To evaluate hematological effects of direct current (DC) and alternating current (AC) extremely low frequency (ELF) electric field exposure, this study investigated red blood cell (RBC) movement in whole blood. Video images of RBCs were recorded under a microscope using specially designed electrode systems. Video analysis software was then used to measure the RBC velocity. The noise level and measurement system stability were confirmed based on results of a no-field exposure experiment. Using the electrode system to produce a non-homogeneous electric field, different movements were found to occur in DC and AC field exposure. The RBCs moved in the directions of the electric field and the gradient of field distribution, respectively, in the DC and AC fields. Dependences of the RBC velocity on the field strength were, respectively, linear and quadratic in the DC and AC fields. These results suggest that electrophoretic and dielectrophoretic movements were, respectively, dominant in the DC and AC fields. The magnitude of the electric field necessary to cause these effects was found to be 10(3)-10(5) times greater than the internationally publicized guideline for human safety. (c) 2022 Bioelectromagnetics Society.

As for the diagnosis and treatment of eye diseases, an ideal fundus imaging system is expected to be portability, low cost, and high resolution. Here, we demonstrate a non-mydriatic near-infrared fundus imaging system with light illumination from an electronic contact lens (E-lens). The E-lens can illuminate the retinal and choroidal structures for capturing the fundus images when voltage is applied wirelessly to the lens. And we also reconstruct the images with a depth-dependent point-spread function to suppress the scattering effect that eventually visualizes the clear fundus images.

Ion carriers play an important role in biology in terms of control of a biological system, whereas electron carriers are key to engineered systems. Therefore, a biodevice requires an electron-ion coupling mechanism that can transmit an ion signal from a device to a biological system via an external voltage. In this study, a protonic biodevice is demonstrated that translates the proton signals from the device into an enzyme cascade by means of an ATP synthase and bioluminescent luciferase. The proton signals can be modulated through the applied potential to a sulfonated polyaniline biotransducer, with such protonic modulation controlling ATP synthesis at the ATP synthase membrane, followed by bioluminescence in the luciferin-luciferase reaction. The present electron-proton-transportation platform provides a new means of controlling biological cascade reactions, with proton signals mediated by an external voltage.

Wearable devices that generate power using sweat have garnered much attention in the field of skin electronics. These devices require high performance with a small volume and low production rate of sweat by living or-ganisms. Here we demonstrate a high-power biofuel cell bracelet based on the lactate in human sweat. The biofuel cell was developed by using a lactate oxidase/osmium-based mediator/carbon nanotube fiber for lactate oxidation and a bilirubin oxidase/carbon nanotube fiber for oxygen reduction; the fibers were woven into a hydrophilic supportive textile for sweat storage. The storage textile was sandwiched between a hydrophobic textile for sweat absorption from the skin and a hydrophilic textile for water evaporation to improve sweat collection. The performance of the layered cell was 74 ?W at 0.39 V in 20 mM artificial sweat lactate, and its performance was maintained at over 80% for 12 h. Furthermore, we demonstrated a series-connection between anode/cathode fibers by tying them up to wrap the bracelet-type biofuel cell on the wrist. The booster six-cell bracelet generated power at 2.0 V that is sufficient for operating digital wrist watches.

To realize direct power generation from biofuels in natural organisms, we demonstrate a needle-type biofuel cell (BFC) using enzyme/mediator/carbon nanotube (CNT) composite fibers with the structure Osmium-based polymer/CNT/glucose oxidase/Os-based polymer/CNT. The composite fibers performed a high current density (10 mA/cm(2)) in 5 mM artificial blood glucose. Owing to their hydrophilicity, they also provided sufficient ionic conductivity between the needle-type anode and the gas-diffusion cathode. When the tip of the anodic needle was inserted into natural specimens of grape, kiwifruit, and apple, the assembled BFC generated powers of 55, 44, and 33 mu W from glucose, respectively. In addition, the power generated from the blood glucose in mouse heart was 16.3 mu W at 0.29 V. The lifetime of the BFC was improved by coating an anti-fouling polymer 2-methacryloyloxyethyl phosphorylcholine (MPC) on the anodic electrode, and sealing the cathodic hydrogel chamber with medical tape to minimize the water evaporation without compromising the oxygen permeability.

Powering an electrical contact lens is a significant challenge for wearable applications such as augmented reality displays and iontophoretic drug delivery to the eye. Here a hybrid power generation device is developed comprising a wireless power transfer system and a bioabsorbable metal-air primary battery, which provides a multifunctional direct current (DC) and/or alternating current (AC) output. The DC power is generated by Zn loop anode and a bilirubin oxidase (BOD) biocathode in an artificial tear. The Zn-based loop anode is also used as the antenna of a wireless power transfer system that results in high power transfer efficiency of 17.6% at 13.56 MHz. The wireless-powered AC voltage is boosted from 1.5 to 1.5 V + 0.5 V-pp by a DC offset, enabling red light-emitting diode (LED) emission. Furthermore, the hybrid AC and DC offset voltages are boosted to 2.3 V + 0.5 V-pp by a capacitive booster circuit that provides blue LED emission. No hydrogen evolution or pH change is observed in the tear electrolyte. The present hybrid power source can potentially power wearable electronics in body fluids.

Wearable biofuel cells with flexible enzyme/carbon nanotube (CNT) fibers were designed on a cotton textile cloth by integrating two components: bioanode fibers for glucose oxidation and O-2-diffusion biocathode fibers for oxygen reduction. The anode and cathode fibers were prepared through modification with glucose dehydrogenase and bilirubin oxidase, respectively, on multi-walled carbon nanotube-coated carbon fibers. Both biofibers woven on the cloth generated a power density of 48 mu W/cm(2) at 0.24 V from 0.1 mM glucose (human sweat amount), and of 216 mu W/cm(2) at 0.36 V, when glucose was supplied from a hydrogel tank containing 200 mM glucose. Our fiber-based biofuel cell deformed to an S-shape without a significant loss in cell performance. Furthermore, we demonstrated a series-connection involving the tying of biofibers on a cloth with batik-based ionic isolation. The booster four cells generate power at 1.9 V that illuminated an LED on the cloth.

Delivering ions and molecules into living cells has become an important challenge in medical and biological fields. Conventional molecular delivery, however, has several issues such as physical and chemical damage to biological cells. Here, we present a method of directly delivering molecules into adhesive cells with an Au-based nanostraw membrane stamp that can physically inject a target molecule into the cytoplasm through a nanostraw duct. We successfully delivered calcein target molecules into adhesive cells with high efficiency (85%) and viability (90%). Furthermore, we modeled the molecular flow through Au nanostraws and then demonstrated the control of calcein flow by changing the concentration and geometry of Au nanostraws. Our Au membrane stamping provides a new way of accessing the cytoplasm to modulate cellular functions via injected molecules.

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.

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.

Breathalyzers estimate Blood Alcohol Content (BAC) from the concentration of ethanol in the breath. Breathalyzers are easy to use but are limited either by their high price and by environmental concerns, or by a short lifetime and the need for continuous recalibration. Here, we demonstrate a proof-of-concept disposable breathalyzer using an organic electrochemical transistor (OECT) modified with alcohol dehydrogenase (ADH) as the sensor. The OECT is made with the conducting polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS), and is printed on paper. ADH and its cofactor nicotinamide adenine dinucleotide (NAD(+)) are immobilized onto the OECT with an electrolyte gel. When the OECT-breathalyzer is exposed to ethanol vapor, the enzymatic reaction of ADH and ethanol transforms NAD(+) into NADH, which causes a decrease in the OECT source drain current. In this fashion, the OECT-breathalyzer easily detects ethanol in the breath equivalent to BAC from 0.01% to 0.2%. The use of a printed OECT may contribute to the development of breathalyzers that are disposable, ecofriendly, and integrated with wearable devices for real-time BAC monitoring.

In Nature, protons (H+) can mediate metabolic process through enzymatic reactions. Examples include glucose oxidation with glucose dehydrogenase to regulate blood glucose level, alcohol dissolution into carboxylic acid through alcohol dehydrogenase, and voltage-regulated H+ channels activating bioluminescence in firefly and jellyfish. Artificial devices that control H+ currents and H+ concentration (pH) are able to actively influence biochemical processes. Here, we demonstrate a biotransducer that monitors and actively regulates pH-responsive enzymatic reactions by monitoring and controlling the flow of H+ between PdHx contacts and solution. The present transducer records bistable pH modulation from an "enzymatic flip-flop" circuit that comprises glucose dehydrogenase and alcohol dehydrogenase. The transducer also controls bioluminescence from firefly luciferase by affecting solution pH.

In 1804, Theodore von Grotthuss proposed a mechanism for proton (H+) transport between water molecules that involves the exchange of a covalent bond between H and O with a hydrogen bond. This mechanism also supports the transport of OH- as a proton hole and is essential in explaining proton transport in intramembrane proton channels. Inspired by the Grotthuss mechanism and its similarity to electron and hole transport in semiconductors, we have developed semiconductor type devices that are able to control and monitor a current of H+ as well as OH- in hydrated biopolymers. In this topical review, we revisit these devices that include protonic diodes, complementary, transistors, memories and transducers as well as a phenomenological description of their behavior that is analogous to electronic semiconductor devices.

Natural biological composites often couple light weight with tunable and spatially controlled mechanical properties including stiffness, toughness, and hardness. Examples include the toughness of seashells, the hardness of the chiton tooth, and the stiffness gradient of the squid beak. While seashells and the chiton tooth have a mineralized inorganic component, the squid beak is entirely organic. The squid beak is known as one of the hardest fully organic materials. The hydrated squid beak has a large stiffness gradient from soft, at the interface with the squid mouth, to hard at the tip. This gradient occurs from the spatially controlled cross-linking of chitin nanofibers with a protein matrix aided by catecholamines. Here, we introduce a water processable deacetylated chitin composite with tunable mechanical properties from spatially controlled cross-linking assisted by catecholamines. Given the natural abundance of chitin and the ease of water processing, this composite can find applications for bridging mechanically mismatched materials.

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.

Translating ionic currents into measureable electronic signals is essential for the integration of bioelectronic devices with biological systems. We demonstrate the use of a Pd/PdHx electrode as a bioprotonic transducer that connects H+ currents in solution into an electronic signal. This transducer exploits the reversible formation of PdHx in solution according to PdH <-> Pd + H+ + e(-), and the dependence of this formation on solution pH and applied potential. We integrate the protonic transducer with glucose dehydrogenase as an enzymatic AND gate for glucose and NAD(+). PdHx formation and associated electronic current monitors the output drop in pH, thus transducing a biological function into a measurable electronic output. C 2014 Author(s).

The ability of bioelectronic devices to conduct protons and other ions opens up opportunities to interface with biology. In this research highlight, we report on our recent efforts in bioprotonic devices. These devices monitor and modulate a current of protons with an applied voltage. Voltage-controlled proton flow mimics semiconductor devices with complementary transistors or biological behaviors such as synaptic-like memories and enzyme logic.

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.

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.

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.

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.

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.

我々はグルコースから発電可能なバイオ燃料電池の高出力化に向けた研究を行っている。本研究では、グルコース酸化酵素（GOD）と電子伝達メディエータ分子（Med）をCarbon Nano Tube Forest（CNTF）内部のナノ構造に高密度かつ均一に固定する手法を開発した。適切な表面処理と分子設計により、これら要素を分子レベルで整然と配列させることに成功し、結果的に100%に近い電子伝達効率と３次元構造に由来する～10 mA cm^-2クラスの大出力の両立に成功した。

我々は、体内埋め込み型電極として生体適合性を持つ導電性高分子を柔軟でウェットなハイドロゲル表面にパターニングした有機電極（ゲル電極）を作製する技術を確立した。本電極を発展させた歪センサの力学特性は母材であるハイドロゲルに依存し、電気特性は歪により大きく変化する。すなわち、測定対象の硬さに合わせた歪センシングが可能であると考えられる。本発表では、ゲル電極を用いた筋収縮の歪センシングについて報告する。

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.

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.

Enzymatic biofuel cells have attracted attention. However, there are drawbacks to be overcome for practical applications: The lower output voltage and shorter life time. In this paper, I will present our recent progress in MEMS techniques-based challenge to these problems. The possible output voltage of a single biofuel cell is generally lower than 1 V. The series connection of biofuel cells requires a system for ionic isolation between each cell. We will report an automatic air-valve to connect biofuel cells in series based on the air-trapping at a lotus leaf-like superhydrophobic structure that is manufactured between fuel cells arrayed in a microfluidic channel. The lifetime of an operating enzymatic fuel cell is limited typically within one week. We have been developing the parallel-connected biological micro fuel cells, each of which is shielded from fuel solutions by using degradable materials such as PLGA. © 2010 International Federation for Medical and Biological Engineering.

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.

Real-time imaging of single-molecule fluorescence with a zero-mode waveguide (ZMW) was achieved. With modification of the ZMW geometry, the signal-to-background ratio is twice that obtainable with a conventional ZMW. The improved signal-to-background ratio makes it possible to visualize individual binding-release events between chaperonin GroEL and cochaperonin GroES at a concentration of 5 mu M. Two rate constants representing two-timer kinetics in the release of GroES from GroEL were measured with the ZMW, and the measurements agreed well with those made with a total internal reflection fluorescence microscopy. These results indicate that the novel ZMW makes feasible the direct observation of protein-protein interaction at an intracellular concentration in real time.

To elucidate the exact role of the C-terminal region of GroEL in its functional cycle, the C-terminal 20-amino acid truncated mutant of GroEL was constructed. The steady-state ATPase rate and duration of GroES binding showed that the functional cycle of the truncated GroEL is extended by similar to 2 s in comparison with that of the wild type, without interfering with the basic functions of GroEL. We have proposed a model for the functional cycle of GroEL, which consists of two rate-limiting steps of similar to 3- and similar to 5-s duration (Ueno, T., Taguchi, H., Tadakuma, H., Yoshida, M., and Funatsu, T. (2004) Mol. Cell 14, 423-434). According to the model, detailed kinetic studies were performed. We found that a 20-residue truncation of the C terminus extends the time until inorganic phosphate is generated and the time for arresting protein folding in the central cavity, i.e. the lifetime of the first rate-limiting step in the functional cycle, to an similar to 5-s duration. These results suggest that the integrity of the C-terminal region facilitates the transition from the first to the second rate-limiting state.

Three types of self-assembled monolayers (SAMs) are employed for the investigation of protein adsorption. An octadecylsilane (ODS) SAM, a fluoroalkylsilane (FAS) SAM, and a polyethylene glycol (PEG) SAM are selected as examples. The amount of adsorbed protein is measured by fluorescent microscopy. A molecular dynamics (MD) simulation is carried out for modeling both the molecular structures of the SAMs and the water structure on the SAMs. A hydrophilic PEG SAM prevents protein adsorption, while a large amount of adsorption is observed on a hydrophobic ODS SAM. In spite of the hydrophobicity, an FAS SAM prevents protein adsorption as well as a PEG SAM. MD calculation suggests that the existence of surface-bound water on an ODS SAM induces protein, adsorption. In the case of the FAS SAM, owing to the electrostatic interaction and the flexibility of the precursor, the water molecule is not bound to the surface and protein adsorption is suppressed.

Protein nanopatterning techniques to fulfil the requirement for reducing nonspecific adsorption are demonstrated. A target protein is selectively immobilized on a hydroxy-terminated self-assembled monolayer template, the pattern of which is modified with streptavidin used as the intermediating molecule. 3-aminopropyltrimethoxysilane (APTES) used as the intermediating molecule induces nonspecific adsorption of proteins due to nonspecific adsorption of APTES itself. The data indicate that reducing nonspecific adsorption of both the target protein and the intermediating molecule is important for selectivity improvement in protein patterning. As a result of the refinement, a protein nanopattern of 250 nm in diameter has been fabricated.

The feasibility of using an octenyltrimethoxysilane (OCS) self-assembled monolayer (SAM) as a high-resolution electron beam (EB) resist is investigated. The vinyl groups of the OCS SAM can be modified into hydroxy groups, which are useful for biochip fabrication. The hydroxy-modified OCS SAM exhibits higher sensitivity than a vinyl-terminated one. By using the hydroxy-modified OCS SAM as an EB resist, a miniaturized pattern of 18 nm is achieved. Since the hydroxy-modified OCS SAM is repellent to many proteins, this methodology can be useful for the fabrication of protein-immobilizing templates of a molecular scale. (c) 2006 Elsevier B.V. All rights reserved.

We propose a novel process for preferential immobilization of deoxyribonucleic acid (DNA) and green fluorescent protein (GFP) onto organosilane self-assembled monolayer (SAM) templates. One of the advantages of using the organosilane SAM as the template is that it is electron-beam sensitive and, by utilizing the SAM as an alternative resist film, we can make nanopatterns on a molecular scale because the proximity effect is negligible owing to the film&apos;s thinness. An other advantage is that the organosilane SAM is repellent to both DNA and GFP. Thus, the patterned SAM can be utilized as the passivation film covering the outside of the pattern while DNA and GFP are immobilized within the pattern. We investigate two kinds of organosilane SAMs for the template. One is n-octadecyltrimethoxysilane (ODS) SAM, and the other is 1H,1H,2H,2H-perfluorodecyltriethoxysilane (FDS) SAM. Our results indicate that the ODS SAM is more repellent to DNA than the FDS SAM and is suitable for DNA immobilization, while the FDS SAM template is suitable for GFP immobilization via biotin-avidin coupling because the FDS SAM surface prevents the nonspecific adsorption of streptavidin. Although the nonspecific adsorption of DNA and GFP on a SAM is also recognized, by controlling both the concentration and the incubation time, we can immobilize DNA and GFP preferentially onto nanopatterns of 100 nm diameter.

A novel approach of fabricating sub-10-mum patterns on silicon surfaces by electron beam (EB) lithography for attachment of oligonucleotides was described. The shape of the microfabricated arrays was observed to be regular by optical microscopy. An octadecyltrmiethoxysilane (ODS) monolayer was deposited on the regions outside the patterned areas to minimize the nonspecific binding of biomolecules. Cy 5-labeled target DNA was hybridized to both complementary and noncomplementary oligonucleotides that were covalently anchored to micropatterns. As a result, the micropatterns where specific binding occurred show strong signals, whereas no signals are observed in the case of nonspecific binding. These data indicate that miniature micro- and nano-arrays will find applications in biochips and biosensors. (C) 2004 Elsevier B.V. All rights reserved.

A novel fabrication process of silicon microstructure array for preferential immobilization of biomolecules is proposed. We perform electron beam lithography on a self-assembled monolayer (SAM), and achieve high-density silicon patterns terminated with both 3-aminopropyltriethoxysilane (APTES) and octadecyltrimethoxysilane (ODS). The amino-terminated surface produces the site-directed covalent immobilization of DNA inside the pattern, while the hydrophobic surface of the ODS-SAM prevents the adsorption. As a result, we have succeeded in immobilizing the DNA within the amino-modified area. By using this methodology, we demonstrate the miniaturization of deoxyribonucleic acid (DNA) chip. After the covalent attachment of the amino-modified oligonucleotides to the microstructures, we hybridize the immobilized DNA with the target DNA labeled with a fluorescent dye. The signals from the DNA chip exhibit the specific binding due to the DNA-DNA interaction. These results show the feasibility of this technique for high-density information storage and biochip miniaturization. (C) 2004 Elsevier B.V. All rights reserved.

We report a result of a feasibility study on the application of an octadecyltrimethoxysilane, self-assembled monolayer to a resist film for electron beam lithography. The self-assembled monolayer deposited on a silicon dioxide surface by chemical vapor deposition is resistant to both sulfuric acid and hydrofluoric acid. By immersing the electron-beam-irradiated surface into both acids, we successfully develop microstructural patterns in the self-assembled monolayer. In particular, we show the effectiveness of immersing the substrate into a sulfuric-acid-based solution for the development of the pattern. The relationship between the required dose and the developing time is estimated by measuring the morphology of the developed patterns by atomic force microscopy. The pattern in the self-assembled monolayer can be transferred into both the underlying silicon dioxide layer and the silicon substrate. These results indicate that the organosilane self-assembled monolayer serves as an alternative resist for electron beam lithography.