Taira Kajisa

Characterization and molecular cloning of cellobiose dehydrogenase from the brown-rot fungus Coniophora puteana

Cellobiose dehydrogenase (CDH) was purified from the brown-rot fungus Coniophora puteana grown in culture containing crystalline cellulose as a carbon source. The purified enzyme gave a single band at 115 kDa on SDS-PAGE and showed a typical flavocytochrome absorption spectrum. The enzyme oxidized both cellobiose and cellooligosaccharides, but not their monomer, glucose, suggesting typical kinetic features of CDH. A cDNA encoding CDH was cloned by RT-PCR using primers designed from the consensus sequences of known CDHs from white-rot fungi. The cDNA consists of 2448 bp, including an open reading frame encoding the 18 amino acids of the putative signal peptide and the 756 amino acids of the mature protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data for tryptic fragments of the purified C. puteana CDH were consistent with partial amino acid sequences of the mature protein deduced from the cloned cDNA. Moreover, the sequences contained common characteristics of CDH, i.e., two possible residues for a heme ligand (Met 64 and His 160), a flavin-binding motif, and two glucose-methanol-choline oxidoreductase motifs. This is the first cloning of CDH from a brown-rot fungus, and the results suggest structural and kinetic similarity of C. puteana CDH to white-rot fungal CDHs.

Self-oriented immobilization of DNA polymerase tagged by titanium-binding peptide motif

We developed a titanium-binding-peptide-1 (TBP-1)-tagged DNA polymerase, for self-oriented immobilization onto a titanium oxide (TiO2) substrate. The enzymatic function of a polymerase immobilized on a solid state device is strongly dependent on the orientation of the enzyme. The TBP-tagged DNA polymerase, which was derived from a hyperthermophilic archaeon, was designed to incorporate the RKLPDA peptide at the N-terminus, and synthesized by translation processes in Escherichia coli (E. coli). The specific binding of the TBP-tagged DNA polymerase onto a TiO2 substrate was clearly monitored by surface plasmon resonance spectroscopy (SPR) and by surface potential detection with an extended-gate field effect transistor (FET). In the SPR analyses, constant quantities of the DNA polymerase were stably immobilized on the titanium substrate under flow conditions, regardless of the concentration of the DNA polymerase, and could be completely removed by a 4 M MgCl2 wash after measurement. The FET signal showed the contribution of the molecular charge in the TBP motif to the binding with TiO2. In addition, the TBP-tagged DNA polymerase-tethered TiO2 gate electrode enabled the effective detection of the positive charges of hydrogen ions produced by the DNA extension reaction, according to the FET principle. Therefore, the self-oriented immobilization platform based on the motif-inserted enzyme is suitable for the quick and stable immobilization of functional enzymes on biosensing devices.

The genes encoding glycoside hydrolase family 6 and 7 cellulases from the brown-rot fungus Coniophora puteana

Four genes encoding glycoside hydrolase (GH) family 6 and 7 cellulases (cel6A, cel6B, cel7A, and cel7B) were obtained from the brown-rot fungus Coniophora puteana by genomic polymerase chain reaction (PCR) using consensus degenerate hybrid oligonucleotide primers (CODEHOPs) designated from the amino acid sequence of cellobiohydrolases (CBHs) from white-rot fungi. The nucleotide sequences of four genes showed high homology with basidiomycetes CBHs, suggesting the fi rst cloning of the genes encoding Cel6 and Cel7 from brown-rot fungi. PCR using CODEHOP pairs at the catalytic domain successfully amplifi ed both cel6A and cel6B, whereas only cel6A fragment was obtained using the primers including the carbohydrate-binding modules (CBMs), suggesting lack of CBM in Cel6B. Moreover, both cel7A and cel7B were amplified by the PCR using CODEHOP pairs at the catalytic domain, but not by those including CBM, suggesting the absence of Cel7 with CBM in the fungus. From these results, three of four cellulases from C. puteana may not carry CBM, which has an important role for the degradation of crystalline cellulose.

Glucose-responsive hydrogel electrode for biocompatible glucose transistor

Abstract In this paper, we propose a highly sensitive and biocompatible glucose sensor using a semiconductor-based field effect transistor (FET) with a functionalized hydrogel. The principle of the FET device contributes to the easy detection of ionic charges with high sensitivity, and the hydrogel coated on the electrode enables the specific detection of glucose with biocompatibility. The copolymerized hydrogel on the Au gate electrode of the FET device is optimized by controlling the mixture ratio of biocompatible 2-hydroxyethylmethacrylate (HEMA) as the main monomer and vinylphenylboronic acid (VPBA) as a glucose-responsive monomer. The gate surface potential of the hydrogel FETs shifts in the negative direction with increasing glucose concentration from 10 μM to 40 mM, which results from the increase in the negative charges on the basis of the diol-binding of PBA derivatives with glucose molecules in the hydrogel. Moreover, the hydrogel coated on the gate suppresses the signal noise caused by the nonspecific adsorption of proteins such as albumin. The hydrogel FET can serve as a highly sensitive and biocompatible glucose sensor in in vivo or ex vivo applications such as eye contact lenses and sheets adhering to the skin.

Ion Sensitive Transparent-Gate Transistor for Visible Cell Sensing

In this study, we developed an ion-sensitive transparent-gate transistor (IS-TGT) for visible cell sensing. The gate sensing surface of the IS-TGT is transparent in a solution because a transparent amorphous oxide semiconductor composed of amorphous In-Ga-Zn-oxide (a-IGZO) with a thin SiO2 film gate that includes an indium tin oxide (ITO) film as the source and drain electrodes is utilized. The pH response of the IS-TGT was found to be about 56 mV/pH, indicating approximately Nernstian response. Moreover, the potential signals of the IS-TGT for sodium and potassium ions, which are usually included in biological environments, were evaluated. The optical and electrical properties of the IS-TGT enable cell functions to be monitored simultaneously with microscopic observation and electrical measurement. A platform based on the IS-TGT can be used as a simple and cost-effective plate-cell-sensing system based on thin-film fabrication technology in the research field of life science.

Characterization of ion-sensitive extended-gate field effect transistor coated with functional self-assembled monolayer

Extended-gate field effect transistors (FETs) were characterized by modifying self-assembled monolayers (SAMs) of alkanethiols with functional groups (–CH3, –COOH, –NH2, and –OH) on the gate surface. The SAMs with a polar group such as –COOH or –NH2 on the Au gate surface of the FET showed potential responses in the pH range of 5 to 10. In particular, the carboxylated SAM-coated gate FET showed higher sensitivities of 42 to 56 mV/pH, which are close to those of a Nernstian response. Moreover, the pH dependence of the carboxylated SAM-coated gate FET was maintained even in a solution with a higher salt concentration of 500 mM, which was used as the measurement solution. The effect of the ion strength in the solution on the pH response using the SAM-coated gate FET should be considered for the change in the concentration of hydrogen ions within the Debye length, which can be explained using the site-binding model for the functional groups at the terminus of the alkyl chains. In this case, the Debye length is assumed to be the distance from the packed alkyl chain layer of SAMs, which are hydrophobic. From these results, the construction of the SAMs with various functional groups on extended-gate FETs enables the FETs to be applied in ion-sensitive FET biosensors depending on the chemical characteristics of biological targets.

Nonoptical Detection of Allergic Response with a Cell-Coupled Gate Field-Effect Transistor

In this study, we report the label-free and reliable detection of allergic response using a cell-coupled gate field-effect transistor (cell-based FET). Rat basophilic leukemia (RBL-2H3) cells were cultured as a signal transduction interface to induce allergic reaction on the gate oxide surface of the FET, because IgE antibodies, which bind to Fcε receptors at the RBL-2H3 cell membrane, are specifically cross-linked by allergens, resulting in the allergic response of RBL-2H3 cells. In fact, the surface potential at the FET gate decreased owing to secretions such as histamine from the IgE-bound RBL-2H3 cells, which reacted with the allergen. This is because histamine, as one of the candidate secretions, shows basicity, resulting in a change in pH around the cell/gate interface. That is, the RBL-2H3-cell-based FET used in this study was originally from an ion-sensitive FET (ISFET), whose oxide surface (Ta2O5) with hydroxyl groups is fully responsive to pH on the basis of the equilibrium reaction. The allergic response of RBL-2H3 cells on the gate was also confirmed by estimating the amount of β-hexosaminidase released together with histamine and was analyzed using the electrical properties based on an inflammatory response of secreted histamine with the vascular endothelial cell-based FET. Thus, the allergic responses were monitored in a nonoptical and real-time manner using the cell-based FETs with the cellular layers on the gate, which reproduced the in vivo system and were useful for the reliable detection of the allergic reaction.

Long-term and real-time monitoring of chondrocyte behavior synthesizing extracellular matrix with biologically coupled field effect transistor

In this study, we report the differential measurement method of accurately monitoring cellular metabolism with a semiconductor-based field effect transistor (FET), focusing on the proliferation potency of chondrocytes utilized in the field of orthopedics. By adding growth factors to chondrocytes on the gate, cellular activity was induced and continuously monitored as a change in pH during a cellular respiration for ten days using the FET biosensor. Moreover, the electrical signal of the FET device reflected the reproduction property of chondrocytes to synthesize extracellular matrix (ECM). A platform based on the FET device is suitable as a noninvasive, real-time and long-term monitoring system for cellular functions; it will contribute to the elucidation of the mechanism of ECM synthesis by chondrocytes.

Biocompatible Poly(catecholamine)-Film Electrode for Potentiometric Cell Sensing

Surface-coated poly(catecholamine) (pCA) films have attracted attention as biomaterial interfaces owing to their biocompatible and physicochemical characteristics. In this paper, we report that pCA-film-coated electrodes are useful for potentiometric biosensing devices. Four different types of pCA film, l-dopa, dopamine, norepinephrine, and epinephrine, with thicknesses in the range of 7-27 nm were electropolymerized by oxidation on Au electrodes by using cyclic voltammetry. By using the pCA-film electrodes, the pH responsivities were found to be 39.3-47.7 mV/pH within the pH range of 1.68 to 10.01 on the basis of the equilibrium reaction with hydrogen ions and the functional groups of the pCAs. The pCA films suppressed nonspecific signals generated by other ions (Na+, K+, Ca2+) and proteins such as albumin. Thus, the pCA-film electrodes can be used in pH-sensitive and pH-selective biosensors. HeLa cells were cultivated on the surface of the pCA-film electrodes to monitor cellular activities. The surface potential of the pCA-film electrodes changed markedly because of cellular activity; therefore, the change in the hydrogen ion concentration around the cell/pCA-film interface could be monitored in real time. This was caused by carbon dioxide or lactic acid that is generated by cellular respiration and dissolves in the culture medium, resulting in the change of hydrogen concentration. pCA-film electrodes are suitable for use in biocompatible and pH-responsive biosensors, enabling the more selective detection of biological phenomena.

Monitoring of hydroxyapatite crystal formation using field-effect transistor

The biomineralization process of hydroxyapatite (HAp) in simulated body fluid (SBF) was monitored in realtime using extended-gate FETs whose gate electrode was modified with a variety of alkanethiol self-assembled monolayers (SAMs). It was found that the gate surface potential of the carboxyl- and amino-group-terminated SAM-coated gate FET was increased in SBF as HAp crystals grew on the gate surface. Moreover, in the carboxyl-group-terminated SAM-coated gate FET, the rate of increase and the shift of gate surface potential of the FET were found to depend on the concentration of calcium ions in the SBF. It was concluded that the process of HAp crystallization at a SAM-modified surface can be detected using FETs. Thus, a FET device that enables the easy detection of ionic charges in a real-time and label-free manner, will be useful for evaluating biomaterials based on biomineralization such as those in the bone regeneration process.