Ever since Clark and Lyons introduced the first enzyme sensor five decades ago, extensive studies have been carried out to develop a range of biosensors employing the combination of biosensing molecules and transducers. The evolution from the early generation through to the current generation of biosensor research has witnessed the appearance of a number of very diversified biosensing molecules. In this review, we summarize the biomolecular engineering technology underlying the development of biosensing molecules. Among the various biosensor research targets, we focused on the development of glycated protein sensing technology. Glycated protein sensing is one of the most emergent and focused on technology in the field of diabetes diagnosis. We especially focused on the recent advances in the development of fructosyl amino acid/fructosyl peptide oxidases, which are the key enzymes in the biochemical measurement of glycated proteins, as the representative biosensing molecules to acknowledge the rewards of the current advanced biomolecular engineering technology.

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The feasibility of using the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) as a novel tool for FACS analysis during the ES cell differentiation process has been discussed. 2-NBDG entered into the ES cells via a D-glucose specific uptake activity and a general metabolic activity. Although the 2-NBDG uptake was not distinct enough for discriminating cell types, changes in the histogram of 2-NBDG loaded cells during differentiation paralleled with their morphological changes. Thus, it was suggested that 2-NBDG could be used for monitoring the metabolic status of cells during differentiation as an additional marker for higher resolution analysis by FACS.

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In the process of molecular modification on an electrode surface, the unspecific adsorption of molecules impedes preparation of ordered molecular interface. In this study, we have investigated the unspecific adsorption of a designed molecule for photo-excited current based affinity sensors on a semiconductor electrode. In preparation process of the molecular interface, we propose an unspecific-adsorption-free process using cyclodextrin, which is able to suppress unspecific molecular adsorption at the solution-solid interface. Using the method, specific reaction of molecular immobilization can be done without disturbance of the unspecific adsorption.

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We developed a 3D interdigitated (IDA) array electrode consisting of Au nano particles, which increase the electrode surface. The electrode was fabricated using microfabrication and printing techniques. Electrochemical impedance spectroscopy was conducted to measure IgG concentration, when anti-IgG was immobilized onto Dithiobis (succinimidyl propionate) (DTSP) SAM/3D-IDA electrode. A linear relationship between charge transfer resistance (R_ct) values and the logarithm of IgG concentration was observed in the range of 1 µg/ml to 1 mg/ml.

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Room-temperature ionic liquid (RTIL) that is a liquid salt at or below room temperature is expected to be an innovative functional solvent and a liquid material due to its anomalous physicochemical properties such as negligible vapor pressure, flame resistance, and relatively-high conductivity. With an eye on what RTIL has negligible vapor pressure, we have created the analytical technique combined with RTIL and secondary electron microscope (SEM). In this paper, we report several RTIL-based SEM techniques that will be a useful analytical method in both electrochemistry and life science. The aim of this study is to show the utility of the RTIL-based SEM techniques.

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Cytochrome c₃, which has four bis-histidinyl coordinated hemes per molecule, is a redox protein, and stores electrons temporarily until recognizing a redox partner. This mechanism is called “electron pool effect”. In this study, highly sensitive EQCM measurement clarified which heme in cytochrome c₃ caused the electron pool effect. To investigate the key heme to the electron pool effect, cytochrome c₃ mutants in which the sixth axial ligand of one heme was changed were prepared, and the intermolecular electron transfer was measured by viologen-immobilized electrode. The kinetics of the electron transfer complex formation between cytochrome c₃ and immobilized viologen indicated that redox of the heme II in cytochrome c₃ caused the electron pool effect.

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The influence of the channel-forming compound (amphotericin B) on the ion transport between two aqueous phases (W1 and W2) across the bilayer lipid membrane was electrochemically investigated. Sodium salts of organic acids (Na⁺Org⁻) were used as electrolytes in W1 and W2. When the concentrations of Na⁺Org⁻ in W1 and W2 are asymmetrically different, the permeabilites of Na⁺ and Org⁻ were estimated from the zero-current membrane potential. In the presence of amphotericin B, the permeability of Org⁻ is larger than that of Na⁺. It is revealed that the permeability of Org⁻ across the BLM decreases with an increase in molecular weight of Org⁻. The upper limit of molecular weight on the permeable anion was estimated to be about 230 by the extrapolation. The limit seems to be ascribed to the pore size of the amphotericin B channel.

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A patch-type oxygen imaging sheet useful for in vitro cellular metabolic assays was developed. Oxygen-responsive fluorescent microbeads were embedded into a biocompatible polyacrylamide gel sheet, which can be directly attached onto target cells for fluorescent imaging of metabolic activity. The sensor beads were directed in a microfluidic device using AC and DC electric manipulation techniques, followed by encapsulation in a hydrogel. Fluorescent imaging of oxygen-consuming activity was demonstrated for glucose oxidase-modified microparticles as cellular models to show the applicability of the imaging sheet to bioassays.

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In order to accelerate cell leading into microwells on single cell based microwell array system, negative dielectrophoresis was employed. Gold thin film was formed onto PDMS microwell array sheet and high frequency sinusoidal voltage was applied between the gold film and ITO electrode which was above microwell array. Optimal conditions for negative dielectrophoresis were 100 MHz and 3 V in this system. Implanted ratio (ratio of cells in wells comparing with total cells in the measured image) was 63.8%, over 4.5 fold higher than without voltage application. The application of optimal sinusoidal voltage gave no damage on animal cells.

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The current density of gas-diffusion biocathodes for the oxygen reduction in biofuel cells was successfully increased by adjusting the hydrophobicity of porous carbon electrodes and by adding small amount of non-ionic surfactant to enzyme solution to be used in the enzyme adsorption process. Optimization of the hydrophobicity was performed with non-glycoprotein cupper efflux oxidase as an electrocatalyst. Carbon slurry was prepared by mixing Ketjen black and polytetrafluoroethylene in 2-propanol at a weight ratio of 3:2. Carbon paper was modified with the slurry and dried at 60°C to remove the solvent. Addition of small amount of non-ionic surfactant such as Triton X-100 (about 0.01%) into the enzyme solution was very effective to adsorb the enzyme on the hydrophobic porous carbon surface. The proposed method is also effective for glycoprotein multi-copper oxidase to fabricate high performance gas-diffusion biocathode.

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This study investigated the ability of the bacterium Acidithiobacillus ferrooxidans to convert CO₂ into organic compounds in batch culture and during electrochemical cultivation. Metabolites secreted into the culture medium were detected and analyzed by HPLC and mass spectroscopy, which revealed that pyruvic acid was one of the organic acids present in both cultures. We found that although pyruvic acid acted as a strong inhibitor for microbial growth in batch cultures, microbial CO₂ fixation activity in electrochemical cultures was not significantly affected by the addition of excess pyruvic acid. Together, these findings indicate that A. ferrooxidans is a promising bioelectrocatalyst for the conversion of CO₂ into extracellular organic compounds.

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Geobacter sulfurreducens has been extensively studied as a model electricity-producing microbes and it is now well known that extracellular electron transfer (EET) to an anode in this microbe is mediated through the expression of abundant outer membrane c-type cytochromes (OMCs). As the electrons need to travel across the biofilm formed on an anode, the macroscopic EET activity depends on the biofilm thickness. In this sense, it is worth investigating how the EET current depends on the optical cell density (OD) in the electrochemical chamber. Here we report that the OD value strongly affects on the relationship between the EET current and the anode potential.

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We propose an electrochemical, low-concentration adenosine triphosphate (ATP) assay combined with enzymatic ATP amplification. The electrochemical ATP assay consists of four parts: (i) myokinase for converting adenosine monophosphate (AMP) + ATP to two adenosine diphosphate (ADP) molecules, (ii) pyruvate kinase for converting two ADP molecules back to two ATP molecules (ATP amplification) and conversion of phosphoenolpyruvate to pyruvate, (iii) pyruvate dehydrogenase for generating H₂O₂ coupling with pyruvate oxidation, and (iv) electrochemical detection of H₂O₂. This system was used to detect ATP with a detection limit of 10⁻⁹ M ATP.

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The direct electron transfer (DET) reactions of proteins (cytochrome c [cyt c], D-fructose dehydrogenase [FDH], and bilirubin oxidase [BOD]) immobilized on electrodes coated with Au nanoparticles (AuNPs) of various sizes (particle sizes: 7 nm [AuNP₇], 15 nm [AuNP₁₅], and 70 nm [AuNP₇₀]) were examined to elucidate the effect of the AuNP size. For cyt c, the reduction and oxidation currents for the AuNP₇-modified electrode were small compared to the other AuNP sizes (AuNP₁₅ and AuNP₇₀) with the same roughness factor (R_f). For BOD and FDH, the AuNP₇₀-modified electrode had the best performance in terms of the catalytic current at the same R_f. The results demonstrate that the size of spaces on the electrode surface is a very important factor governing the electron transfer reactions of proteins on an AuNP-modified electrode.

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Flow injection analysis with electrochemical detection (FIA-ECD) using α-tocopheol (α-TOH) as a reagent was developed for determining ammonia in exhaled breath. An ethanol-water (4:1, v/v) mixture containing 3 mM α-TOH and 50 mM NaCl was used as the carrier solution. The FIA response at +0.70 V vs. Ag/AgCl in the flow cell was linear over a range of the ammonia concentration from 0.11 to 1.1 ppmv (r = 0.999, n = 7). The lower limit of detection for ammonia was 130 pg (S/N = 3), and the relative standard deviation (RSD) was 2.1% (17 ng, n = 10). The collected exhaled breath in a Tedlar^® bag was mixed with water containing NaCl to dissolve ammonia in exhaled breath, and then it was diluted with ethanol containing α-TOH to the same composition of the carrier solution to be injected into the FIA-ECD. The present FIA-ECD method required simple sample preparation and was applied to the determination of ammonia in exhaled breath from a healthy human and gerbils.

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Single nucleotide polymorphisms (SNPs) are important biomarkers for evaluating sensitivity to drugs and for predicting whether people might have a disease in the future. In this study, we constructed an electrochemical detection system of an SNP of peroxisome proliferator-activated receptor γ₂ (PPARγ₂) (C34G) using glucose dehydrogenase (GDH) based on a single base extension method. Target DNA was hybridized with capture DNA immobilized on a gold electrode. Biotinylated dCTP was inserted to probe DNA if target DNA had the SNP. Avidin conjugated GDH can bind to one base extended DNA resulting in increased response current after washing of redundant avidin conjugated GDH and addition of glucose. In this system, PPARγ₂ (C34G) was detected specifically with 10⁻⁸ mol dm⁻³ detection limit based on amperometric sensing system.

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Vascular endothelial growth factor (VEGF) is expected to find application as a prognostic biomarker in cancer diagnosis. In this study, we measured VEGF using a simple bound/free separation system that utilizes an aptamer, whose capacity for hybridization with capture DNA immobilized on beads changes in the presence or absence of target molecules. Two systems were constructed using a VEGF aptamer selected by in vitro screening and a VEGF aptamer improved by a dimerization strategy. We can detect VEGF based on electrochemistry using both aptamers. We previously reported the detection of two proteins in addition to VEGF—IgE and thrombin—and the success of VEGF detection with this system suggests that this is a versatile system for the detection of various molecules.

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An amperometric sensor based on a soluble molecularly imprinted catalyst (MIC) has been developed for the detection of fructosyl amine compounds. A soluble MIC containing water-soluble functional monomers, an imidazole catalyst, and small amounts of a hydrophilic cross-linker is developed and used as a fructosyl amine oxidase mimic and for amperometric sensor construction. Fructosyl valine (Fru-val), a model compound of glycated hemoglobin, HbA1c, is used as the template. The MIC specifically oxidizes Fru-val in the presence of 1-methoxyphenazine methosulfate (electron acceptor) and reacts with the glycated peptide, fructosyl-valine-histidine sequence at the N-terminal of the β-globin in HbA1c. The biosensor was fabricated by immobilizing the soluble MIC on Au electrodes via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-mediated amidation coupling. Using the soluble MIC-based sensor, 0.05 to 0.6 mM Fru-val could be determined at 40°C and neutral pH. The steady-state current increase for this sensor was 33 nA in the presence of 0.05 mM Fru-val. The sensor showed 1.4 times higher sensitivity to Fru-val than to Fru-ε-lys, the competitor in HbA1c detection.

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Hemoglobin (Hb) was adsorbed onto a carbon-felt (CF), which is a microelectrode ensemble of microcarbon fiber (ca. 7 µm diameter) and possesses a random three-dimensional structure. Direct electron transfer between Hb heme and the CF was achieved without any electron mediating species and special materials. The Hb-adsorbed CF (Hb-CF) showed a pair of well-defined cyclic voltammetric peaks with the formal potential of −0.221 V (vs. Ag/AgCl) at pH 5.0 (0.1 M phosphate/citrate buffer), which is attributed to Hb Fe(III)/Fe(II) redox couple. The apparent heterogeneous electron-transfer rate constant (k_s) was estimated to be 14.6 s⁻¹. Furthermore, the Hb-CF exhibited an excellent bioelectrocatalytic activity for the reduction of O₂. This bioelectrocatalytic activity was inhibited by azide, which binds to active heme center of Hb.

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We developed an electrochemical vascular endothelial growth factor (VEGF) detection system using two VEGF-binding aptamers. Sensitive detection systems of VEGF are highly required for cancer diagnosis. In this study, VEGF-A was detected using a sandwiching method with a pyrroquinoline quinone glucose dehydrogenase (PQQ-GDH)-labeled VEGF-binding aptamer and another aptamer immobilized onto a gold wire electrode. We evaluated various combinations of VEGF-binding aptamers to be employed in the process. In addition, we examined dose dependency for the electric current generated by PQQ-GDH, measured in the constructed VEGF detection system. Using this newly constructed system, we successfully detected VEGF₁₆₅ at 15 nM (M = mol dm⁻³) concentration.

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A stand-alone wireless glucose-sensing system can be constructed by combining a biofuel cell and a transmitter that sends signals to the receiver using only the power generated in the biofuel cell without any external power source. However, the inherent low power supply of biofuel cells limits its application as a power source for signal transmittance. We have previously reported a novel device, called a “BioCapacitor.” In this study, we report a stand-alone, self-powered, wireless glucose sensing system called a “BioLC-Oscillator.” A BioLC-Oscillator is composed of a BioCapacitor and voltage controlled oscillator (VCO) circuits, whose resonance frequencies depend on the input voltage level. We succeeded in constructing a stand-alone, self-powered, wireless glucose sensing system called the BioLC-Oscillator by using a radio transmitter in which the radio wave resonance frequency changes according to glucose concentration within the range from 0.86 to 10.1 mM, which covers from the hypoglycemic range to a part of the hyperglycemic range.

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Nitrous oxide (N₂O) is known as a greenhouse gas and a dominant ozone-depleting substance. It is is released mainly from agricultural processes. Therefore, the development of an on-site monitoring system is required to measure N₂O concentration and control the release from the source. Using oxygen-insensitive cytochrome c-type nitrous oxide reductase, wNosZ, from Wolinella succinogenes, we have developed an electrochemical enzyme sensor. The sensor signal depends on the concentration of N₂O in a reaction cell containing methoxy-5-methylphenazinium methylsulfate (mPMS) as an electron mediator under Ar atmosphere and, surprisingly, under air. Moreover, in the absence of addition of an electron mediator to the reaction cell, we observe that the reduction current depends on the concentration of N₂O, which implies direct electron transfer. The wNosZ electrode is stable when stored at 4°C for 2 weeks and is specific to N₂O. These results suggest that wNosZ holds great promise as a component of a novel direct-electron-transfer-type electrochemical sensing system for N₂O.

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A direct electron transfer-type glucose sensor was constructed using a bacterial membrane-bound thermostable periplasmic glucose dehydrogenase complex (FADGDH), which was composed of a FAD-containing catalytic subunit, a cytochrome c subunit containing heme c as the electron transfer unit, and a chaperone-like subunit. To allow for subcutaneous insertion of the electrode, a stainless-based needle-type miniaturized electrode having the same diameter as a 30-G needle (0.3 mm) was designed. To achieve high current density, we investigated the use of carbon nanoparticles with various surface areas as sensor components. The current density correlated well with the surface area of the carbon nanoparticles, and Ketjen Black was found to be the best carbon nanoparticle in combination with FADGDH. The sensor responded quickly to glucose and demonstrated potential application for monitoring glucose levels in vivo.

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