Tomohiko Yamazaki

Construction of a molecular imprinting catalyst using target analogue template and its application for an amperometric fructosylamine sensor.

Molecular imprinting technology is becoming a versatile tool for preparing tailor-made molecular recognition elements. However, inherent problems of the molecular imprinting technology include the availability and preparation of template molecules. We recently reported artificial enzyme sensors for fructosylamines constructed by imprinting with fructosyl valine (Fru-val), a model compound for HbA1c (Anal. Lett., 2003). However, because the availability of Fru-val is limited, we attempted to construct a Fru-val-oxidizing molecularly imprinted catalyst (MIC) utilizing the analogue molecule methyl valine (m-val) as template molecule. An electrode employing the m-val-imprinted polymer showed 1.2-fold higher sensitivity toward Fru-val compared with the control polymer-employing electrode. We also used the positively charged functional monomer allylamine as functional monomer in order to increase the selectivity of the MIC toward Fru-val. The selectivity of the electrode immobilizing the allylamine-containing polymer showed 1.7-fold higher response toward Fru-val than toward Fru-epsilon-lys. By combining the use of both allylamine as the functional monomer and m-val as the template molecule, an even better MIC-immobilized electrode was produced with a Fru-val selectivity comparable to that constructed by imprinting with Fru-val.

Towards the use of molecularly imprinted polymers containing imidazoles and bivalent metal complexes for the detection and degradation of organophosphotriester pesticides

Abstract For the preparation of an enzyme mimic for phosphotriester hydrolysis, we have introduced catalytic groups in a polymer, mimicking the structure of the catalytic center of the enzyme phosphotriesterase. The polymers containing Co 2+ –imidazole complexes show a hydrolyric activity which was 20 times higher than polymers containing only imidazole or a solution containing only cobalt ions. Additionally, a molecularly imprinted polymer that was synthesized using a paraoxon analogue as template showed higher paraoxon hydrolysis activity than a control polymer prepared in the same way but without the template molecule.

A novel thermostable glucose dehydrogenase varying temperature properties by altering its quaternary structures

Abstract A novel thermostable glucose dehydrogenase was purified from a Gram-negative moderate thermophilic bacterium isolated from soil near a hot spring. The homogeneously purified enzyme sample showed two peaks at the optimum temperature for reaction, i.e., at around 45 and 70°C. However after 30 min of incubation at 70°C, the enzyme showed only one peak at 75°C. From the results of native and SDS-PAGE of this enzyme, it was suggested that at temperatures below 45°C, this enzyme was a hetero-oligomeric complex constructed from two distinct peptides with MWs of 67,000 and 43,000, thereby showing that the optimum temperature for reaction was 45°C. Incubation at 70°C of this hetero-oligomer dissociated each subunit and resulted in a single peptide enzyme with a MW of 67,000 that showed GDH activity with optimal temperature only at 75°C. This single peptide enzyme retained more than 80% of its initial activity even after 30 min of incubation at 60°C. Therefore, we concluded that this novel enzyme showed a different optimum temperature for reaction according to its quaternary structure.

Highly Ordered 1D Fullerene Crystals for Concurrent Control of Macroscopic Cellular Orientation and Differentiation toward Large‐Scale Tissue Engineering

A highly aligned 1D fullerene whisker (FW) scaffold in a centimeter area is fabricated by interfacial alignment. The resulting aligned FW scaffold enables concurrent control over cellular orientation and differentiation to muscle cells. This aligned FW scaffold is made by a facile method, and hence the substrate is a promising alternative to other cell scaffolds for tissue engineering.

Cloning and expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase from Burkholderia cepacia in Escherichia coli

We have cloned a 1620-nucleotide gene encoding the catalytic subunit (alpha subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia. The FAD binding motif was found in the N-terminal region of the alpha subunit. The deduced primary structure of the alpha subunit showed about 48% identity to the catalytic subunits of sorbitol dehydrogenase (SDH) from Gluconobacter oxydans and 2-keto-D-gluconate dehydrogenases (2KGDH) from Erwinia herbicola and Pantoea citrea. The alpha subunit of B. cepacia was expressed in Escherichia coli in its active water-soluble form, showing maximum dye-mediated GDH activity at 70 degrees C, retaining high thermal stability. A putative open reading frame (ORF) of 507 nucleotides was also found upstream of the alpha subunit encoding an 18-kDa peptide, designated as gamma subunit. The deduced primary structure of gamma subunit showed about 30% identity to the small subunits of the SDH from G. oxydans and 2KGDHs from E. herbicola and P. citrea.

BioCapacitor—A novel category of biosensor

This research reports on the development of an innovative biosensor, known as BioCapacitor, in which biological recognition elements are combined with a capacitor functioning as the transducer. The analytical procedure of the BioCapacitor is based on the following principle: a biocatalyst, acting as a biological recognition element, oxidizes or reduces the analyte to generate electric power, which is then charged into a capacitor via a charge pump circuit (switched capacitor regulator) until the capacitors attains full capacity. Since the charging rate of the capacitor depends on the biocatalytic reaction of the analyte, the analyte concentration can be determined by monitoring the time/frequency required for the charge/discharge cycle of the BioCapacitor via a charge pump circuit. As a representative model, we constructed a BioCapacitor composed of FAD-dependent glucose dehydrogenase (FADGDH) as the anodic catalyst, and attempted a glucose measurement. Charge/discharge frequency of the BioCapacitor increased with the increasing glucose concentration, exhibiting good correlation with glucose concentration. We have also constructed a wireless sensing system using the BioCapacitor combined with an infrared light emitting diode (IRLED), an IR phototransistor system. In the presence of glucose, the IRLED signal was observed due to the discharge of the BioCapacitor and detected by an IR phototransistor in a wireless receiver. Therefore, a BioCapacitor employing FADGDH as its anodic catalyst can be operated as a self-powered enzyme sensor.

Vortex-Aligned Fullerene Nanowhiskers as a Scaffold for Orienting Cell Growth

A versatile method for the rapid fabrication of aligned fullerene C60 nanowhiskers (C60NWs) at the air-water interface is presented. This method is based on the vortex motion of a subphase (water), which directs floating C60NWs to align on the water surface according to the direction of rotational flow. Aligned C60NWs could be transferred onto many different flat substrates, and, in this case, aligned C60NWs on glass substrates were employed as a scaffold for cell culture. Bone forming human osteoblast MG63 cells adhered well to the C60NWs, and their growth was found to be oriented with the axis of the aligned C60NWs. Cells grown on aligned C60NWs were more highly oriented with the axis of alignment than when grown on randomly oriented nanowhiskers. A study of cell proliferation on the C60NWs revealed their low toxicity, indicating their potential for use in biomedical applications.

Subunit analyses of a novel thermostable glucose dehydrogenase showing different temperature properties according to its quaternary structure

We previously reported a novel glucose dehydrogenase (GDH) showing two peaks in the optimum temperature for the reaction at around 45°C and at around 75°C. Each peak derived from hetero-oligomeric enzyme, constructed from two distinct peptides with an α-subunit (MWs 67,000) and β-subunit (MWs 43,000), and a single peptide enzyme containing an α-subunit alone. The function of the two subunits in the thermostable co-factor binding GDH was investigated. The results of spectroscopic analyses indicated that the α-subunit contained an unknown co-factor showing specific fluorescence spectra like pyrroloquinoline quinone (PQQ), and the β-subunit was cytochrome c. Moreover, the results of a urea denaturation and reconstitution experiment suggested that the dissociation of the hetero-oligomeric complex to a single peptide was reversible. The kinetic parameter analyses for glucose and the electron mediator also suggested that the β-subunit was responsible for electron transfer from the catalytic center of the α-subunit to the electron mediator.

BioRadioTransmitter: A Self-Powered Wireless Glucose-Sensing System

Background: Although an enzyme fuel cell can be utilized as a glucose sensor, the output power generated is too low to power a device such as a currently available transmitter and operating system, and an external power source is required for operating an enzyme-fuel-cell-based biosensing system. We proposed a novel biosensor that we named BioCapacitor, in which a capacitor serves as a transducer. In this study, we constructed a new BioCapacitor-based system with an added radio-transmitter circuit and a miniaturized enzyme fuel cell. Methods: A miniaturized direct-electron-transfer-type compartmentless enzyme fuel cell was constructed with flavin adenine dinucleotide-dependent glucose dehydrogenase complex-based anode and a bilirubin-oxidase-based cathode. For construction of a BioRadioTransmitter wireless sensing system, a capacitor, an ultra-low-voltage charge-pump-integrated circuit, and Hartley oscillator circuit were connected to the miniaturized enzyme fuel cell. A radio-receiver circuit, comprising two field-effect transistors and a coil as an antenna, was used to amplify the signal generated from the biofuel cells. Results: Radio wave signals generated by the BioRadioTransmitter were received, amplified, and converted from alternate to direct current by the radio receiver. When the capacitor discharges in the presence of glucose, the BioRadioTransmitter generates a radio wave, which is monitored by a radio receiver connected wirelessly to the sensing device. Magnitude of the radio wave transmission frequency change observed at the radio receiver was correlated to glucose concentration in the fuel cells. Conclusions: We constructed a stand-alone, self-powered, wireless glucose-sensing system called a BioRadioTransmitter by using a radio transmitter in which the radio wave transmission frequency changes with the glucose concentration in the fuel cell. The BioRadioTransmitter is a significant advance toward construction of an implantable continuous glucose monitor.

Molecular Imprinting Catalyst Based Artificial Enzyme Sensor for Fructosylamines

Abstract The molecular imprinting technology is becoming a versatile tool in constructing tailor-made molecular recognition elements. However, no oxidoreductase mimics which can be utilized for the amperometric sensor has ever been developed using molecularly imprinting technology. Glycated hemoglobin, HbA1c, is an important indicator for diabetic control. In order to develop a novel diagnostic system for the measurement of HbA1c, we developed molecularly imprinting technology based artificial enzyme which can catalyze the oxidative cleavage of fructosyl valine, a model compound for HbA1c. Based on our previous report, that polyvinylimidazole functioned as the catalyst for this reaction, we developed the molecularly imprinted catalyst (MIC). Molecularly imprinted catalyst was synthesized by imprinting polyvinylimidazole based polymer using fructosyl valine (Fru-val) that is the model compound for HbA1c as template molecules, based on both typical principles for molecular imprinting, noncovalent and covalent approaches. Fructosyl valine bound to the imprinted polymer more than two times higher than the control polymer, which was prepared in the same way but without the template molecule. The MIC-employing sensor showed high current response to Fru-val compared with the sensor employing the control polymer, due to the increase in the affinity toward cis-diol harboring compound. The amperometric sensor employing MIC showed the selectivity toward Fru-val compared with the sensor signal toward fructosyl-ε-lysine (Fru-ε-lys), while the sensor employing the control polymer showed almost identical responses to Fru-val as that to Fru-ε-lys.