controlled drug release. (C) 2010 Elsevier Ltd. All rights reserved.

We have developed gel sheet-supported C(2)C(12) myotube micropatterns and combined them with a microelectrode array chip to afford a skeletal muscle cell-based bioassay system. Myotube line patterns cultured on a glass substrate were transferred with 100% efficiency to the surface of fibrin gel sheets. The contractile behavior of each myotube line pattern on the gel was individually controlled by localized electrical stimulation using microelectrode arrays that had been previously modified with electropolymerized poly(3,4-ethylenedioxythiophene) (PEDOT). We successfully demonstrated fluorescent imaging of the contraction-induced translocation of the glucose transporter, GLUT4, from intracellular vesicles to the plasma membrane of the myotubes. This device is applicable for the bioassay of contraction-induced metabolic alterations in a skeletal muscle cell.

A porous membrane-based cell culture device was developed to electrically stimulate a confluent monolayer of C2C12 myotubes. The device&apos;s cell culture substrate is a microporous alumina membrane-modified by attaching an atelocollagen membrane on the upperside and a hole-spotted poly(dimethylsiloxane) (PDMS) film on the underside. When electric current is generated between the device&apos;s Pt ring electrodes - one of which is placed above the cells and the other below the PDMS layer - the focused current at the PDMS hole can electrically stimulate the cells. C2C12 myoblasts were cultured on the substrate and differentiated into myotubes. When the electrical pulses were applied, myotubes started to contract slightly in and near the hole, and that the continuous stimulation increased both the number of stimuli-responding myotubes and the magnitude of the contraction considerably owing to the underlying atelocollagen membrane with muscle tissue-like stiffness. Also, the generation of contractile myotubes on a wider region of the membrane substrate was possible by applying the electrical pulses through the array of holes in the PDMS film. Using the present system, the glucose uptake by contractile myotubes was examined with fluorescence-labeled glucose, 2-NBDG, which displayed a positive correlation between the contractile activity of myotubes and the uptake of 2-NBDG. (C) 2010 Elsevier Ltd. All rights reserved.

We report herein the micropatterning of poly(3,4-ethylenedioxythiophene) (PEDOT) on a hydrogel, agarose, to provide a fully organic, moist, and flexible electrode. The PEDOT/agarose electrodes were prepared through two electrochemical processes: electropolymerization of PEDOT into the hydrogel and electrochemical-actuation-assisted peeling. We also present a typical application of the PEDOT/agarose electrode to the cultivation of contractile myotubes.

We have combined a topographically patterned agarose microstamp with an electrode substrate to develop a novel printing device that internally contains an electrochemical system for a controlled supply of reactive ink to the stamp surface. The 10 wt % agarose gel containing 0.1 M PBS + 25 mM K Br showed suitable elasticity for forming stamps and served as the electrolytic medium for the electrochemical oxidation of Br- to generate H BrO. The electrode substrate patched with an agarose stamp having 50-mu m-high bumps was used for the spatially confined detachment of heparin/polyethyleneimine precoated on glass substrates, followed by micropatterned adsorption of fibronectin. Using the microelectrode array, the addressable micropatterning of protein by the controlled delivery of H BrO to each bump was demonstrated.

In this study, we prepared injectable collagen microspheres for the sustained delivery of recombinant human vascular endothelial growth factor (rhVEGF) for tissue engineering. Collagen solution was formed into microspheres under a water-in-oil emulsion condition, followed by crosslinking with water-soluble carbodiimide. Various sizes of collagen microspheres in the range of 1-30 mu m diameters could be obtained by controlling the surfactant concentration and rotating speed of the emulsified mixture. Particle size proportionally decreased with increasing the rotating speed (1.8 mu m per 100 rpm increase in the range of 300-1,200 rpm) and surfactant concentration (3.1 mu m per 0.1% increase in the range of 0.1-0.5%). The collagen microspheres showed a slight positive charge of 8.86 and 3.15 mV in phosphate-buffered saline and culture medium, respectively. Release study showed the sustained release of rhVEGF for 4 weeks. Released rhVEGF was able to induce capillary formation of human umbilical vein endothelial cells, indicating the maintenance of rhVEGF bioactivity after release. In conclusion, the results suggest that the collagen microspheres have potential for sustained release of rhVEGF.

Contractile C2C12 myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C2C12 myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adhered on a conventional culture dish. Biotechnol. Bioeng. 2010; 105: 1161-1167. (C) 2009 Wiley Periodicals, Inc.

Two-dimensional cell patterns prepared on substrate surfaces by an electrochemical-based biolithography method have been transferred into fibrin gels prepared in situ. Line patterns of human umbilical vein endothelial cells (HUVECs) in the gel that was strained after the transfer formed a linear vessel-like structure within 8 days.

A microfluidic device was integrated with a controlled coculture system of HeLa cells and human umbilical vein endothelial cells (HUVECs). This integrated assembly allowed control of the direction of flow of medium (along with signaling factors secreted from cells) across the cultured cells. We grew HeLa cells and HUVECs to confluency on separate substrates and then joined the two substrates. A microfluidic device was then assembled onto the substrates and a cell coculture was initiated with controlled perfusion of the medium. When the medium flow was directed from the HeLa side to the HUVEC side, the HUVECs retreated and the HeLa cells migrated into the newly vacated areas. By contrast, when the medium flow was in the opposite direction, there was essentially no net movement of either cell type. Our results suggest that the migration of HeLa cells and HUVECs in coculture was likely mediated by soluble factors produced by HeLa cells.

We present a device enabling impedance measurements that probe the motility and mitosis of a single adherent cell in a controlled way. The micrometre-sized electrodes are designed for adhesion of an isolated cell and enhanced sensitivity to cell motion. The electrode surface is switched electro-chemically to favour cell adhesion, and single cells are attracted to the electrode using positive dielectrophoresis. Periods of linear variation in impedance with time correspond to the motility of a single cell adherent to the surface estimated at 0.6 mu m h(-1). In the course of our study we observed the impedance changes associated with mitosis of a single cell. Electrical measurements, carried out concomitantly with optical observations, revealed three phases, prophase, metaphase and anaphase in the time variation of the impedance during cell division. Maximal impedance was observed at metaphase with a 20% increase of the impedance. We argue that at mitosis, the changes detected were due to the charge density distribution at the cell surface. Our data demonstrate subtle electrical changes associated with cell motility and for the first time with division at the single-cell level. We speculate that this could open up new avenues for characterizing healthy and pathological cells.

A sequential power generation system for prolonging the net lifetime of a miniature biofuel cell stack has been developed. The system consists of layered chambers of enzyme fuel cells designed to be exposed sequentially to fuel solution by automatically switched fuel flow. The cell chambers were initially separated by magnetized plastic covers sealed with a degradable glue, poly(lactic-co-glycolic acid) (PLGA). The time that the cover was opened by attraction with an external magnet, thereby activating the following cell, was adjustable from a few hours to a few weeks by controlling the weight ratio of Fe3O4 in the covers and the molecular weight of PLGA. By using sequential power generation in this way, the power output of the system was stable for longer periods, and therefore the net lifetime of the stack has been extended as compared with that of a single biofuel cell.

This paper reports integration of quartz-crystal microbalance (QCM) sensors on a monolithic quartz crystal plate without using microfabrication. The QCM sensor used for this study is specially developed for sensing within a microfluidic channel. In this study, the distance between QCMs was optimized by monitoring frequency change caused by a mass load onto a neighboring QCM. It was found that circular shape QCM is suitable for the integration. The optimized QCM array was used for monitoring an etching of a gold electrode of the QCM by electrochemically generated oxidant. The measured phenomenon is practically useful for the cleaning of an integrated sensor within a packaged microfluidic channel.

The integration of electrochemical-based biolithography (ECBL) with an ordinary atomic force microscope (AFM) enables in situ lithography adjacent to a single, cultured cell, consequently allowing the morphological shape of the cell to be manipulated. The tip of a commercially available AFM cantilever was modified to serve as an electrode that could generate the oxidant HBrO for local, controlled etching of a cytophobic material (heparin or albumin) previously layered adjacent to a living cell. A NIH-3T3 fibroblast, initially confined to a patterned area, extended along a bioadhesive surface that had been newly exposed using the ECBL-AFM system. (C) 2009 Elsevier B.V. All rights reserved.

We have studied the coimmobilization of glucose dehydrogenase (GDH) and its cofactor, oxidized nicotinamide adenine dinucleotide (NAD(+)), on a ketjenblack (KB) electrode as a step toward a biofuel cell anode that works without mediators. A KB electrode was first treated with a sulfuric acid/nitric acid/water mixture to lower the overvoltage for NADH oxidation, and was next chemically modified with NAD(+) and GDH. The improved GDH/NAD(+)/KB electrode is found to oxidize glucose around 0 V vs. Ag/AgCl. A biofuel cell constructed with a bilirubin oxidase-immobilized KB cathode showed a maximum power density of 52 mu/cm(2) at 0.3 V. (C) 2009 Published by Elsevier B.V.

The current microdevices used for biomedical research are often manufactured using microelectromechanical systems (MEMS) technology. Although it is possible to fabricate precise and reproducible rectangular microchannels using soft lithography techniques, this kind of geometry may not reflect the actual physiology of the microcirculation. Here, we present a simple method to fabricate circular polydimethysiloxane (PDMS) microchannels aiming to mimic an in vivo microvascular environment and suitable for state-of-the-art microscale flow visualization techniques, such as confocal mu PIV/PTV. By using a confocal mu PTV system individual red blood cells (RBCs) were successfully tracked trough a 75 mu m circular PDMS microchannel. The results show that RBC lateral dispersion increases with the volume fraction of RBCs in the solution, i.e. with the hematocrit.

We describe a microfluidic method to selectively capture a type of leucocytes (neutrophils or eosinophils) from a suspension of mixed leucocytes in a specific region of a microchannel whose upper wall is equipped with an array of microband electrodes. One of the microelectrodes is used for locally electrogenerating hypobromous acid (HBrO) that renders the opposite face of the channel protein-adsorptive. Since the patterned layer of poly(ethylene glycol)dimethacrylate (PEGDM), impervious to HBrO, is previously introduced at the channel bottom, the region of protein adsorption can be precisely defined even in the presence of fluid-flow. After capture antibody was immobilized on the treated region, an AC voltage (1 MHz, 20 V(pp)) was applied to the microelectrode array for negative dielectrophoresis (DEP) to quickly concentrate cells from the flowing cell suspension. By immobilizing neutrophil- or eosinophil-specific antibodies onto the created region, we demonstrated that the corresponding leucocyte type can be captured from the concentrated leucocytes. (C) 2009 Elsevier B.V. All rights reserved.

We investigated the behavior of red blood cells (RBCs) in a microchannel with stenosis using a confocal micro-PTV system. Individual trajectories of RBCs in a concentrated suspension of up to 20% hematocrit (Hct) were measured successfully. Results indicated that the trajectories of healthy RBCs became asymmetric before and after the stenosis, while the trajectories of tracer particles in pure water were almost symmetric. The asymmetry was greater in 10% Hct than in 20% Hct. We also investigated the effect of deformability of RBCs on the cell-free layer thickness by hardening RBCs using a glutaraldehyde treatment. The results indicated that deformability is the key factor in the asymmetry of cell-free layer thickness. Therefore, the motions of RBCs are influenced strongly by the Hct, the deformability, and the channel geometry. These results give fundamental knowledge for a better understanding of blood flow in microcirculation and biomedical microdevices. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

We report a porous membrane-based cell culture device that can conduct localized electrical stimulation of a cell monolayer. The device&apos;s cell culture substrate is a microporous alumina membrane with an underlying thin poly(dimethylsiloxane) (PDMS) film spotted with holes. When electric current is generated between the device&apos;s Pt ring electrodes-one of which is placed above the cells and the other below the PDMS layer-the current density condenses at the holes in the PDMS film, and cells located above the holes can be electrically stimulated. C2C12 cells were confluently cultured on the substrate and were differentiated to myotubes. To control the stimulated area in the substrate, we attempted to seal and reopen the holes of the PDMS film by using an air bubble. Since the current pulse could be effectively blocked at the sealed holes, fluorescent Ca(2+) transients, indicative of cellular excitation, were observed from the myotubes located above holes in the open state.

We report the construction of microfluidic biofuel cells connected in series by semiautomatic air valves, in which a lotus leaf-like superhydrophobic structure (a micropillar array) traps air and ionically isolates each biofuel cell aligned within the channel. The micropillar array was prepared by conventional photolithography and reactive ion etching (RIE) techniques, and the structural parameters of the micropillars (size and spacing) were optimized as to obtain the highest degree of hydrophobicity. The open-circuit voltage measured at the ends of three aligned biofuel cells was three times larger than that of a single cell using the semiautomatic air valve system. ©2009 IEEE.

We report two novel techniques "Electrochemical Bio-Lithography" that enables in-situ cellular micropatterning, and "Ultra Anisotropic Electrodeposition" for making micropatterns of conducting polymers. The former technique for cellular micropatterning was combined with dielectrophoresis (DEP) and AFM, and we have realized the in-situ spatiotemporal cellular micropatterning in various environments. On the other hand, we have found the micropatterned hydrophobic area around electrode serves as a template for in-situ circuit formation with conducting polymers. Importantly, since the electropolymerization can be conducted without significant damage to the existing cell cultures, these two techniques can be combined to form hybrid micropatterns of cells and conducting polymers.

We investigated the interactions between HeLa cells and human umbilical vein endothelial cells (HUVECs) by monitoring their movements in a controllable coculture system. Two complementary, detachable, cell-substrates, one of polystyrene (PS) and the other of poly(dimethylsiloxane) (PDMS), were fabricated by replica molding. Coculturing was started by mechanically assembling two complementary substrates. One substrate was covered with a confluent layer of HeLa cells and its complement covered with confluent HUVECs. Using this coculture system as a tumor/endothelium model, we found that the HeLa cells migrated towards the HUVECs, while, simultaneously, the HUVECs retreated and that both types of cells migrated approximately twice as rapidly (two hundred microns per twenty-four hours) as they did alone. Additionally, when direct contact between the two cell types was prevented, the HUVECs initially migrated towards the HeLa cells and then retreated. The characteristics of the cell movements, i.e. direction and speed, probably are consequences of cell-cell signaling, with such signals possibly important during tumor cell intra- and extravasation.

An ordinary atomic force microscopy (AFM) was functionalized and applied to electrochemically draw micropatterns of biomolecules. To fabricate an electrochemical AFM probe having an electrode at the tip, a metal-coated AFM probe was first insulated with Parylene C, and then the apex of the tip was ground mechanically to expose the electrode. The effective electrode diameter was estimated to be ca. 500 nm. The electrode probe was positioned close to a heparin-coated antibiofouling substrate and used to locally generate hypobromous acid from a dilute Br(-) solution to render the substrate surface protein-adhesive. In situ topographical imaging after the electrochemical treatment suggested the heparin layer became detached to allow the adsorption of proteins, in this case fibronectin. The diameter of the drawn fibronectin pattern was 2 mu m, which is one order of magnitude smaller than we achieved previously using a microdisk electrode (tip diameter 10 mu m).

An electrode was coated by double layer of electrodeposited polypyrroles (PPy). The inner thicker PPy was doped with p-toluene sulfonate to ensure larger electrical capacity that is required for less-invasive stimulation of neural cells. The outer thinner PPy was doped with polyacrylate to present carboxyl group on the surface. Nerve Growth Factor was immobilized by utilizing the carboxyl groups, and successfully induced the differentiation of neuronal PC12 cells and their neurite growth. The present layered structure is one of effective approach to modify the high capacity PPy-based stimulation electrode with biomolecules for controlling cellular functions.

We describe a method to create cell-adhesive regions and to position adherent cells on the newly created regions in sequence within a microfluidic channel. One of the microelectrodes fabricated at the channel wall was used for locally electrogenerating hypobromous acid that renders the opposite face of the channel protein-adsorptive. After the fibronectin was immobilized on the treated region, an ac voltage (1 MHz, 20 Vpp) was applied to the microelectrodes array in the presence of suspended HeLa cells. Since a repulsive force of negative dielectrophoresis (DEP) directs the cells toward the weakest region of the nonuniform electric field, the cells were positioned on the fibronectin-patterned region to allow the cell adhesion, even in the presence of fluid flow (&lt; 0.1 mu L min(-1)). By repeating the above process, two types of cells could be patterned in the microchannel.

An enzyme-based glucose/O(2) biofuel cell was constructed within a microfluidic channel to study the influence of electrode configuration and fluidic channel height on cell performance. The cell was composed of a bilirubin oxidase (BOD)-adsorbed O(2) cathode and a glucose anode prepared by co-immobilization of glucose dehydrogenase (GDH), diaphorase (Dp) and VK(3)-pendant poly-L-lysine. The consumption of O(2) at the upstream cathode protected the downstream anode from interfering O(2) molecules, and consequently improved the cell performance (maximum cell current) ca. 10% for the present cell. The cell performance was also affected by the channel height. The output current and power of a 0.1 mm-height cell was significantly less than those of a 1 mm-height cell because of the depletion of O(2), as determined by the shape of the E-I curve at the cathode. On the other hand, the volume density of current and power was several times higher for the narrower cell. (C) 2007 Elsevier B.V. All rights reserved.

We describe a method to selectively immobilize a type of leucocytes (neutrophils or eosinophils) from a suspension of mixed leucocytes on the site-specific region inside a microchannel. One of the microelectrodes fabricated at the upper wall of the channel was used for locally electrogenerating hypobromous acid that renders the opposite face of the channel protein-adsorptive. Since the patterned layer of poly(ethylene glycol)dimethacrylate, impervious to HBrO, is previously introduced at the channel bottom, the region of protein adsorption can be precisely defined even in the presence of fluid-flow. After antibody was immobilized on the treated region, an ac voltage (1 MHz, 20 Vpp) was applied to the microelectrodes array in the presence of cell suspension flowing through the channel in order to concentrate cells onto the antibody-immobilized region by negative dielectrophoresis force. The results show that the immobilized antibody would selectively recognize the corresponding leucocyte type from the trapped cells to allow their adhesion. ©The Electrochemical Society.

We describe herein a method for the site-specific immobilization of proteins on a three-dimensional polydimethylsiloxane (PDMS) microstructure, i.e., an array of microposts, within the channel of a microfluidic device. The protein-adhesive sites in the preassembled device are protected by a layer of heparin, an antibiofouling agent. To ready a device for experimentation, electrical pulses from microelectrodes within the channel spatiotemporally generate hypobromous acid that quickly removes the heparin, exposing protein-adhesive surface. To prove that, for assays performed within microfluidic channels of identical dimensions, their relative sensitivities can be increased if the inner channel surface areas are increased by the presence of PDMS microstructures, a glutathione peroxidase (GPX) sandwich immunoassay was performed within a microfluidic channel that had a region without and a region with a microstructure. Even though both regions had the same two-dimensional areas, the added dimension of the PDMS microstructure significantly increased the sensitivity of the GPX assay. Finally, irregularly shaped, protein-adsorptive regions occur upon electrochemical treatment when there is fluid-flow even in the absence of moving liquid. We found that the shape of protein-adsorptive regions can be completely controlled, even in the presence of fluid-flow, when the protein-adsorptive regions are delineated by regions of poly(ethylene glycol)dimethacrylate. (C) 2007 Elsevier B.V. All rights reserved.

We report herein the invention of and proof of function for a porous membrane-based electroporation device that can deliver molecules into spatially restricted and predefined areas of a cell monolayer. The device's cell culture substrate is a microporous alumina membrane (pore size 0.02 mu m), with an underlying thin poly-(dimethylsiloxane) (PDMS) film that has one or more holes with diameters in the one-tenth millimeter range. When a transient electric field is generated between the device's two planar electrodes - one of which is placed above the cells and the other below the PDMS layer - the filed condenses only in the volume defined by hole in the PDMS film and therefore localized electroporation can occur. We demonstrate that Lucifer Yellow (LY) and plasmid DNA are selectively introduced into only those HeLa cells located above the holes. Using the device containing a PDMS film with multiple holes, a patterned array of LY-stained cells was resulted. Compared with the operation of and the results obtained from a conventional cuvette electroporation device, our device greatly decreases the necessary operating voltage, can be used with cells attached to a substrate, and increases the number of conditions that can be screened in a single experiment. Finally, since a PDMS film with different sized holes, produces different localized electric field strengths, it is possible to determine the optimum electroporetic conditions in a single experiment. (c) 2007 Elsevier B.V. All rights reserved.

cell adhesion inside silicone tubing. A platinum (Pt) needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with layer-by-layer deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The utility of this approach was demonstrated by patterning co-cultures of hepatocytes or endothelial cells with fibroblasts. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.

We have studied the possibility of making biocompatible, conductive patterns on a substrate by controlling the lateral growth rate of conducting polymers upon electropolymerization. Surface modification with heparin was found to enhance the lateral growth of polypyrrole, especially in the presence of dodecylbenzenesulfonate, and thus the micropatterning of heparin around electrodes leads to the formation of polypyrrole patterns.

Viamin K-3-modified poly-L-lysine (PLL-VK3) was synthesized and used as the electron transfer mediator during catalytic oxidation of NADH by diaphorase (Dp) at the anode of biofuel cell. PLL-VK3 and Dp were co-immobilized on an electrode and then coated with NAD(+)-dependent glucose dehydrogenase (GDH). The resulting enzymatic bilayer (abbreviated PLL-VK3/Dp/GDH) catalyzed glucose oxidation. Addition of carbon black (Ketjenblack, KB) into the bilayer enlarged the effective surface area of the electrode and consequentially increased the catalytic activity. An oxidation current of ca. 2 mA cm(-2) was observed when the electrochemical cell contained a stirred 30 mM glucose, 1.0 mM NAD(+), pH 7.0 phosphate-buffered electrolyte solution. The performance of glucose/O-2 biofuel cells, constructed as fluidic chips with controllable fuel flow and containing a KB/PLL-VK3/Dp/GDH-coated anode and an Ag/AgCl or a polydimethylsiloxane-coated Pt cathode, were evaluated. The open circuit voltage of the cell with the PDMS-coated Pt cathode was 0.55 V and its maximum power density was 32 LW cm(-2) at 0.29 V when a pH 7.0-buffered fuel containing 5.0 mM glucose and 1.0 mM NAD(+) was introduced into the cell at a flow rate of 1.0 mL min(-1). The cell's output increased as the flow rate increased. During 18 h of continuous operation of the cell with a load of 100 k Omega, the output current density declined by ca. 50%, probably due to swelling of the enzyme bilayer. (c) 2007 Elsevier Ltd. All rights reserved.

The electrically conducting polymer polypyrrole (PPy) was electrochemically deposited onto Pt microelectrodes on a polyimide (PI) substrate. Pre-modification of the PI surface with a self-assembled monolayer of octadecyltrichlorosilane-induced anisotropic lateral growth of PPy along the P1 surface and enhanced adhesive strength of the PPy film. The lateral growth of PPy film around the electrode anchored the whole film to the substrate. External stimulation of cultured cardiac myocytes was carried out using the PPy-coated microelectrode. The myocytes on the microelectrode substrate were electrically conjugated to form a sheet, and showed synchronized beating upon stimulation. The threshold charge for effective stimulation of a 0.8cm(2) sheet of myocytes was around 0.2 mu C, roughly corresponding to a membrane depolarization of 250 mV. (c) 2006 Elsevier Ltd. All rights reserved.

We report a method for producing patterned cell adhesion inside silicone tubing. A platinum needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with electrostatic deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors. © 2007 IEEE.

We report a method for producing patterned cell. adhesion inside silicone tubing. A platinum needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with electrostatic deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The controilled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.

We report a method for producing patterned cell co-cultures inside silicone tubing. A platinum needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with electrostatic deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.

Single crystals of Li(0.68)CoO(2), Li(0.48)CoO(2) and Li(0.35)CoO(2) were successfully synthesized for the first time by means of electrochemical and chemical delithiation processes using LiCoO(2) single crystals as a parent compound. A single-crystal X-ray diffraction study confirmed the trigonal R (3) over barm space group and the hexagonal lattice parameters a = 2.8107(5) angstrom, c = 14.2235(6) angstrom, and c/a = 5.060 for Li(0.68)CoO(2); a = 2.8090(15) angstrom, c = 14.3890(17) angstrom, and c/a = 5.122 for Li(0.49)CoO(2); and a = 2.8070(12) angstrom, c = 14.4359(14) angstrom, and c/a 5.143 for Li(0.35)CoO(2). The crystal structures were refined to the conventional values R = 1.99% and wR = 1.88% for Li(0.68)CoO(2); R = 2.40% and wR = 2.58% for Li(0.48)CoO(2); and R = 2.63% and wR = 2.56% for Li(0.35)CoO(2). The oxygen-oxygen contact distance in the CoO(6) octahedron was determined to be shortened by the delithiation from 2.6180(9) angstrom in LiCoO(2) to 2.5385(15) angstrom in Li(0.35)CoO(2)). The electron density distributions of these Li(x)CoO(2) crystals were analyzed by the maximum entropy method (MEM) using the present single-crystal X-ray diffraction data at 300 K. From the results of the single-crystal MEM, strong covalent bonding was clearly visible between the Co and O atoms, while no bonding was found around the Li atoms in these compounds. The gradual decrease in the electron density at the Li site upon delithiation could be precisely analyzed. (c) 2006 Elsevier Inc. All rights reserved.

We describe herein a method for controlling the pattern of permissible cell migration and proliferation on a substrate in time and space. Using this method, a confluent monolayer of cells that is confined within a defined region is released into a neighboring region. Incorporated into the method is an electrochemical technique that uses a scanning microelectrode to draw regions on the surface of the system that thereafter can support cell migration and growth. The supporting glass substrate is patterned with regions of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer that are not affected by the electrochemical treatment and also robustly resist cellular overgrowth as well as regions that can be individually switched when electrochemically treated from cell repellent to cell adhering. It is therefore possible to strictly define the areas into which cells can migrate. We found that HeLa cells migrate more rapidly as the width of cell-adhering lanes increases until a width of ca. 50 mu m is reached, at which point the migration rate is roughly constant. We also designed a drug assay using our cell migration technique. The technique allows for cell migration only into defined region(s) and therefore may become an important tool for evaluating the biological activity of potential drugs because drug activity and cell motility often directly correlate.

On-demand immobilization of proteins at specific locations in a microfluidic device would advance many types of bioassays. We describe a strategy to create a patterned surface within a microfluidic channel by electrochemical means, which enables site-specific immobilization of protein matrixes and cells under physiological conditions, even after the device is fully assembled. By locally generating hypobromous acid at a microelectrode in the microchannel, the heparin-coated channel surface rapidly switches from antibiofouling to protein-adhering. Since this transformation allows compartmentalizing of multiple types of antibodies into distinct regions throughout the single microchannel, simultaneous assay of two kinds of complementary proteins was possible. This patterning procedure can be applied to conventional microfluidic devices since it requires only some electrodes and a voltage source (1.7 V, DC).

The ability to control the behavior of living cells in vitro is critical in a wide range of research fields including fundamental cell biology, tissue engineering, and cell-based chip devices. Patterning cells on substrate surfaces is one of the most important approaches to modulate cellular behaviors and functions in culture. Here, we describe a cell patterning technique based on an electrochemical method, "electrochemical biolithography", which enables the localized immobilization of living cells under typical physiological conditions such as those found in culture media. The present technique is applicable even for a previously cell-patterned substrate and for a grooved substrate, opening new possibilities for the cell-based studies. Also, the integration of this electrochemical technique into a microfluidic device will provide a novel type of cell-chip which enables on-demand immobilization of cells just prior to the use of the device.

The ability to control of the behavior of living cells in vitro is critical in fundamental cell biology as well as applied biotechnology including tissue engineering and cell-based chip devices. Patterning cells on surfaces is one of the most important approaches to modulate cell behaviors and functions in culture. Here, we describe ongoing work on the newly developed surface patterning technique, "electrochemical bio-lithography", with a focus on the following two subjects: 1) patterning cells within preassembled microfluidic devices and 2) dynamic control of cellular motility.

To create an enzyme-based biological fuel cell generating electricity from glucose and 02, we modified a glassy carbon electrode with a bi-layer polymer membrane, the inner layer containing diaphorase (Dp) and the outer, glucose dehydrogenase (GDH, an NAD(+)-dependent enzyme). The Dp membrane was formed from a newly synthesized 2-methyl-1,4-naphthoquinone (Vitamin K-3; VK3)-based polymer. This polymer showed reversible redox activity at a potential close to that of free VK3 (-0.25 V vs. Ag/AgCl sat. KCl), and served as an electron mediator of Dp for the electrocatalytic oxidation of NADH to NAD(+). The addition of Ketjenblack into the Dp/VK3 film enhanced the generation of NAD(+). The outer GDH membrane oxidized glucose continuously using NAD(+) generated at the inner Dp film. To construct the glucose/O-2 biological fuel cell, we coupled the enzyme-modified anode with a polydimethylsiloxane-coated Pt cathode. The cells open circuit voltage was 0.62 V and its maximum power density was 14.5 mu W/cm(2) at 0.36 V in an air-saturated phosphate buffered saline solution (pH 7.0) at 37 degrees C containing 0.5 mM NADH and 10 mM glucose. Although its performance deteriorated to ca. 4 mu W/cm(2) over 4 days, the cell subsequently maintained this power density for more than 2 weeks.

Two-dimensional cross-linked polysiloxane Langmuir-Blodgett (LB) films were prepared and applied to nitric oxide (NO)-permselective membranes in order to block other electroactive interfering species. The cross-linked siloxane LB films deposited on platinum micro-disc electrodes (10 μm in diameter) offered revealing high performances as a permselective membrane for NO sensor such as high sensitivity to NO (detection limit, 40 nM) and high selectivity (e.g., the ratio of current response for acetaminophen or uric acid on the modified electrode to that on the bare electrode, less than 10-3). Furthermore, the permselective membrane could be easily deposited irrespective of the size and shape of electrode. © 2005 Elsevier Ltd. All rights reserved.

We have investigated a technique for the local delivery of chemicals to part of a cellular network. The device for the local stimulation was prepared by binding a permeable substrate made of a porous polycarbonate membrane and a micromask made of an elastomeric poly(dimethylsiloxane) (PDMS) film having a patterned hole (diameter, 50-200 mu m). As the model of the cellular network, primary cardiac myocytes were cultured on the membrane as a monolayer sheet and a line pattern, and then octanol (a gap junctional inhibitor) was locally delivered to the cells from the opposite phase of the porous substrate through a hole in the micromask. Intercellular communications of these cells were evaluated by observing the cytosolic Ca2+ transients. The synchronous Ca2+ transition on a myocyte sheet was stopped at only the area defined by the micromask. Based on the experiments using the line-patterned myocytes, it was proved that the electrically conjugating cellular network was divided into independent parts by the octanol stimulation across the cellular network. This research was carried out with the objective of developing a biosensing system based on a cellular network. (c) 2005 Elsevier B.V. All rights reserved.

An amperometric nitric oxide (NO)-sensing microelectrode was constructed using two-dimensional (213) cross-linked Langmuir-Blodgett (LB) films of siloxane copolymer as permselective membranes in order to block other electroactive interfering species. The 2D cross-linked siloxane LB films deposited on platinum micro-disc electrodes (10 mu m in diameter) were highly effective in the elimination of the interfering responses (e.g., the ratio of current response for dopamine on the modified electrode to that on the bare electrode, less than 10(-4)). Furthermore, high sensitivity to NO (detection limit, 40 nM) was also remained with modified LB films consisting of monolayers. (c) 2005 Elsevier B.V. All rights reserved.

The oxygen consumption rate of three-dimensional cultured cells integrated in a silicon chip was non-invasively quantified by scanning electrochemical microscopy (SECM). The cells were embedded in a collagen gel matrix and entrapped in silicon microstructures. The oxygen concentration profile near the cell panel agreed well with the theoretically determined spherical diffusion. The oxygen consumption rate (F) of a single cell was calculated as (1.0 +/- 0.19) x 10(-16) mol s(-1). The oxygen consumption of a cell panel increased along with the cellular proliferation; the increase in respiration activity was in accordance with the increase in cell numbers. Our three-dimensional micro-culture system combined with SECM enables continuous quantification of the oxygen consumption rate using very small amounts of sample. (c) 2004 Elsevier B.V. All rights reserved.

To create an enzyme-based biological fuel cell generating electricity from glucose and O-2, we modified a glassy carbon electrode with a bi-layer polymer membrane, the inner layer containing diaphorase (Dp) and the outer, glucose dehydrogenase (GDH, an NAD(+)-dependent enzyme). The Dp membrane was formed from a newly synthesized 2-methyl-1,4-naphthoquinone (Vitamin K-3; VK3)-based polymer. This polymer showed reversible redox activity at a potential close to that of free VK3 (-0.25 V vs. Ag/AgCl sat. KCl), and served as an electron mediator of Dp for the electrocatalytic oxidation of NADH to NAD(+). The addition of Ketjenblack into the Dp/VK3 film enhanced the generation of NAD(+). The outer GDH membrane oxidized glucose continuously using NAD(+) generated at the inner Dp film. To construct the glucose/O-2 biological fuel cell, we coupled the enzyme-modified anode with a polydimethylsiloxane-coated Pt cathode. The cell's open circuit voltage was 0.62 V and its maximum power density was 14.5 mu W/cm(2) at 0.36 V in an air-saturated phosphate buffered saline solution (pH 7.0) at 37 degrees C containing 0.5 mM NADH and 10 mM glucose. Although its performance deteriorated to ca. 4 mu W/cm(2) over 4 days, the cell subsequently maintained this power density for more than 2 weeks. (C) 2005 Elsevier B.V. All rights reserved.

Spatiotemporal control of surface properties under physiological conditions such as those found in culture media is an important technique in fundamental cell biology, tissue engineering, and cell-based bioelectronics. To this end, we have developed a mild, wet cellular micropatterning technique. The principle of the technique is based on the fact that the cell-repellant property of the albumin-coated substrate rapidly switches to cell-adhesive upon exposure to the reactive oxidizing agent, electrochemically generated hypobromous acid. Herein, we report the effect of the hypobromous acid on serum albumin physisorbed on a hydrophobic substrate. It was found that albumin molecules detach from the substrate by application of the oxidizing agent, resulting in exposure of the underlying hydrophobic surface to the liquid phase. The adsorption of extracellular matrix proteins such as fibronectin onto the hydrophobic surface induces cell adhesion and growth.

A novel three-dimensional cell culture system was constructed with an array of cell panels (4 x 5) in a silicon chip. together with multi-channel drug containers. Human breast cancer (MCF-7) cells were embedded in a collagen-gel matrix and entrapped in a pyramidal-shaped silicon hole. Each cell panel can be isolated by a channel composed of a microfluid part and a reservoir. A cell panel was exposed to 200 mm KCN for 2 days to demonstrate that each cell panel could be independently evaluated under various stimulation conditions. Based on the cellular respiration activity, the proliferation behavior was continuously monitored on the silicon-based cell array for 5 days using scanning electrochemical microscopy (SECM). The cells entrapped in the device (3-D culture) proliferated normally, and the proliferation rate was lower than that of cells grown in a monolayer cell culture (2-D culture). The effects of three anticancer drugs measured simultaneously on the cell chip were in good agreement with those obtained by a conventional colorimetric assay. Our results suggest that the silicon-based device for 3D culture is appropriate for a chemosensitivity assay involving multi-chemical stimulation. (C) 2004 Elsevier Ltd. All rights reserved.

The use of microfluidic system is a recent trend in diagnostic assays and bioreactors based on the protein and cellular engineering. Most of microfluidic biodevices would require the combination with a technique to pattern proteins and living cells within microchannels just prior to the use of the devices, since these bio-elements are delicate against desiccation, oxidation and beat. Here, we present a simple, electrochemical method to locally immobilize proteins and cells on substrate surface, easily applicable to the microfluidic system. The integration of the technique into a microfluidic device and the on-demand immobilization of proteins and cells were achieved. The sandwich immunoassay within the microchannel was demonstrated, in which a series of protocols from immobilization to measurement can be carried out under physiochemical conditions.

Controlling the interfaces between cells and materials is important subject for a wide range of research fields such as cell biology, tissue engineering, and biomedical devices. Here we report a novel electrochemical method to direct the adhesion and growth of mammalian cells on a substrate during cultivation in situ, named "Electrochemical Bio-Lithography". We found that the cell-repellent nature of the albumin- or heparin-coated substrates can be locally switched to cell-adhesive, by treatment with hypobromous acid electrochemically generated at the tip of the scanning microelectrode. Since this technique can be conducted under typical physiological conditions, we were able to direct cellular proliferation and migration by drawing adhesive micropatterns over the preexisting cellular pattern. The integration of this electrochemical system into a microfluiclic device will provide a novel type of cell-chip which enables on-demand immobilization of cells just prior to the use of devices.

Two-dimensional (2D) cross-linked polysiloxane Langmuir-Blodgett (LB) films, which were prepared by polymer-polymer cross-linking on an airwater interface, were applied to selective permeable membranes for nitric oxide (NO) sensor. The 2D cross-linked siloxane LB films deposited on platinum electrodes were remarkably effective in the elimination of the interfering responses (e.g., acetaminophen, L-cysteine, and L-ascorbic acid) and a rapid response to NO remained with modified LB films consisting of monolayers.

The microcontact printing (muCP) is a well-established method to pattern a material of interest using an elastomeric stamp. We have developed two techniques which assist the: muCP-based cell-patterning for the controlled growth guidance of cultured neuronal cells on, substrates. (i) Contact-transfer of extracellular matrix (ECM) protein on a microelectrode array substrate was achieved by spatially designing the microstamp to allow printing proteins even on the surface having uneven structures, and the differentiated PC12 cells showed selective adhesion and growth in the pre-determined locations on the electrode array. (ii) Cell alignment onto the pre-patterned ECM protein was also succeeded by using the microstructured silicon wafer having a band array of microholes, and the placed PC12 cells extended their axons along the protein pattern. These researches were carried out with the objective to developing a cell-based device based on a cellular network.

We have investigated a technique for the local stimulation of cultured cells by considering the combination of electrochemical bioassay using scanning electrochemical microscopy (SECM). The device for the local stimulation was prepared by binding a permeable substrate made of a microporous alumina membrane (pore size 0.02 mum, surface porosity 50%) and a micromask made of an elastomeric poly(dimethylsiloxane) (PDMS) (40 mum thickness having a 50 mum diameter hole). HeLa cells were cultured on the membrane and a chemical (ethanol, in this study) was locally delivered from the opposite phase. The live-dead staining experiments showed that the ethanol stimulation was applied only to the cells in the area defined by the micromask. The electroactive species around the stimulated and non-stimulated cells were simultaneously measured by SECM using a 5.0 mum-radius Pt microelectrode as a probe at -0.5 V versus Ag/AgCl and comparatively evaluated. (C) 2004 Elsevier B.V. All rights reserved.

We describe a novel anticancer drug sensitivity assay on a silicon chip applicable for tumors extirpated from in vivo mammalians. Human promyelocytic leukemia (HL-60) cells were subcutaneously (s.c.) inoculated in SCID mice, then removed 31 days after the inoculation. The cells were embedded in a small volume (18 nL) of a collagen-gel matrix on a pyramid-shaped silicon microstructure for further cultivation. The respiration activity of the cells on the chip was measured by scanning electrochemical microscopy (SECM). The proliferation behavior was continuously monitored for 6 days. It seemed that the proliferation rate of the cells removed from the mice was lower than that cultured in a flask and conformed to that in mice. The effects of cisplatin (CDDP) and etoposide (VP-16) on the HL-60 cultured in vivo were in good agreement with those obtained by a conventional colorimetric assay. Our results suggest that the SECM-based assay is appropriate for biopsy specimens in a relatively short-time evaluation. (C) 2003 Wiley-Liss, Inc.

The nature of an albumin-coated substrate that blocks protein adsorption and cell adhesion was rapidly switched to cell-adhesive by exposure to an oxidizing agent such as HBrO. This finding has enabled cellular pattern drawing even on a single-cell level by closely scanning a microelectrode above the substrate and electrochemically producing the agent at the tip of the electrode. The present microelectrochemical cell patterning is applicable even for a previously cell-patterned substrate and for a grooved substrate. These unique technical features will have impacts on a variety of cell-based studies that require the analysis of heterotypic cell-cell interactions and cellular arrangement on an uneven surface such as semiconductor devices.

The cardiac myocytes patterned on a single cell level were prepared by microcontact printing, and the response to chemical stimuli was studied using confocal fluorescent Ca2+ imaging. The patterned iriyocytes were found to conjugate by forming gap junction. It was confirmed for the patterned myocytes that gap junction communication was reversibly inhibited by 1-octanol, but activated by caffeine. Localized stimulation with chemicals was also attempted using a microinjection system for the myocyte patterns formed by single cell alignment. This research was carried out with the objective of developing a bioassay system based on a cellular network. (C) 2003 Elsevier Science Ltd. All rights reserved.

The respiratory activities of cultured HeLa cells were monitored at a single cell level using scanning electrochemical microscopy (SECM) that produces images of the localized distribution of oxygen around the cell. The change in the cellular activity was traced after exposures to KCN, ethyl alcohol and the antibiotic drug, Antimycin A. The results were compared with those from the conventional fluorescence monitoring using Calcein-AM that is sensitive to deformation of the cell membrane. The SECM-based measurement follows the decrease in the cellular activity upon exposure to KCN and Antimycin A more rapidly than the fluorescence-based measurements, demonstrating that SECM is suitable for studying the cellular influence of respiration inhibitors. (C) 2003 Elsevier Science B.V. All rights reserved.

Scanning electrochemical microscopy (SECM) was applied to the Study of the spatial distribution of the respiratory activity in a PC12 neuronal cell. The SECM imaging of the oxygen concentration around the cells indicated that the axon of PC12 as well as its cell body consumed oxygen as the result of mitochondrial respiration. The variation in respiratory activity during NGF-induced axonal growth was monitored at the growth cone of the axon. The respiratory activity at the growth cone Was found to be particularly activated by the NGF treatment. (C) 2003 Elsevier Ltd. All rights reserved.

The enzymatic activity of diaphorase (Dp) immobilized on a solid substrate was characterized using a scanning electrochemical microscope (SECM) with shear force feedback to control the substrate-probe distance. The shear force between the substrate and the probe was monitored with a tuning fork-type quartz crystal and used as the feedback control to set the microelectrode probe close to the substrate surface. The sensitivity and the contrast of the SECM image were improved in the constant distance mode (distance, 50 nm) with the shear force feedback compared to the image in the constant height mode without the feedback. By using this system, the SECM and topographic images of the immobilized diaphorase were simultaneously measured. The microelectrode tip used in this study was ground aslant like a syringe needle in order to obtain the shaper topographic images. This shape was also effective for avoiding the interference during the diffusion of the enzyme substrates. (C) 2003 Elsevier B.V. All rights reserved.

A microbial chip for bioassay was fabricated and its performance was characterized by scanning electrochemical microscopy (SECM). The microbial chip was fabricated by filling the micropores on a glass substrate with collagen gel embedding Escherichia coli (E.coli) cells. For measuring the activity of E.coli cells, the SECM measurement using ferricyanide as an electron mediator showed that the respiration activity of the immobilized E.coli cells increased with the concentration of glucose in the solution. The results suggested that the microbial chip can be used as a chemical sensor for nutritive species.

The immobilization of two enzymes on a pair of Au microband electrodes was performed by using electrochemical desorption of self-assembled monolayer (SAM) of alkanethiol. Both of the An electrodes were coated with the SAM of n-octadecanethiol (ODT-SAM) at first. The ODT-SAM on one of the Au electrodes was electrochemically removed by applying reductive potential. The resulting naked Au surface was re-coated with the SAM of 2-aminoethanethiol (AET). These treatments resulted in a couple of Au electrodes coated with the ODT-SAM and AET-SAM, respectively. Horseradish peroxidase. (HRP) was selectively immobilized on the AET-SAM by using crosslinking agent, glutaraldehyde. On the other hand, diaphorase (Dp) was immobilized on the surface of ODT-SAM by hydrophobic interaction. The imaging of the resulting substrate with scanning electrochemical microscope (SECM) demonstrated the enzymatic activities of HRP and Dp at the AET- and ODT- treated An electrodes, respectively.

A hybrid system consisting of a scanning electrochemical microscope (SECM) and scanning confocal microscope (SCFM) was fabricated and used for micropatterning and imaging of diaphorase immobilized on a glass substrate. Simultaneous imaging of the diaphorase spots demonstrated that the SECM can provide information on a localized enzyme reaction, while the SCFM affords information on the location of the active enzyme. By using this SECM/SCFM system, spatially selective deactivation of diaphorase was performed by inducing a local electrochemical reaction or by irradiating with the focused laser. The resulting patterns of diaphorase were simultaneously imaged with the SECM/SCFM system. 2003 Elsevier Science B.V. All rights reserved.

The respiratory activity of collagen-embedded living cells was imaged by scanning electrochemical microscopy (SECM) with the objective to study anticancer drug sensitivity. Two kinds of cancer cells, the human erythro-leukemia cell line (K562) and its adriamycin-resistant subline (K562/ADM), were immobilized at the array of microholes micromachined on a silicon wafer for comparative characterization of their sensitivity to the anticancer drug, ADM. The results obtained by the SECM method showed correspondence to a conventional colorimetric assay (SDI assay). Furthermore, since the SECM assay is based on the noninvasive measurement of the respiration activity, continuous monitoring of a dose response was possible.

Scanning chemiluminescence microscopy (SCLM) with electrophoretic injection was developed and applied to visualize enzyme reactions localized in an enzyme microspot. The SCLM uses a tapered glass capillary as a probe for injecting a small amount of luminol onto the substrate to generate localized chemiluminescence. The electrophoretic injection by application of a constant current between the inside and outside of the capillary enabled the continuous and controllable injection of a minute quantity of luminol in the range of 0.1 pmol/s. The image of enzyme activity in a monolayer spot of horseradish peroxidase was obtained by using the electrophoretic injection-based SCLM system. (C) 2003 Elsevier Science B.V. All rights reserved.

The patterning of cardiac myocytes on a micron scale (similar to5 mum) was achieved by microcontact printing of fibronectin onto a hydrophobically pretreated glass substrate. The patterned cardiac myocytes conjugated with each other by forming a gap junction, as judged from the synchronized Ca2+ transition over the pattern, and thus simultaneously contracted. The dynamic change of the Ca2+ concentration within the patterned tissue was analyzed quantitatively during successive contraction and relaxation using a Nipkow-type high-speed confocal microscope. (C) 2003 Wiley Periodicals, Inc.

A simply designed valveless switch for microparticle sorting was fabricated on a glass chip. A successful sorting of 10 mum diameter polystyrene latex beads was performed by the microfluidic system consisted of a unique electrophoretic switch and pair of parallel laminar flow streams. In applying the voltage to the electrodes placed on the banks of the flow through channel, microparticles were electrophoretically diverted across the boundary between two distinct laminar flows. (C) 2003 Elsevier Science B.V. All rights reserved.

Micropatterns of the electrically conjugating cardiomyocytes were prepared on a single cell level by microcontact printing (mCP), and the localized chemical stimulations were applied to the cellular pattern using multiple laminar flows. The locally delivered 1-octanol inactivated part of the myocyte patterns, while the other areas retained the activity showing spontaneous and synchronous pulsatility. Since both the cells and flows were well defined as micropatterns and each integrated on a chip, the obtained results simply demonstrate the cellular responses in a single-cell network.

A microbial chip was fabricated by filling the micropores on a glass substrate with collagen-embedded Escherichia coli ( E. coli) cells, and characterized by scanning electrochemical microscopy (SECM) in a solution containing ferricyanide. The activity of the E. coli cells in the collagen gel microstructure was imaged and characterized with SECM by mapping the localized concentration of ferrocyanide produced by the respiration of the cells. The SECM-based activity measurement detected as low as approximately 100 E. coli cells. Furthermore, the optical-microscopic observation indicated that the E. coli cells on the chip proliferated during the incubation. The sequential SECM measurements were performed for the same E. coli chip to obtain the microbial growth curve for a small number of microorganisms.

The micropatterns (similar to5 mum) of HeLa cells were maintained for more than 4 days on a glass surface modified by the corresponding pattern of a covalently attached polyethylene glycol (PEG) monolayer, even in the presence of serum proteins.

Scanning electrochemical microscopy (SECM) and scanning chemiluminescence microscopy (SCLM) were used for imaging an enzyme chip with spatially-addressed spots for glucose oxidase (GOD) and uricase microspots. For the SECM imaging, hydrogen peroxide generated from the GOD and/or uricase spots was directly oxidized at the tip microelectrode in a solution containing glucose and/or uric acid (electrochemical (EC) detection). For the SCLM imaging, a tapered glass capillary (i.d. of 1 similar to 2 mum) filled with luminol and horseradish peroxidase (HRP) was used as the scanning probe for generating the chemiluminescence (CL). The inner solution was injected from the capillary tip at 78 pl s(-1) while scanning above the enzyme-immobilized chip. The CL generated when the capillary tip was scanned above the enzyme spots was detected using a photon-counter (CL detection). Two-dimensional mapping of the oxidation current and photon-counting intensity against the tip position affords images of which their contrast reflects the activity of the immobilized GOD and uricase. For both the EC and CL detections, the signal responses were plotted as a function of the glucose and uric acid concentrations in solution. The sensitivities for the EC and CL detection were found to be comparable. (C) 2002 Elsevier Science B.V. All rights reserved.

The micropatterns of mammalian cells (HeLa cells) were prepared on glass substrates, and the respiration of the patterned cells was studied by microelectrode techniques, mainly by scanning electrochemical microscopy (SECM). The cellular patterns on a micrometer scale were prepared by microcontact printing of an extracellular matrix protein, fibronectin, onto a hydrophobic glass plate. The oxygen concentration in the vicinity of the patterned cells was mapped by scanning a Pt microelectrode, and the obtained SECM images proved that the cells in patterns were living with the uptake of oxygen. HeLa cells in the band patterns were well spread, while the cells in the small island patterns were restricted in their shape. The respiratory activities of these cells were evaluated by measuring the difference in the oxygen concentration between the bulk solution and the cell surface, and it was shown that a spreading cell consumed a significantly larger amount of oxygen than a round cell.

Electrochemical studies of spherical nickel hydroxide Ni(OH)(2) with high density were carried out by using a microelectrode technique. A carbon fiber microelectrode was contacted with a single particle of nickel hydroxide in 5 M KOH aqueous solution. In this way, cyclic voltammetry and galvanostatic charge/discharge experiments were carried out. The Ni(OH)(2); NiOOH redox reaction, oxygen evolution reaction and charge/discharge capacity were examined without any dilution with a binder and a conductive additive. Data were compared with those of the composite electrode consisting of the nickel hydroxide particle and a polymer binder, which is the practical form in the Ni-MH batteries. The potential-step technique was also performed to evaluate the apparent chemical diffusion coefficient of the proton (D-app) in the nickel hydroxide. Since the nickel hydroxide particle was a dense, conductive sphere, the spherical diffusion model was employed for the analysis. The D-app was found to be around 10(-9) cm(2) s(-1). (C) 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.

A microelectrode technique was applied to investigate the electrochemical properties of LiMn2O4 particles at elevated temperatures. Cyclic voltammograms of LiMn2O4 were measured after the particles were exposed to the electrolytes. This technique results in rapid and precise evaluation of the redox behavior of the materials. A significant capacity fading was observed in 1 M LiPF6/EC + PC electrolytic solution, which indicates that both LiMn2O4 and LiPF6 participate in the reaction to produce an inert material on the particle surface. Next, the capacity fading for two different BET surface area particles were compared using 1 M LiPF6/ EC+ PC at 50 degreesC. The reaction was found not to be controlled by the surface area. Finally, a Li1.1Mn1.9O4 particle was employed. The fading in discharge was ca. 10% for 50 cycles even at 50 degreesC, which means that the partial substitution of Mill in LiMn2O4 by Li substantially enhanced the capacity stability.

Scanning chemiluminescence microscopy (SCLM) was applied to visualize bienzyme reaction at a microspot (50 mum in radius) composed of glucose oxidase (GOD) and horseradish peroxidase (HRP). In an aqueous solution Containing glucose, a glass capillary tip was scanned over the bienzyme spot with luminol injected continuously from the tip to induce local chemiluminescence. The GOD catalyzes the oxidation of glucose by oxygen to form H2O2 which oxidizes luminol in the presence of HRP to generate chemiluminescence. Since the enzymatic luminescence reaction proceeds only within a bienzyme spot, even an array of spots was clearly imaged by SCLM without any significant interference between neighbor spots.

A microbial chip for bioassay was fabricated and its performance was characterized by scanning electrochemical microscopy (SECM). The microbial chip was prepared by spotting a suspension of Escherichia coli on a polystyrene substrate by using a glass capillary pen. The respiration activity of the E. coli spot was imaged with SECM by mapping the oxygen concentration around the spot. The SECM images of the microbial chips clearly showed spots with lower reduction currents, indicating that E. coli in the spots uptake oxygen by respiration. The bactericidal effects of antibiotics (streptomycin and ampicillin) were measured using the E. coli-based microbial chip, and discussed in comparison with the minimum inhibitory concentration (MIC) determined by an agar plate dilution method. (C) 2001 John Wiley & Sons, Inc.

A single crystal of LiMn2O4 (similar to 35 X 20 X 12 mum) was prepared by a flux method. The electrochemical properties of this material were investigated in a 1 M LiClO4/propylene carbonate + ethylene carbonate solution using a microelectrode technique. A Pt microfilament was brought into contact with the single crystal, and cyclic voltammetry and potential step chronoamperometry measurements were successfully performed on this electrode. The single crystal of LiMn2O4 exhibited reversible behavior during the Li-ion extraction/insertion. A new peak was observed at 3.86 V vs. Li/Li+ which was not clearly reported so far on polycrystalline of LiMn2O4. The chemical diffusion coefficient of Li-ion into the LiMn2O4 crystal was estimated to be 10(-11) cm(2)/s. (C) 2001 The Electrochemical Society.

We have developed a new class of synthetic membranes that consist of a porous polymeric support. This support contains an ensemble of gold nanotubules that span the complete thickness of the support membrane. The support is a commercially available microporous polycarbonate filter with cylindrical nanoscopic pores. The gold nanotubules are prepared via electroless deposition of Au onto the pore walls, and tubules that have inside diameters of molecular dimensions (&lt;1 nm) call be prepared. Hence, these membranes are a new class of molecular sieves. We review in this paper the ion-transport properties of these Au nanotubule membranes. We will show that these membranes call be cation-permselective or anion-permselective, and that the permselectivity can be reversibly switched between these two states. Ion permselectivity can be introduced by two different routes. The first entails chemisorption of all ionizable thiol, e.g., a carboxylated ammonium-containing thiol to the Au tubule walls. If the thiol contains both of these functionalities (e.g., the amino acid cysteine), the permselectivity call be reversibly switched by varying the pH of the contacting solution phase. Ion permselectivity, can also he introduced by, potentiostatically charging the membrane in all electrolyte solution. By applying excess negative charge, cation permselective membranes are obtained, and excess positive large yields anion permselective membranes. In this case the permselectivity can be reversibly, switched by changing the potential applied to the membrane.

Surface enhanced Raman scattering (SERS) has been applied to study the lithium intercalation/deintercalation process at the interface of a pyrolytic graphite electrode with propylene and ethylene carbonate containing organic solutions. We have focused on the lattice vibration of the most outer graphite surface layer simultaneously with cyclic voltammetric measurements. In situ Raman spectroscopy performed in this way allowed us to determine the La value that describes the size of graphitic microcrystallites along the a-axis. It was found that the La value decreases when the electrode is polarized to potentials between 0.02 and 1.0 V. This phenomenon can be correlated with the intercalation of lithium ions into the graphene structure. According to the spectral change, the size of the graphitic microcrystallites shows reversible behavior with potential cycling at the surface of the electrode.

Various aspects in the lithium battery field have been explored in our group: (i) uniform and dense thin films of LiMn2O4, up to 0.5 mum thickness have been synthesized by electrostatic spray deposition (ESD). The electrochemical properties of these films were investigated by cyclic voltammetry and impedance spectroscopy under a variety of experimental conditions. (ii) The kinetic and transport properties of Lithium insertion/extraction of numerous sphere-shaped single particles have been also evaluated by transient techniques as well as by ac-impedance spectroscopy. (iii) Electrochemical quartz crystal microbalance (EQCM) technique was employed to study the low capacity fading of LiMn2O4 at elevated temperatures in LiPF6 containing solutions. It has been confirmed that this phenomenon is due to the manganese dissolution promoted by acidic species (HF) originated from the reaction of LiPF6 with water. (iv) Alternative materials were sought to replace the actual LiCoO2 or LiMn2O4. Among them: Li1.1Mn1.9O4. LiNi0.85Co0.15O2, and Li,Li1.10Mn1.852Cr0.048O4. Interestingly, Li1.10Mn1.852Cr0.048O4 exhibited no significant capacity fading even in the 1 M LiPF6/PC-EC (1:1) solution at 50 degreesC upon 50 cycles of charge/discharge. (C) 2001 Elsevier Science B.V. All rights reserved.

This is the first report of impedance technique run on single particle LiCoO2 electrodes with the aim of clarifying its electronic and ionic transport properties. Measurements were successfully conducted on a LiCoO2 particle of 15 mum diam resulting in impedance magnitude on the order of M Omega. The impedance spectra exhibited (i) one semicircle in the high frequency region, (ii) Warburg impedance in low frequencies, and finally, (iii) a limiting capacitance in the very low frequencies. The spectra were analyzed using a modified Randles-Ershler circuit, so that the reaction kinetics could be precisely evaluated. The charge transfer resistance decreased as the potential increased, whereas the double layer capacitance was almost invariant with the potential. Thus, the apparent chemical diffusion coefficient (D-app) of lithium ions was determined to be 10(-11) to 10(-7) cm(2)/s as function of electrode potential. These results are in agreement with those obtained by potential step chronoamperometry technique. (C) 2001 The Electrochemical Society.

We have developed a new class of synthetic membranes that consist of a porous polymeric support that contains an ensemble of gold nanotubules that span the complete thickness of the support membrane. The support is a commercially available microporous polycarbonate filter with cylindrical nanoscopic pores. The gold nanotubules are prepared via electroless deposition of Au onto the pore walls; i.e., the pores acts as templates for the nanotubules. We have shown that by controlling the Au deposition time, Au nanotubules that have effective inside diameters of molecular dimensions (&lt;1 nm) can be prepared. Hence, these membranes are a new class of molecular sieves. In addition, because these membranes are composed of an electronically conductive material, excess charge can be applied to the tubules by electrochemical charging in an electrolyte solution. We have shown that this allows for control of ion-transport selectivity in these membranes. Finally, because the tubules are composed of gold, well-known Au-thiol chemistry can be used to change the chemical environment within the tubules. Via this route chemical transport selectivity can be introduced into these membranes. This paper reviews progress on size-based, charge-based, and chemical-interaction-based transport selectivity in this new class of membranes.

A uniform, dense film of spinel LiMn2O4 (0.1 mum thick) has been prepared by the electrostatic spray deposition (ESD) technique. The electroanalytical behavior of this electrode is elucidated by application of electrochemical impedance spectroscopy (EIS). The data have been modeled using an equivalent circuit approach. An excellent fit was found between measured data and an equivalent circuit, comprising Li+ migration through surface him, potential-dependent charge transfer resistance, semiinfinite Warburg-type element, reflecting solid state Li+ ion diffusion and a finite space Warburg-type element, describing both diffusion and accumulation of lithium at the very low frequency. The apparent chemical diffusion coefficient of lithium in the spinel phase was found within 10(-12) &lt; &lt;(D)over tilde&gt;(Li) &lt; 10(-9) cm(2) s(-1) as a function of electrode potential with minima at the potentials corresponding to the voltammetric peaks. The intercalation capacitance was found 0.7 &lt; C-L &lt; 47 mF cm(-2) exhibiting maxima at the potentials corresponding to the voltammetric peaks. (C) 2001 Elsevier Science B.V. All rights reserved.

Tin oxide thin films were prepared by the electrostatic spray deposition at 400 degreesC, followed by annealing at 500 degreesC in air. X-ray diffraction and X-ray photoelectron spectroscopy revealed that the resulting films were amorphous SnO2. The electrochemical properties of the SnO2 films with lithium were studied by in situ conductivity measurements using an interdigitated microarray electrode as well as by cyclic voltammetry, galvanostatic cycling measurements and ac-impedance spectroscopy in 1 M LiClO4/(PC + EC). The SnO2 film electrodes exhibited reversible capacities greater than 1300 mA h g(-1) when cycled between 0.05 and 2.5 V at 0.2 mA cm(-2). However, this capacity faded rapidly after repeated cycling. If the electrode was cycled between 0 and 1.0 V, a reversible capacity of 600 mA h g(-1) was maintained for more than 100 cycles. In addition, a stable reversible capacity of about 500 mA h g(-1) was obtained even at current density as high as 2 mA cm(-2). Thus it is suggested that a higher potential than 1.5 V would cause reformation of tin oxide, which may destroy the stable nanocomposite structure of metallic tin and lithium. These arguments were supported by in situ conductivity measurements with microarray electrodes. (C) 2001 Elsevier Science Ltd. All rights reserved.

Scanning electrochemical microscopy (SECM) was applied to the immunoassay of leukocidin, which is a toxic protein produced by methicillin-resistant Staphylococcus aureus (MRSA), with the intention of developing and early diagnostic for MRSA infection. An antibody-chip for leukocidin was prepared by self-assembling of anti-leukocidin on a protein A-coated glass substrate. A sample solution containing leukocidin was spotted onto the antibody-chip, followed by labeling with horseradish peroxidase (HRP) via a sandwich method. The reduction current of the oxidized form of ferrocenylmethanol generated by the HRP reaction was monitored to view SECM images of the spot of captured leukocidin, The amplitude of reduction current depended on the concentrations of sample solutions used for making spots. This SECM-based immunoassay detects as low as 5.25 pg mL(-1) leukocidin.

The electrochemical stability of single particles of cathode materials (LiMn2O4, Li1.10Cr0.048Mn1.852O4, LiCoO2 and LiNi0.85Co0.15O2) was investigated by means of a microelectrode technique at 25 degrees C and 50 degrees C. The cycle stability was evaluated by multi-cyclic voltammetry. LiMn2O4 showed good cycle stability in LiClO4/propylene carbonate (PC) + ethylene carbonate (EC) and LiBF4/PC + EC solutions even at 50 degrees C. On the contrary, in LiPF6/PC + EC, significant capacity fading during charge-discharge was observed at 50 degrees C. The cycle stability of LiMn2O4 in the latter solution was improved by partial substitution of Mn by Cr and Li. Regarding LiCoO2, its cycle life in LiClO4/PC + EC at 50 degrees C was unsatisfactory when the potential was scanned between 3.60 and 4.30 V. On the other hand, LiCoO2 retained 90% of its capacity when the potential scan was limited to 4.00 V. LiNi0.85Co0.15O2 showed similar trend at 50 degrees C. (C) 2000 Elsevier Science S.A. All rights reserved.

We have investigated the electric field dependence of the carrier photogeneration efficiency in an organic photoconductor with high sensitivity. The rate-determining step to generate photocarriers was considered to be the electron transfer between the neighboring two-redox molecules, which is influenced by the electric field. The overall carrier photogeneration efficiency was expressed by employing the electron-transfer velocity at the rate-determining step as a function of electric-field-dependent activation energy. This model successfully fits the experimental results for an organic photoconductor over a wide range of field strengths. (C) 2000 Published by Elsevier Science B.V.

Electrochemical properties of a LiMn2O4 Blm in 1 M LIPF6/PC + EC solution was studied by means of the electrochemical quartz crystal microbalance (QCM) at an elevated temperature (50 degrees C). The film preparation was conducted by an electrostatic spray deposition, which enabled us to obtain a uniform and dense film of LiMn2O4 on a QCM electrode at low temperature (400 degrees C) to avoid the thermal damage of the quartz. During the galvanostatic charge-discharge experiment, the mass of electrode specimen increased, most likely due to the formation of a passivation film. If an as-prepared electrode was only immersed in the LiPF6 solution at 50 degrees C, the mass of electrode decreased steadily, resulting eventually in extinction of the manganese film. Such manganese dissolution was attended by a shift of open circuit voltage from 3.8 to 4.3 V vs. Li, suggesting a formation:for lambda-MnO2 as an intermediate in the dissolution reaction. (C) 2000 The Electrochemical Society. S0013-4651(99)09-053-9. All rights reserved.

Electrical conductivity of LiMn2O4, which is a promising cathode active material for lithium ion batteries, was monitored in situ during electrochemical lithium extraction and reinsertion reactions in 1 M LiClO4 propylene carbonate solution. The in-situ conductivity measurement was achieved by means of an interdigitated microarray electrode coated with a uniform and dense film of LiMn2O4 The conductivity of Li1-xMn2O4 was found to exhibit a peak-shaped profile as a function of lithium content. The conductivity of Cr3+-doped spinel, LiMn1.95-Cr0.05O4, decreased monotonically with decreasing lithium content. These results are discussed by considering the effects of phase transformation on the conductivity of these materials.

Electrochemical lithium-ion extraction/insertion properties of Li1-xNiO2 single particles were investigated by attaching a filament microelectrode to the particle in 1 mol/dm(3) LiClO4/ethylene carbonate + propylene carbonate electrolyte. High-resolution cyclic voltammograms and galvanostatic chronopotentiograms were recorded. In addition, we observed in situ particle fracture during charge-discharge using an optical microscope equipped with a charge-coupled device camera. We found that the particle fractures when it is polarized above 4.2 V vs. Li/Li+. This phenomenon was explained by the change in crystal parameters expected to occur for x &gt; 0.75 in Li1-xNiO2. (C) 2000 The Electrochemical Society. S1099-0062(99)11-042-3. All rights reserved.

The microelectrode technique was applied to investigate the electrochemical properties of LiCo1-xMnxO2 (x = 0, 0.01, 0.05, 0.2, or 0.5) synthesized using the citrate process. From the X-ray diffraction measurements, an expansion of the c-axis and a decrease in the crystal size of the materials were observed on substitution of Mn into LiCoO2 In the electrochemical measurements, the high-speed cyclic voltammogram for the Mn-substituted materials gave one set of peaks at 3.9 V vs. Li/Li+. The apparent chemical diffusion constant (D-app) of LiCo0.8Mn0.2O2 obtained from the potential step experiment was 6.4 x 10(-8) cm(2)/s, which is larger than that of LiCoO2. The increase in D-app is attributable to the expansion of the c-axis and/or the decrease in the crystal size. In addition, the increase in Mn substitution up to 20% lead to an improvement in the kinetic reversibility and the cycle stability of LiCoO2.

In situ Raman spectra of active oxygen species present in solutions of potassium superoxide (KO2) in (62 + 38) mol% (Li + K)(2)CO3 eutectic at 650 degrees C were observed. We have attempted to obtain vibrational spectra of these entities by surface enhanced Raman scattering (SERS) technique.

Electrical de conductance of a composite electrode for a lithium-ion battery was monitored in situ during successive charge/discharge cycles, in order to investigate functions of conductive additives. Such measurement was achieved by means of an interdigitated microarray electrode with a bipotentiostat. The composite films of mesocarbon microbeads ( MCMBs) heat-treated at 1000 degrees C were studied with conductive additives of acetylene black or a synthetic graphite in a 1 M LiClO4, non-aqueous carbonate solution. The role of the conductive additives is discussed in connection with their type and content within the composites. In addition, we propose a modified preparation procedure for the composites having a high retention of conductance. (C) 1999 Elsevier Science S.A. All rights reserved.

Electrochemical preparation of polymerized self-assembled monolayers on an Au(lll) substrate has been attempted by using aminobenzenethiol and 3-aminophenethylthiol. A self-assembled monolayer of aminobenzenethiol was easily polymerized if it consisted of ortho and meta isomers in their molar ratio of 1:1 but not at a single component monolayer of each isomer. In contrast, a self-assembled monolayer of a single component of 3-aminophenethylthiol allowed its easy electrochemical polymerization. The polymerized monolayers gave electrochemical activities very similar to those of conventional polyaniline and its derivatives. Furthermore, the self-assembled monolayer of these thiols got high stability against reductive desorption in an alkaline solution when polymerized. Underpotential deposition of Cu gave Cu coverage of 41% and 65% on the electrodes coated with the polymerized monolayers of aminobenzenethiol and 3-aminophenethylthiol, respectively.

The effects of surface treatments on the hydrogen storage properties of a Mg2Ni alloy particle were investigated by the microvoltammetric technique, in which a carbon-filament microelectrode was manipulated to make electrical contact with the particle placed in a KOH aqueous solution. It was found that the hydrogen storage properties of Mg2Ni at room temperature were improved by the surface treatments with a nickel plating solution. The sodium salts (sodium phosphinate and sodium dihydrogen citrate) contained in the nickel plating solution made the alloy form an amorphous-like state, resulting in an improved hydrogen absorption/desorption capacity at room temperature as high as about 150 mA hg(-1) from the original value of 17 mA hg(-1).

The electrochemical quartz crystal microbalance (EQCM) technique was successfully used to investigate the lithium insertion/extraction reaction in LiMn2O4 spinel. A uniform and dense film of LiMn2O4 was prepared by electrostatic spray deposition (ESD) onto an Au-coated quartz plate, which was used as an electrode for the EQCM experiments. The ESD method enabled us to perform the low-temperature spinel synthesis at 400 degrees C td avoid the thermal damage of the quartz. The electrochemical properties of the LiMn2O4 film in a 1 M LiClO4 carbonate solution were studied at 50 degrees C. The data obtained for the lithium insertion/extraction were discussed for the first time in connection with the mass change of the LiMn2O4 specimen.

The electrical de conductance (S) and the solid-state diffusion coefficient of the lithium ion (D-Li(+)) were evaluated for a single spherical particle of mesocarbon microbead (MCMB) heat-treated at 1000 degrees C. The measurements were conducted in situ during the electrochemical lithium insertion into the MCMB by using microelectrode-based techniques. The data obtained are discussed as functions of the electrode potential and of the amount of inserted lithium. Both parameters (S and D-Li(+)) were found to depend on the lithium content, and their variation manner changed around 0.6 V vs Li/Li+ at which the inserted lithium amount was ca. 25% of the total insertion. The results would arise from the difference between the first and the second half of the lithium insertion reaction mechanisms.

Some of recent topics on the use of microelectrodes for the characterization of solid materials, especially the active materials of lithium ion batteries, were overviewed based mainly on our research. The microelectrodes used here can be classified to the microdisk and interdigitated array. The systems based on the microdisk electrodes were established for the characterization of a single particle of active materials, On the other hand, the interdigitated array microelectrodes were used for measuring conductivity changes of materials' film in situ during the lithium insertion / extraction reaction. It is demonstrated hers that the microelectrode-based characterization techniques bring unique information, which will contribute to the design of high performance lithium ion batteries.

Cadmium sulfide nanoparticles (Q-CdS) modified with 2-mercaptoethanesulfonate and 2-amiaoethanethiol in a molar ratio of 2:1 were covalently immobilized onto-an Au surface covered with a self-assembled monolayer of 3,3'-dithiobis(succinimidylpropionate), and the resulting electrodes were further immobilized with Q-CdS using glutaraldehyde as a binding agent. The degree:of anodic photocurrents was greatly influenced by charged conditions of hole scavengers used because of the presence of sulfonate groups on the Q-CdS surfaces; triethylamine having positive charges gave large photocurrents, triethanolamine medium photocurrents, and formate small photocurrents. If Q-CdS having a large emission from their surface trap states was used, anodic photocurrents were depressed with increasing anodic polarization from the onset potentials which were ca. -1.1 V vs SCE for the use of any kinds of hole scavengers, and the greatest depression appeared at -0.25 V, beyond which a steep increase in anodic photocurrents was seen. In contrast, no significant depression in photocurrents was observed and anodic photocurrents were monotonically increased, in the case of using Q-CdS having an intense band-gap emission. When the energetic position at the emission maximum is correlated to the potential at which the greatest photocurrent depression appeared, photocurrent-potential characteristics are discussed in terms of involvements of surface states in the photoelectrode reactions.

Two-dimensionally organized CdS nanoparticle films were prepared with the use of the Langmuir-Blodgett (LB) technique. 2-Aminoethanethiol-modified CdS nanoparticles were spread on a water subphase which contained glutaraldehyde as a cross-linking agent for binding the surface-modified CdS nanoparticles with each other, and the cross-linking reaction was undertaken under several different compressions of the CdS nanoparticle layer spread on the water subphase. Surface pressure-area isotherms of the CdS monoparticulate layer taken after the cross-linking were largely different depending on the area used for the cross-linking. By transfer of the CdS monoparticulate film prepared at the air-water interface to the 2-aminoethanethiol-modified gold substrate using the LB technique, CdS monoparticulate films containing different surface concentrations were prepared on the gold substrate. It was easily done to cumulate the monoparticulate film on the gold substrate using the same transfer technique. The prepared CdS nanoparticles films showed n-type photosensitivities which were enhanced by increasing the number of cumulation of monoparticulate films.

Mesophase pitch carbon (MPC) is one of the possible carbonaceous materials as an anode for rechargeable lithium ion batteries. The MPC thin film electrodes were fabricated by modifying reticulated-nickel substrates with a quinoline-soluble part of MPC. We have studied the electrochemical insertion/extraction properties of lithium ion into/from the MPC-based carbon, focusing on the effects of heat-treatment temperature (700-1000 degrees C). Potential excursion experiments show that there are two different mechanisms for extraction of lithium ions; one can be attributed to deintercalation from a graphitized part and the other to extraction from the ungraphitized part of the carbon film. (C) 1999 Elsevier Science Ltd. All rights reserved.

Some of the recent topics on the use of microelectrodes for the materials characterization, especially for the materials in battery and sensor applications, were overviewed. Results were presented for conducting polymers and for battery active materials such as a graphitized carbon. The filament-type and the interdigitated-array-type microelectrodes were prepared, and used respectively for the particle-level characterization and for the in situ conductivity measurement. It is demonstrated that the microelectrode-based characterization techniques bring unique information, which will contribute to the design of high performance micro devices. (C) 1999 Elsevier Science Ltd. All rights reserved.

Novel electrochemical techniques based on a filament-type and an array-type microelectrodes were developed to study in situ the conductance change of a carbon particle and its composite film during electrochemical lithium insertion/extraction reactions. Mesocarbon microbeads (MCMB, Osaka Gas Co.) heat treated at 1000 degrees C were investigated in an organic solution containing 1 M LiClO4 as the electrolyte. Measurements focusing on a single MCMB particle were achieved by attaching a molybdenum-filament microelectrode to the particle; they have shown that both the voltammogram and conductance profile of MCMB itself were stable in amplitudes and shapes for successive lithium insertion. Another kind of measurement using an array-type microelectrode was performed on a composite film consisting of MCMBs and poly(vinylidene fluoride), which is an actual form of MCMB in the use for lithium secondary batteries. The composite film was prepared on an interdigitated array of nickel microelectrodes to measure in situ its conductance change. It was found that the conductance of the composite film decreased rapidly, being accompanied by a decrease of redox capacity of the film. By considering the stable behavior of MCMB itself, we concluded that the electrical contact between MCMBs was broken due probably to the volume change of MCMB induced by the lithium insertion/extraction reactions. Addition of acetylene black to the composite greatly improved the interparticle connection. In addition to these practically important results, it is also suggested that there exist at least two different insertion sites within the MCMB. As demonstrated here, microelectrode-based techniques are unique and an effective approach to study battery active materials from both fundamental and practical standpoints.

Electrochemical hydrogenation/dehydrogenation properties were studied for a single particle of a AB(5)-type Mm-based (Mm: misch metal) hydrogen storage alloy, MmNi(3.55)Co(0.75)Mn(0.4)Al(0.3). A carbon fiber microelectrode was manipulated to make electrical contact with an alloy particle, and the cyclic voltammetry and the galvanostatic charge/discharge experiments were performed. A single particle of the alloy showed the discharge capacity of 280 mAhg(-1), the value being 90% of the theoretical capacity. In addition, the hydrogenation and the anodic oxidation of Co element in the alloy were clearly separated to be able to discuss in detail. Data were compared with that of the composite film consisting of the alloy particles and a polymer binder, which is more practical form for Ni-MH batteries. The potential-step experiment was also carried out to determine the apparent chemical diffusion coefficient of hydrogen atom (D-app) in the alloy. Since the ahoy particle we used here was a dense, conductive sphere, the spherical diffusion model was employed for data analysis. D-app was found to vary the order between 10(-9) and 10(-10) cm(2) s(-1) over the course of hydrogenation/dehydrogenation process. (C) 1999 Elsevier Science Ltd. All rights reserved.

A carbon thin film (thickness, ca. 1 mu m) was prepared by the electrostatic spray deposition onto a Ni-substrate at 400 degrees C and heat-treated at 500 degrees C in vacuum. Electrochemical lithium ion insertion/extraction properties of the resulting carbon film electrodes were studied by cyclic voltammetry and electrochemical impedance spectroscopy in IM LiClO4/ (PC + EC). In the case of slow scan rates less than 10mV / 22sec in step potential electrochemical spectroscopy, two anodic peaks appeared, indicating that there are two different insertion / extraction mechanisms. Cole-Cole plots of impedance spectra showed two well-separated semicircles and a linear locus. The first semicircle at higher frequency range seems to correspond to the migration of lithium ions through passivating film at carbon surface, and the second semicircle and linear locus arise from carbon thin film itself.

Lithium-ion insertion/extraction at a single particle (10-30 mu m in diam.) of LiMn2-xNixO4 (x = 0, 0.2, 0.5) was characterized by a microelectrode technique, in which a fine metal filament was manipulated to make electrical contact with the particle placed in 1 M LiClO4/PC+EC solution. By using this technique, we can evaluate the correct current-potential behavior of LiMn2-xNixO4 without any dilution due to an organic binder and a conductive additive. The cyclic voltammetry (CV) was performed for the particles up to a high anodic potential limit of 5.2 V vs. Li/Li+. Undoped LiMn2O4 shows two current peaks around 4.1 V due to the redox reaction of Mn3+/4+ in the solid state phase, while the CV of Ni-doped LiMn2O4 was strongly deformed, showing new current peaks in a high potential region (similar to 4.7 V) at the expense of the 4.1 V wave. The 4.7 V wave would arise from the redox reaction of Ni2+4+. The cyclability of LiMn2-xNixO4 was evaluated by multi-scan cyclic voltammetry.

Size-quantized CdS thin films were prepared by sequential underpotential deposition of S and Cd on Au(lll). The prepared films in an aqueous solution containing triethanolamine as a sacrificial electron donor showed n-type photoelectrode behaviors. Action spectra of anodic photocurrents were red-shifted with an increase of the film thickness toward characteristic values of those of bulk materials. The energy gap of the film determined from the action spectra showed a remarkable tendency of decrease with an increase of the film thickness up to 3.5 nm. Beyond 3.5 nm the rate of decrease with increase in the film thickness declined. An energy gap value characteristic of bulk materials was obtained with CdS films greater than 6.8 nm.

An uniform, dense film of spinel LiMn2O4 (thickness, about 0.5 mu m) was prepared by the electrostatic spray deposition method onto a Pt film electrode on Si substrate kept at 400 degrees C. Electrochemical properties of the LiMn2O4 film in a solution of 1 M LiClO4/propylene carbonate were studied by cyclic voltammetry and potential-step chronoamperometry. The oxidation and reduction of the film, which are accompanied by the extraction/insertion of lithium ions, proceeded almost quantitatively around 4 V vs. Li/Li+ with excellent coulombic reversibility. The apparent chemical diffusion coefficient of Li+ in Li1-xMn2O4 spinel phase varied on the order between 10(-10) and 10(-12) cm(2) s(-1) as a function of electrode potentials with minima at the potentials corresponding to the voltammetric current peaks.

Electrical conductivity of a sputter-deposited LiCoO2 thin film was studied in situ during electrochemical lithium extraction/insertion, revealing that the metallic behavior of Li1-xCoO2 induced by the initial lithium extraction did not revert to its original insulating state in the following successive reduction/oxidation cycles.

Thin films of LiMn2O4 have been prepared by RF magnetron sputtering on interdigitated microarray electrodes. In situ conductivity-potential profiles and cyclic voltammograms during extraction/insertion processes of Li ions were obtained simultaneously in nonaqueous and aqueous electrolyte solutions (1 M LiClO4/propylene carbonate and 1 M LiCl/water). The electronic conductivity of Li1-xMn2O4 was found not to show metallic transition and maintain its semiconducting state during the extraction/insertion of Li ion. A slight decrease in conductivity was observed with increasing the anodic potential, i.e., with increasing x (lithium extraction) and recovered reversibly when the potential returned to the cathodic side (re-insertion of Li ions). Similar results were obtained in both aqueous and nonaqueous electrolyte solutions.

The solid-state diffusion of lithium ion in carbon was directly investigated in single spherical particles of graphitized mesocarbon microbeads (MCMB, Osaka Gas Co.). In order to achieve electrochemical measurements focusing on a single particle, a molybdenum-filament microelectrode was maneuvered to make electrical contact with a single MCMB placed in an organic solvent mixture containing lithium ions as an electrolyte. This novel microelectrode technique gives electrochemical information about a MCMB particle, itself, without any dilution with additives such as organic binders. The chemical diffusion coefficient (D) of lithium ion was determined from potential-step chronoamperograms assuming spherical diffusion of lithium ions in the MCMB particle. The values of D were found to change the order between 10(-9) and 10(-11) cm(2)/s depending on the electrode potential. Plots of D showed pronounced minima at the potentials where the transformation of staging level of lithium intercalation compounds occurred. (C) 1998 The Electrochemical Society, Inc. S1099-0062(98)01-019-0.

Photoelectrochemical properties of spinel Li1-xMn2O4 (0 &lt; x &lt; 1) thin film on ITO electrodes were studied in propylenecarbonate (PC) containing 1 M LiClO4. Cathodic photocurrents were clearly observed, suggesting the occurrence of photo-assisted lithium insertion reaction at the Li1-xMn2O4 film electrode.

Underpotential deposition (UPD) of Ag on Au(111)/mica electrodes coated with a self-assembled monolayer (SAM) of propanethiol or octanethiol and reductive desorption of the SAM after conducting UPD have been studied using voltammetry, XPS measurement, and scanning tunneling microscopy (STM). The reductive desorption potential of the SAM was changed by UPD of Ag from a characteristic value obtained at Au to that at Ag, indicating that the UPD of Ag took place through the SAM layer in such a way as to intervene between the SAM and the Au electrode. No significant loss of thiol molecules occurred during the Ag deposition. The rate of UPD through the SAM of propanethiol was so fast as to be completed within 10 s, while that for the SAM of octanethiol took ca. 50 min or more to build up the SAM/Ag/Au structure. Voltammetric results indicated that the UPD of Ag proceeded initially at molecular defects in the SAM of octanethiol and that the resulting Ag islands grew laterally to limiting coverage. Ex situ STM observations showed clearly the presence of such Ag islands on terraces of the Au(111) electrode surface.

Electrodeposition of Cu on an Au(111)/mica electrode surface coated with a self-assembled monolayer (SAM) of propanethiol has been studied in a potential region comprising the underpotential deposition (UPD). The UPD of Cu and stripping of the deposited Cu took place reversibly through the SAM layer, and furthermore it was found that the SAM was still present on the electrode surface without significant changes in its amount and structure even after repeating Cu deposition/stripping cycles. The Cu adlayer stabilized the SAM completely toward its reductive desorption in an alkali solution, suggesting that a homogeneous Cu adlayer was formed on the entire electrode surface. A surface structure of the resulting electrode is discussed on the basis of these electrochemical results.

A self-assembled mixed-monolayer of 3-mercaptopropionic acid (MPA) and hexadecanethiol (KDT) was prepared on a gold electrode substrate by the co-adsorption from a mixed ethanol solution of these thiols, and the electrochemical reductive desorption behavior of the resulting mixed monolayer was studied in 0.5 M KOH aqueous solution. If ethanol containing 1 mM MPA and 0.2 mM HDT was used for preparation of monolayers, two cathodic waves due to desorption of the thiolates appeared at different potentials which accorded with reductive desorption potential of each component, suggesting strongly that a phase segregation occurred in the mixed monolayer. The integration of each current peak indicated that the fraction of HDT in the mixed monolayer tended to increase with increasing the soaking time of the gold electrode in the mixed solutions of thiols. The selective desorption of MPA was accomplished successfully from the mixed monolayer of MPA and HDT, and the resulting electrode showed electrochemical behavior characteristics of an array of microelectrodes, suggesting that domains of MPA were dispersed in the original mixed monolayer. By analyzing voltammograms taken in a Ru(NH3)(6)(3+) solution, assuming that a hexagonally arranged array of circular microdisk electrodes was formed by the desorption, the average radius of a domain of MPA was estimated to be ca. 25 nm for the mixed monolayer containing MPA with its molar fraction of 0.06. (C) 1997 Elsevier Science S.A.

Tubules of 200 nm outer diameter spinel LiMn2O4 were prepared by thermal decomposition of an aqueous solution containing lithium nitrate and manganese nitrate at 1:2 molar ratio using a nanoporous alumina membrane as a template. After dissolving the template membrane, the resulting nanotubule array of LiMn2O4 was coated with polypyrrole to investigate the galvanostatic charge-discharge characteristics. The polypyrrole-coated LiMn2O4 tubule electrodes exhibited higher capacities than the polypyrrole-coated LiMn2O4 thin-film electrode prepared under the same conditions, except for the use of the alumina membrane; it was ca. 2.5 times greater at 0.1 mA cm(-2) and became ca. 12 times greater with an increase in current density to 1.0 mA cm(-2). The observed high capacity of the tubule electrode seems to have resulted from two kinds of effects: a decrease in real current density of high specific surface area at the tubule electrode and a decrease in thickness of LiMn2O4 solid phase for Li+ ions to diffuse through during the charging and discharging reactions.

Size-quantized CdS particles (Q-CdS) capped with 2-aminoethanethiol and 2-mercaptoethanesulfonate were prepared by means of the AOT/heptane inverse micelles method. The resulting CdS particles were covalently immobilized in a high dispersion on an Au(lll) surface coated previously with a self-assembled monolayer of 3,3'-dithiobis(succinimidyl propionate). The immobilized Q-CdS particles were stable against tip scanning in scanning tunneling microscopy (STM). Tunneling spectroscopy (TS) of a single particle whose size was determined from a STM image allowed successfully the determination of the band-gap energy of the size-quantized particle, and the band-gap value obtained was in agreement with that predicted from the tight-binding approximation.

An indium-tin oxide (ITO) electrode coated with prussian blue (PB) film containing TiO2 particles (PB[TiO2]/ITO electrode) was prepared by electrochemical deposition of PB in the presence of suspended TiO2 particles. Irradiation of the prepared film with a high pressure mercury are lamp in a solution containing methanol as an electron donor caused photoinduced reduction of PB to colorless prussian white (PW). Irradiation of a spot on the PB[TiO2] film, however, did not restrict the reduction of PB to the irradiated part, rather the reduction area spread beyond the irradiation zone, the extent being larger with increasing irradiation time. The electron transport from the photoproduced PW to the surrounding PB through the conducting ITO substrate was found to be responsible for the observed spread. Area-selective photoreduction of PB to PW was successfully achieved by inserting a thin ferrocenylmethyltrimethylammonium (FA)-bound Nafion between the PB[TiO2] and ITO to give a PB[TiO2]/Nafion[FA]/ITO electrode. The preparation of this bilayer electrode was accomplished by electrochemically depositing a PB[TiO2] layer onto a Nafion[FA]-coated ITO electrode. FA in the Nafion layer blocked electrochemical reduction of PB to PW, but mediated oxidation of the photoproduced PW. White images were successfully formed in the blue films of the bilayer electrodes by irradiation under open-circuit condition.

This paper describes the sensing characteristics of conductive polymer/enzyme-coated microarray electrodes consisting of assembly of microband electrodes with a few mu m width and a few mm length. The sensing principle of the array devices is based on detection of conductometric changes induced by chemical reactions of analytes. The followings are the contents of this paper: 1. Fabrication of ultrathin conductive polymers at hydrophobically-pretreated microarray electrodes. 2. In situ conductivity measurements using microarray electrodes coated with conductive polymers. 3. Redox behavior of polypyrrole-coated microarray electrodes. 4. NADH switching behavior of polypyrrole/ diaphorase-coated microarray electrodes. 5. pH sensing behavior of polypyrrole- or polyaniline-coated microarray electrodes. 6. Penicillin sensing behavior of polypyrrole/penicillinase-coated microarray electrodes. 7. Glucose sensing behavior of polyaniline/glucose oxidase-coated microarray electrodes.

We fabricated an electrochemical photo memory device using a Pt-TiO₂-Pt microarray electrode coated with a polypyrrole film. The TiO₂ microband electrode, positioned between two comb-shaped Pt electrodes, was an n-type semiconductor to oxidize the polypyrrole film under light irradiation. Because oxidation enhances the conductivity of polypyrrole, light irradiation is detected by the increase in the conductivity of polypyrrole between the two Pt comb electrodes. Enhanced conductivity remained even after light irradiation was terminated. This photo memory behavior was observed when the potential of the titanium oxide electrode was set to -0.3V vs. SCE.

Membranes containing cylindrical metal nanotubules that span the complete thickness of the membrane are described. The inside radius of the nanotubules can be varied at will; nanotubule radii as small as 0.8 nanometer are reported. These membranes show selective ion transport analogous to that observed in ion-exchange polymers. Ion permselectivity occurs because excess charge density can be present on the inner walls of the metal tubules, The membranes reject ions with the same sign as the excess charge and transport ions of the opposite sign. Because the sign of the excess charge on the tubule can be changed potentiostatically, a metal nanotubule membrane can be either cation selective or anion selective, depending on the potential applied to the membrane.

The electrochemical behavior and electrical conductivity of C-60 thin films were studied in acetonitrile containing M(bpy)(3)(2+) (M = Fe, Ni, Ru or Os; bpy = 2,2'-bipyridine). Cyclic voltammetry of C,, films showed stable reduction-reoxidation waves. In situ conductivity measurements indicated that an enhanced conductivity (on the order of 10(-2) S cm(-1)) appeared reversibly on electrochemical reduction accompanied by doping of M(bpy)(3)(2+) ions into the C-60 films. The electrochemical reduction also induced some structural change.

A conductometric glucose sensor was fabricated by coating a twin-microband electrode with a bilayer of a membrane composed of polyaniline and glucose oxidase/gluconolactonase films. The enzyme-catalyzed hydrolysis of glucose induces a change in the conductivity of the pH-sensitive polyaniline film. The conductivity change was detected by the current flowing between the two-band electrode. The sensor demonstrates clear responses to glucose up to 1 mM.

Crystallographic changes of C-60 thin film during electrochemical reduction and reoxidation in acetonitrile containing of tetra-n-butylammonium (TBA(+)) were studied by X-ray diffraction (XRD) and scanning electron microscope (SEM). XRD spectra showed a reversible structure change due to the electrochemical doping of TBA(+).

Polypyrrole grows effectively along the glass substrates pretreated with n-alkylsilane reagents in electropolymerization from aqueous solutions, resulting in the formation of an ultrathin polypyrrole film at twin-microband electrodes. However, no effective promotion of lateral growth of polypyrrole was observed when the substrate was pretreated with 3-aminopropyltriethoxysilane or when the electrolysis was conducted in acetonitrile, suggesting that the hydrophobicity of the substrate is essential for promotion of the lateral growth of polypyrrole. The promotion effect is accounted for by adsorption of pyrrole monomers and selective deposition of intermediate oligomers at the hydrophobic surfaces. The polymerization anisotropy (the ratio of lateral growth rate to vertical growth rate) was ca. 25. The partial pretreatment provided a way of micropatterning with polypyrrole on an insulating substrate. Such a promotion effect at hydrophobic surfaces is amplified in the presence of a small amount of an anionic surfactant. Polyaniline also grows effectively along the hydrophobic surface with the polymerization anisotropy of &gt;100.

Electrosynthesized polypyrrole grows effectively at a hydrophobic substrate. Hydrophobic pretreatment of interdigitated microarray electrodes with silanization reagents prompts the lateral growth of polypyrrole film, resulting in the interconnection of arrays with a thin, uniform film. On the basis of this phenomenon, the micropatterning with polypyrrole can successfully be carried out by electropolymerization at a substrate with a silanized pattern.

Electrosynthesized copolymers of pyrrole and N-methylpyrrole were characterized by in situ conductivity measurements at microarray electrodes and cyclic voltammetry in aqueous solution. The redox properties were easily controlled by changing the monomer ratio in the polymerization solution. The microarray electrode coated with polypyrrole or the copolymer served as redox switching devices showing the rapid 'on' and 'off' responses (typically, within a few seconds) to redox reagents in solutions with high reproducibility. The copolymerization was effective to give selectivity and sensitivity to this device.

A microarray electrode coated with pyrrole-N-methylpyrrole copolymer containing diaphorase shows an on-off response upon addition of NADH, since diaphorase catalyses the reduction of the polymer by NADH from a conductive to an insulating state.