Atsunori Hiratsuka

Problems associated with the thin-film Ag/AgCl reference electrode and a novel structure with improved durability

Abstract We have developed a novel thin-film Ag/AgCl reference electrode which shows dramatic improvement in its durability. To make the electrode resistant to heat treatment in its fabrication, a metal adhesive layer in the currently used thin-film Ag/AgCl electrodes was replaced by a polyimide layer, and a gold backbone structure was employed. Unlike the currently used structure, the entire surface of the silver layer was covered with a polyimide layer and AgCl was grown from the periphery of the pattern. The structure was resistant to baking at 300°C. Whereas the currently used Ag/AgCl electrode has a very short lifespan in concentrated KCl solutions, the novel electrode showed surprising resistance in these solutions. In a KCl and AgCl saturated solution, the novel electrode gave a stable potential for approximately 8 h, while the currently used electrode functioned for only 1.5 min. A dramatic difference was also observed in the resistance to interfering materials. Although 1 mM KI or K 2 S was enough to break the currently used electrode, the novel electrode was resistant to these materials and showed much smaller potential shift. In the novel Ag/AgCl structure, the rate of AgCl formation was significantly delayed and the spread of the AgCl layer was visualized. Therefore, any of the electrochemical and chemical methods of AgCl formation could be easily applied.

Plasma Polymerized Films for Sensor Devices

Miniaturization with semiconductor microfabrication or micromachining techniques is one of the main concerns in sensor technologies. For the combination of sensor and microelectronics technology, polymers should be fabricated in a mass-producible and miniaturization-conscious manner as an interface. The polymers fabricated in conventional manners could have potential problems, e.g., obtaining thin (<1 µm) and homogeneous films. Plasma-polymerized films, which are made in a glow discharge or plasma in a vapor phase, offer a new alternative. This review describes the several characteristics, properties, and applications of plasma-polymerized films in both chemical and biological fields.

Application of plasma-polymerized films for isoelectric focusing of proteins in a capillary electrophoresis chip

The first use of plasma polymerization technique to modify the surface of a glass chip for capillary isoelectric focusing (cIEF) of different proteins is reported. The electrophoresis separation channel was machined in Tempax glass chips with length 70 mm, 300 microm width and 100 microm depth. Acetonitrile and hexamethyldisiloxane monomers were used for plasma polymerization. In each case 100 nm plasma polymer films were coated onto the chip surface to reduce protein wall adsorption and minimize the electroosmotic flow. Applied voltages of 1000 V, 2000 V and 3000 V were used to separate mixtures of cytochrome c (pI 9.6), hemoglobin (pI 7.0) and phycocyanin (pI 4.65). Reproducible isoelectric focusing of each pI marker protein was observed in different coated capillaries at increasing concentration 2.22-5 microg microL(-1). Modification of the glass capillary with hydrophobic HMDS plasma polymerized films enabled rapid cIEF within 3 min. The separation efficiency of cytochrome c and phycocyanin in both acrylamide and HMDS coated capillaries corresponded to a plate number of 19600 which compares favourably with capillary electrophoresis of neurotransmitters with amperometric detection.

Mass transport behavior of electrochemical species through plasma-polymerized thin film on platinum electrode

Abstract We propose a novel method for generating thin film coating for use on a platinum (Pt) electrode. This is accomplished by a plasma-polymerized film (PPF), which is deposited directly onto the substrate under dry condition. The resulting films are extremely thin (

Integration of microfabricated needle-type glucose sensor devices with a novel thin-film Ag/AgCl electrode and plasma-polymerized thin film: mass production techniques.

We developed an integrated array of needle-type biosensors employing a novel process of fabrication, comprising conventional semiconductor fabrication and micromachining technology. Amperometric sensing electrodes with plasma-polymerized films and a thin-film Ag/AgCl reference electrode were directly integrated on a glass substrate with thin-film process, e.g., sputtering. An enzyme was immobilized on the electrode via the plasma-polymerized film, which was deposited directly on the substrate using a dry process. The novel thin-film Ag/AgCl reference electrode showed stable potentials in concentrated chloride solutions for a long period. The plasma-polymerized film is considered to play an important role as an interfacial design between the sensing electrode and the immobilized enzyme considering that the film is extremely thin, adheres well to the substrate (electrode) and has a highly cross-linked network structure and functional groups, such as amino groups. The results showed increments of the sensor signal, probably because the plasma-polymerized film allowed a large amount of enzyme to be immobilized. The greatest advantage is that the process can permit the mass production of high-quality biosensors at a low cost.

Design and synthesis of new fluorescent probe for rapid and highly sensitive detection of proteins via electrophoretic gel stain.

A new fluorescent molecular probe, 2,2′‐(1E,1′E)‐2,2′‐(4‐(dicyanomethylene)‐4H‐pyrane‐2,6‐diyl)bis(ethene‐2,1‐diyl)bis(sodium benzenesulfonate) salt (1), possessing the cyanopyranyl moieties and two benzene sulfonic acid groups was designed and synthesized to detect proteins in solution and for high‐throughput SDS‐PAGE. Compound 1 exhibited no fluorescence in the absence of proteins; however, it exhibited strong fluorescence on the addition of bovine serum albumin as a result of intramolecular charge transfer. Compared with the conventional protocols for in‐gel protein staining, such as SYPRO Ruby and silver staining, 1 achieves higher sensitivity, even though it offers a simplified, higher throughput protocol. In fact, the total time required for protein staining was 60–90 min under optimum conditions much shorter than that required by the less‐sensitive silver staining or SYPRO Ruby staining protocols. Moreover, 1 was successfully applied to protein identification by mass spectrometry via in‐gel tryptic digestion, Western blotting, and native PAGE together with protein staining by 1, which is a modified protocol of blue native PAGE (BN‐PAGE). Thus, 1 may facilitate high‐sensitivity protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.

Thermophilic Talaromyces emersonii Flavin Adenine Dinucleotide-Dependent Glucose Dehydrogenase Bioanode for Biosensor and Biofuel Cell Applications

Flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) was identified and cloned from thermophilic filamentous fungi Talaromyces emersonii using the homology cloning method. A direct electron transfer bioanode composed of T. emersonii FAD-GDH and a single-walled carbon nanotube was produced. Enzymes from thermophilic microorganisms generally have low activity at ambient temperature; however, the T. emersonii FAD-GDH bioanode exhibits a large anodic current due to the enzymatic reaction (1 mA cm–2) at ambient temperature. Furthermore, the T. emersonii FAD-GDH bioanode worked at 70 °C for 12 h. This is the first report of a bioanode with a glucose-catalyzing enzyme from a thermophilic microorganism that has potential for biosensor and biofuel cell applications. In addition, we demonstrate how the glycoforms of T. emersonii FAD-GDHs expressed by various hosts influence the electrochemical properties of the bioanode.

Fully automated two-dimensional electrophoresis system for high-throughput protein analysis.

A fully automated two-dimensional electrophoresis (2DE) system for rapid and reproducible protein analysis is described. 2DE that is a combination of isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein expression analysis. Here, all the operations are achieved in a shorter time and all the transferring procedures are performed automatically. The system completed the entire process within 1.5 h. A device configuration, operational procedure, and data analysis are described using this system.

Comparison of Direct and Mediated Electron Transfer in Electrodes with Novel Fungal Flavin Adenine Dinucleotide Glucose Dehydrogenase

Direct and mediated electron transfer (DET and MET) in enzyme electrodes with a novel flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from fungi are compared for the first time. DET is achieved by placing a single-walled carbon nanotube (CNT) between GDH and a flat gold electrode where the CNT is close to FAD within the distance for DET. MET is induced by using a free electron transfer mediator, potassium hexacyanoferrate, and shuttles electrons from FAD to the gold electrode. Cyclic voltammetry shows that the onset potential for glucose response current in DET is smaller than in MET, and that the distinct redox current peak pairs in MET are observed whereas no peaks are found in DET. The chronoamperometry with respect to a glucose biosensor shows that (i) the response in DET is more rapid than in MET; (ii) the current at more than +0.45V in DET is larger than the current at the current-peak potential in MET; (iii) a DET electrode covers the glucose concentration range for clinical requirements and is not susceptible to interfering agents at +0.45 V; and (iv) a DET electrode with the novel fungal FAD-GDH does not affect sensing accuracy in the presence of up to 5 mM xylose, while it often shows a similar response level to glucose with other conventionally used fungus-derived FAD-GDHs. It is concluded that our DET system overcomes the disadvantage of MET.