Drastically Increase in Atomic Nitrogen Production Depending on the Dielectric Constant of Beads Filled in the Discharge Space.

Nitrogen activation, especially dissociation (production of atomic nitrogen), is a key step for efficient nitrogen fixation, such as nitrogen reduction to produce ammonia. Nitrogen reduction reactions using water as a direct hydrogen source have been studied by many researchers as a green ammonia process. We studied the reaction mechanism and found that the nitrogen reduction could be significantly improved via efficient production of atomic nitrogen through electric discharge. In the present study, we focused on packed-bed dielectric barrier discharge (PbDBD) using dielectric beads as the packing material. The experimental results showed that more atomic nitrogen was produced in the nitrogen activation by the discharge in which the discharge space was filled with the dielectric beads than in the nitrogen activation by the discharge without using the dielectric beads. Then, it was clarified that the amount of atomic nitrogen increased as the dielectric constant of the beads to be filled increased, and the amount of atomic nitrogen produced increased up to 13.48 times. Based on the results, we attempted ammonia synthesis using water as a direct hydrogen source with the efficiently generated atomic nitrogen. When the atomic nitrogen gas generated by the PbDBD was sprayed onto the surface of the water phase and subsequently reacted as a plasma/liquid interfacial reaction, the nitrogen fixation rate increased by 7.26-fold compared to that when using the discharge without dielectric beads, and the ammonia production selectivity increased to 83.7%.

Reactive Oxygen Species Penetrate Persister Cell Membranes of Escherichia coli for Effective Cell Killing.

Persister cells are difficult to eliminate because they are tolerant to antibiotic stress. In the present study, using artificially induced Escherichia coli persister cells, we found that reactive oxygen species (ROS) have greater effects on persister cells than on exponential cells. Thus, we examined which types of ROS could effectively eliminate persister cells and determined the mechanisms underlying the effects of these ROS. Ultraviolet (UV) light irradiation can kill persister cells, and bacterial viability is markedly increased under UV shielding. UV induces the production of ROS, which kill bacteria by moving toward the shielded area. Electron spin resonance-based analysis confirmed that hydroxyl radicals are produced by UV irradiation, although singlet oxygen is not produced. These results clearly revealed that ROS sterilizes persister cells more effectively compared to the sterilization of exponential cells (**p < 0.01). These ROS do not injure the bacterial cell wall but rather invade the cell, followed by cell killing. Additionally, the sterilization effect on persister cells was increased by exposure to oxygen plasma during UV irradiation. However, vapor conditions decreased persister cell sterilization by reducing the levels of hydroxyl radicals. We also verified the effect of ROS against bacteria in biofilms that are more resistant than planktonic cells. Although UV alone could not completely sterilize the biofilm bacteria, UV with ROS achieved complete sterilization. Our results demonstrate that persister cells strongly resist the effects of antibiotics and starvation stress but are less able to withstand exposure to ROS. It was shown that ROS does not affect the cell membrane but penetrates it and acts internally to kill persister cells. In particular, it was clarified that the hydroxy radical is an effective sterilizer to kill persister cells.

Contribution of Discharge Excited Atomic N, N2 *, and N2+ to a Plasma/Liquid Interfacial Reaction as Suggested by Quantitative Analysis.

Electric-discharge nitrogen comprises three main types of excited nitrogen species-atomic nitrogen (Natom ), excited nitrogen molecules (N2 *), and nitrogen ions (N2+ ) - which have different lifetimes and reactivities. In particular, the interfacial reaction locus between the discharged nitrogen and the water phase produces nitrogen compounds such as ammonia and nitrate ions (denoted as N-compounds generically); this is referred to as the plasma/liquid interfacial (P/L) reaction. The Natom amount was analyzed quantitatively to clarify the contribution of Natom to the P/L reaction. We focused on the quantitative relationship between Natom and the produced N-compounds, and found that both N2 * and N2+ , which are active species other than Natom , contributed to P/L reaction. The production of N-compounds from N2 * and N2+ was enhanced upon UV irradiation of the water phase, but the production of N-compounds from Natom did not increase by UV irradiation. These results revealed that the P/L reactions starting from Natom and those starting from N2* and N2+ follow different mechanisms.

Formation Mechanism of Flattened Top HFBI Domical Droplets.

A water droplet assumes a spherical shape because of its own surface tension. However, water droplets containing dissolved hydrophobin (HFBI) have flat surfaces. In our previous study, the mechanism of this unique phenomenon was revealed. HFBI forms a self-organized membrane that has a densely packed and honeycomb-like structure. Furthermore, the buckling strength of the membrane is higher than the surface tension of the HFBI droplet. Therefore, an HFBI domical droplet has a flat surface. However, it was not clear why only the top of the domical droplet was flattened while other areas such as the side face were not. In this study, we observed HFBI domical droplets to investigate this phenomenon. The flat top area (self-organized HFBI membrane) remained parallel to the ground even if the substrate was tilted. Therefore, buoyancy was thought to be a factor affecting the HFBI membrane. In addition, the side face of the HFBI domical droplet was analyzed by atomic force microscopy and electrochemical impedance spectroscopy, and it was found that the sides of the HFBI droplet were not composed of densely packed HFBI membranes.

Flattened-Top Domical Water Drops Formed through Self-Organization of Hydrophobin Membranes: A Structural and Mechanistic Study Using Atomic Force Microscopy.

The Trichoderma reesei hydrophobin, HFBI, is a unique structural protein. This protein forms membranes by self-organization at air/water or water/solid interfaces. When HFBI forms a membrane at an air/water interface, the top of the water droplet is flattened. The mechanism underlying this phenomenon has not been explored. In this study, this unique phenomenon has been investigated. Self-organized HFBI membranes form a hexagonal structured membrane on the surface of water droplets; the structure was confirmed by atomic force microscopy (AFM) measurement. Assembled hexagons can form a planar sheet or a tube. Self-organized HFBI membranes on water droplets form a sheet with an array of hexagonal structures or a honeycomb structure. This membrane, with its arrayed hexagonal structures, has very high buckling strength. We hypothesized that the high buckling strength is the reason that water droplets containing HFBI form flattened domes. To test this hypothesis, the strength of the self-organized HFBI membranes was analyzed using AFM. The buckling strength of HFBI membranes was measured to be 66.9 mN/m. In contrast, the surface tension of water droplets containing dissolved HFBI is 42 mN/m. Thus, the buckling strength of a self-organized HFBI membrane is higher than the surface tension of water containing dissolved HFBI. This mechanistic study clarifies why the water droplets formed by self-organized HFBI membranes have a flattened top.

Electrochemical properties of honeycomb-like structured HFBI self-organized membranes on HOPG electrodes.

HFBI (derived from Trichoderma sp.) is a unique structural protein, which forms a self-organized monolayer at both air/water interface and water/solid interfaces in accurate two-dimensional ordered structures. We have taken advantage of the unique functionality of HFBI as a molecular carrier for preparation of ordered molecular phase on solid substrate surfaces. The HFBI molecular carrier can easily form ordered structures; however, the dense molecular layers form an electrochemical barrier between the electrode and solution phase. In this study, the electrochemical properties of HFBI self-organized membrane-covered electrodes were investigated. Wild-type HFBI has balanced positive and negative charges on its surface. Highly oriented pyrolytic graphite (HOPG) electrodes coated with HFBI molecules were investigated electrochemically. To improve the electrochemical properties of this HFBI-coated electrode, the two types of HFBI variants, with oppositely charged surfaces, were prepared genetically. All three types of HFBI-coated HOPG electrode perform electron transfer between the electrode and solution phase through the dense HFBI molecular layer. This is because the HFBI self-organized membrane has a honeycomb-like structure, with penetrating holes. In the cases of HFBI variants, the oppositely charged HFBI membrane phases shown opposite electrochemical behaviors in electrochemical impedance spectroscopy. HFBI is a molecule with a unique structure, and can easily form honeycomb-like structures on solid material surfaces such as electrodes. The molecular membrane phase can be used for electrochemical molecular interfaces.

Boost protein expression through co-expression of LEA-like peptide in Escherichia coli.

The boost protein expression has been done successfully by simple co-expression with a late embryogenesis abundant (LEA)-like peptide in Escherichia coli. Frequently, overexpression of a recombinant protein fails to provide an adequate yield. In the study, we developed a simple and efficient system for overexpressing transgenic proteins in bacteria by co-expression with an LEA-like peptide. The design of this peptide was based on part of the primary structure of an LEA protein that is known hydrophilic protein to suppress aggregation of other protein molecules. In our system, the expression of the target protein was increased remarkably by co-expression with an LEA-like peptide consisting of only 11 amino acid residues. This could provide a practical method for producing recombinant proteins efficiently.

Solid-support immobilization of a "swing" fusion protein for enhanced glucose oxidase catalytic activity.

The strategic surface immobilization of a protein can add new functionality to a solid substrate; however, protein activity, e.g., enzymatic activity, can be drastically decreased on immobilization onto a solid surface. The concept of a designed and optimized "molecular interface" is herein introduced in order to address this problem. In this study, molecular interface was designed and constructed with the aim of attaining high enzymatic activity of a solid-surface-immobilized a using the hydrophobin HFBI protein in conjunction with a fusion protein of HFBI attached to glucose oxidase (GOx). The ability of HFBI to form a self-organized membrane on a solid surface in addition to its adhesion properties makes it an ideal candidate for immobilization. The developed fusion protein was also able to form an organized membrane, and its structure and immobilized state on a solid surface were investigated using QCM-D measurements. This method of immobilization showed retention of high enzymatic activity and the ability to control the density of the immobilized enzyme. In this study, we demonstrated the importance of the design and construction of molecular interface for numerous purposes. This method of protein immobilization could be utilized for preparation of high throughput products requiring structurally ordered molecular interfaces, in addition to many other applications.

Structure-function relationships in hydrophobins: probing the role of charged side chains.

Hydrophobins are small fungal proteins that are amphiphilic and have a strong tendency to assemble at interfaces. By taking advantage of this property, hydrophobins have been used for a number of applications: as affinity tags in protein purification, for protein immobilization, such as in foam stabilizers, and as dispersion agents for insoluble drug molecules. Here, we used site-directed mutagenesis to gain an understanding of the molecular basis of their properties. We especially focused on the role of charged amino acids in the structure of hydrophobins. For this purpose, fusion proteins consisting of Trichoderma reesei hydrophobin I (HFBI) and the green fluorescent protein (GFP) that contained various combinations of substitutions of charged amino acids (D30, K32, D40, D43, R45, K50) in the HFBI structure were produced. The effects of the introduced mutations on binding, oligomerization, and partitioning were characterized in an aqueous two-phase system. It was found that some substitutions caused better surface binding and reduced oligomerization, while some showed the opposite effects. However, all mutations decreased partitioning in surfactant systems, indicating that the different functions are not directly correlated and that partitioning is dependent on finely tuned properties of hydrophobins. This work shows that not all functions in self-assembly are connected in a predictable way and that a simple surfactant model for hydrophobin function is insufficient.

Gold nanoparticles functionalized with peptides for specific affinity aggregation assays of estrogen receptors and their agonists.

Nuclear receptors regulate the transcription of genes and various functions such as development, differentiation, homeostasis, and behavior by formation of complexes with ligand and co-activator. Recent findings have shown that agonists of a ligand may have a toxic effect on cellular/tissular function through improper activation of nuclear receptors. In this study, a simple assay system of hetero-complexes of three different molecules (estrogen receptor, ligand, and co-activator peptide) has been developed. This assay system employs functionalized gold nanoparticles (GNPs: 15 nm in diameter). The surfaces of the GNPs were modified by a 12- or 20-amino-acid peptide that contains the sequence of co-activator for activating nuclear receptor by an agonist ligand. Owing to the affinity of the peptide, the functionalized GNPs aggregate faster when the nuclear receptor and the agonist ligand are also present. The aggregation of GNPs can be identified by shifts in adsorption spectrum, which give information about the specificity of agonist ligands. Similarly, this spectrum shift can measure concentration of known agonist ligand. This simple agonist screening will be employed as high through-put analysis (HTA) in the discovery of drugs that act through nuclear receptors.

Post-synapse model cell for synaptic glutamate receptor (GluR)-based biosensing: strategy and engineering to maximize ligand-gated ion-flux achieving high signal-to-noise ratio.

Cell-based biosensing is a "smart" way to obtain efficacy-information on the effect of applied chemical on cellular biological cascade. We have proposed an engineered post-synapse model cell-based biosensors to investigate the effects of chemicals on ionotropic glutamate receptor (GluR), which is a focus of attention as a molecular target for clinical neural drug discovery. The engineered model cell has several advantages over native cells, including improved ease of handling and better reproducibility in the application of cell-based biosensors. However, in general, cell-based biosensors often have low signal-to-noise (S/N) ratios due to the low level of cellular responses. In order to obtain a higher S/N ratio in model cells, we have attempted to design a tactic model cell with elevated cellular response. We have revealed that the increase GluR expression level is not directly connected to the amplification of cellular responses because the saturation of surface expression of GluR, leading to a limit on the total ion influx. Furthermore, coexpression of GluR with a voltage-gated potassium channel increased Ca(2+) ion influx beyond levels obtained with saturating amounts of GluR alone. The construction of model cells based on strategy of amplifying ion flux per individual receptors can be used to perform smart cell-based biosensing with an improved S/N ratio.

Ordered nano-structure of a stamped self-organized protein layer on a HOPG surface using a HFB carrier.

A groundbreaking method for ordered molecular layer preparation on a solid surface employing the drop-stamp method has been developed by us taking advantage of the characteristics of the HFB molecule as a self-organizer/adsorption carrier. It is a smart method which can be used to prepare a self-organized protein layer on a solid surface without unspecific adsorption or defects. In our previous report, we clarified the self-organizing nature of HFB-tagged protein molecules on a surface of a solution droplet. In this report, a protein layer was prepared on a HOPG surface by using the drop-stamp method with a maltose binding protein (MBP)-tagged HFBII molecule. The structure of the stamped protein layer was investigated using frequency modulation atomic force microscopy (FM-AFM) in a liquid condition. The FM-AFM images show that the drop-stamp method can prepare an ordered protein layer on a solid surface smartly. The drop-stamp method using a HFB carrier is a practical method which can be used to prepare an ordered protein layer on a solid substrate surface without unspecific adsorption defects.

Electrochemical preparation of junction between a molecule and solid surface through a metal coordinative peptidic tag.

Molecular immobilization is a way of making a set molecules function as an interface. In order to fabricate a molecular interface, it is necessary to preserve the physical/chemical structure of the molecular layer. Almost all immobilization methods are based on chemical cross-linking reactions that lack orientation and reflect the original structure of the molecules. In order to fabricate a molecular interface which can be used in practical applications, we developed a novel method of molecular immobilization through electrochemical reactions. Our method is based on about the discovery of a novel interfacial phenomenon, i.e., that the coordinate metal ion in the peptidic ligand can be reduced and deposited on an electrode while preserving the bond between the metal and the ligand. The metal ion coordinative peptide tag (designed to contain less than 6 amino acids) can be deployed to objective molecule (e.g., proteins, peptides) in advance, either genetically or chemically. In the present electrochemical immobilization process, the coordinated Cu(2+) ion is reduced (deposited) on an electrode surface electrochemically, and the deposited metal remains bounded to the objective molecule-tagged peptidic ligand on a solid (electrode) surface. Using the EC tag tagged molecules, electrochemical molecular immobilization was performed, and an investigation of the surface properties was undertaken through both chemical quantifications and X-ray photoelectron spectroscopy (XPS). The complex formation between the ECtag tagged molecule and the metal ion was also investigated by photospectromitric analysis. All results suggest that ECtag tagged molecular immobilization is an interfacial phenomenon inducing neutral metal complex formation.

[Cellular engineering and biosensor technology for high through-put analysis on drug discovery].

Sensors have been developed to determine the concentration of specific compounds in situ. They are already widely employed as a practical technological tool in the clinical and healthcare fields. Recently, another concept of biosensing has been receiving attention: biosensing for the evaluation of molecular potency. The author described the idea as qualified analysis. The development of this novel concept has been supported by the development of related technologies, such as electrochemistry, molecular interface science, molecular design, molecular biology (genetic engineering), and cellular/tissual engineering. This study addresses this new concept of biosensing and its application to the evaluation of the potency of chemicals in biological systems, in the field of cellular/tissual engineering. Cellular biosensing will provide valuable information for both pharmaceutical research and chemical safety, and be applicable in drug discovery in vitro as a screening tool.

Engineered synapse model cell: genetic construction and chemical evaluation for reproducible high-throughput analysis.

Bioassay models of neural functions must lend themselves to high-throughput analysis in neural drug discovery. However, smart analysis methods for these functions have not yet been fully established. Here, we describe the development of a synapse model for cell-based biosensing. The engineered synapse model cell expresses ionotropic glutamate receptor on its surface, like the neural postsynaptic membrane. The advantages of the model cell are the ease of handling and reproducibility as compared with the cultured neural cell, and it can be employed to evaluate receptor function through ion flux analysis. The agonist-induced sodium influx was monitored as an agonist concentration-dependent increase in the observed fluorescence signal. Furthermore, we found that our model cell enables the correction of uneven cellular signal levels using a reporter system. Our engineered synapse model cell can be employed as a powerful tool for the screening of lead substances in pharmaceutical high-throughput analysis.

The amphiphilic protein HFBII as a genetically taggable molecular carrier for the formation of a self-organized functional protein layer on a solid surface.

A "drop-stamp method" has been developed for the design and fabrication of molecular interfaces. The amphiphilic protein HFBII, isolated from filamentous fungi, was employed as a genetically taggable molecular carrier for the formation of a structrally ordered layer of functional protein molecules on a solid surface. In this study, the interfacial behavior of maltose-binding protein tagged with HFBII (MBP-HFBII fusion protein) at both the air/water and water/solid interfaces was investigated. A rigid molecular layer of MBP-HFBII fusion protein was successfully formed through the drop-stamp procedure by employing an intermixed system, in which HFBII molecules are intermingled as nanospacers to prevent the intermolecular steric hindrance of the fusion protein. The results show that the drop-stamp method can be utilized in the high-throughput fabrication of structurally ordered molecular interfaces.

Artificial-enzyme gel membrane-based biosurveillance sensor with high reproducibility and long-term storage stability.

We propose that the most sophisticated strategy for primary biosurveillance is to exploit structural commonality through the detection of biologically relevant phosphoric substances. A novel assay, an artificial-enzyme membrane was designed and synthesized for sensor fabrication. This artificial-enzyme catalyzes the hydrolysis of the diphosphoric acid anhydride structure. This structure-selective, albeit not molecule-selective, catalytic hydrolysis was successfully coupled with amperometric detection. Since the catalytic reaction produces a dephosphorylation product (PO(4)(3-)), it can be reduced by an electrode potential of -250 mV vs. Ag/AgCl. Owing to the structural selectivity of the artificial-enzyme membrane, the sensor can detect biological phosphoric substances comprehensively that have the diphosphoric acid anhydride structure. The sensor successfully determined various biological phosphoric substances at concentrations in the micromolar (microM) to millimolar (mM) range, and it showed good functional stability and reproducibility in terms of sensor responses. This sensor was used to detect Escherichia coli lysed by heat treatment, and the response increased with increasing bacterial numbers. This unique technique for analyzing molecular commonality can be applied to the surveillance of biocontaminants, e.g. microorganisms, spores and viruses. Artificial-enzyme-based detection is a novel strategy for practical biosurveillance in the front line.

Seamless signal transduction from live cells to an NO sensor via a cell-adhesive sensing matrix.

A smart live-cell assay was developed as a cellular biosensing system. This system is based on novel tactics: the direct assembly of human cultured cells onto a cell-adhesive sensing matrix. This novel design provides considerable advantages, among them the possibility of capturing molecular signals immediately after they are secreted from living cells. The design also helps preserve all cellular characteristics intact. In this study, a cell-adhesive NO sensing matrix, acting as both an NO-permeable membrane and a cell-adhesive scaffold, was designed using functional polymers and a short peptide sequence derived from extracellular matrix (ECM) proteins. Using the cell-adhesive NO sensing matrix, we constructed a cellular biosensing system based on in situ monitoring of NO released from a human umbilical vein endothelial cell (HUVEC) layer. HUVECs were employed as an organ-functional model of a blood vessel in view of screening vasodilatory substances for clinical purposes. In our novel system, the electrochemical NO sensor is adjacent to the NO-producing cells, which allows the sensing device to achieve superior sensitivity and precise response to a very low number of NO molecules. Our design enables the fixing of the exact distance between the organ-functional model and the chemical sensor without cumbersome manipulations. Consequently, this cellular biosensing system may be readily applicable to high-throughput analysis in the field of drug screening.

Smart Immobilization of oligopeptides through electrochemical deposition onto surface.

A novel method for electrochemical molecular immobilization has been developed. Molecular immobilization on an electroconductive material surface is achieved by genetic and chemical introduction of a tag. The immobilization reaction is based on the remarkable phenomenon of neutral metal complex formation on a redox interface. For electrochemical immobilization, a metal coordinative peptide (EC tag) is introduced to the target molecule and is coordinated with a divalent metal ion. In the electrochemical immobilization process, the coordinated metal in the oligopeptide is reduced to the zero-valent metal state and is deposited on the electroconductive substrate. In the present study, we exploit our previous findings to carry out electrochemical peptide immobilization. This immobilization process can be modulated by an applied potential. Although the immobilized peptide is tightly attached the substrate, it can be removed by oxidation of deposited metal though application of an oxidation potential. The method can be employed for the immobilization of various molecules, e.g. proteins, peptides, and nano-materials, on electroconductive solid surfaces. The unique advantages of the present molecular immobilization method are the ease of application and the novel molecular modulations that are achievable.

Molecular commonality detection using an artificial enzyme membrane for in situ one-stop biosurveillance.

Biodetection and biosensing have been developed based on the concept of sensitivity toward specific molecules. However, current demand may require more levelheaded or far-sighted methods, especially in the field of biological safety and security. In the fields of hygiene, public safety, and security including fighting bioterrorism, the detection of biological contaminants, e.g., microorganisms, spores, and viruses, is a constant challenge. However, there is as yet no sophisticated method of detecting such contaminants in situ without oversight. The authors focused their attention on diphosphoric acid anhydride, which is a structure common to all biological phosphoric substances. Interestingly, biological phosphoric substances are peculiar substances present in all living things and include many different substances, e.g., ATP, ADP, dNTP, pyrophosphate, and so forth, all of which have a diphosphoric acid anhydride structure. The authors took this common structure as the basis of their development of an artificial enzyme membrane with selectivity for the structure common to all biological phosphoric substances and studied the possibility of its application to in situ biosurveillance sensors. The artificial enzyme membrane-based amperometric biosensor developed by the authors can detect various biological phosphoric substances, because it has a comprehensive molecular selectivity for the structure of these biological phosphoric substances. This in situ detection method of the common diphosphoric acid anhydride structure brings a unique advantage to the fabrication of in situ biosurveillance sensors for monitoring biological contaminants, e.g., microorganism, spores, and viruses, without an oversight, even if they were transformed.