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In this study, we were challenging to identify characteristic compounds in breast cancer cell lines. GC analysis of extracts from the culture media of breast cancer cell lines (MCF-7, SK-BR-3, and YMB-1) using a solid-phase Porapak Q extraction revealed that two compounds of moderate volatility, 1-hexadecanol and 5-(Z)-dodecenoic acid, were detected with markedly higher amount than those in the medium of fibroblast cell line (KMST-6). Furthermore, LC-TOF/MS analysis of the extracts clarified that in addition to the above two fatty acids, the amounts of five unsaturated fatty acids [decenoic acid (C10:1), decadienoic acid (C10:2), 5-(Z)-dodecenoic acid (C12:1), 5-(Z)-tetradecenoic acid (C14:1), and tetradecadienoic acid (C14:2)] in MCF-7 medium were higher than those in medium of KMST-6. Interestingly, H2O2-oxidation of 5-(Z)-dodecenoic acid and 5-(Z)-tetradecenoic acid produced volatile aldehydes that were reported as specific volatiles in breath from various cancer patients, such as heptanal, octanal, nonanal, decanal, 2-(E)-nonenal, and 2-(E)-octenal. Thus, we concluded that these identified compounds over-produced in breast cancer cells in this study could serve as potential precursors producing reported cancer-specific volatiles.

The saltiness enhancement effect is the effect whereby saltiness is enhanced by adding specific substances to salt (sodium chloride). Since this effect can be used in the development of salt-reduced foods, a method to objectively evaluate the saltiness with this effect is required. A taste sensor with lipid/polymer membranes has been used to quantify the taste of food and beverages in recent years. The sensor electrodes of this taste sensor have the feature of selectively responding to each of the five basic tastes, which is realized by the lipid/polymer membranes. In this study, we developed a new saltiness sensor based on the lipid/polymer membrane with the aim of quantifying the saltiness enhancement effect. In addition to the conventional components of a lipid, plasticizer, and polymer supporting reagent, the membrane we developed comprises ionophores, which selectively capture sodium ions. As a result, the response of the sensor increased logarithmically with the activity of NaCl in measured samples, similarly to the taste response of humans. In addition, all of the sensor responses increased upon adding saltiness-enhancing substances, such as citric acid, tartaric acid and branched-chain amino acids (BCAAs), to NaCl samples. These findings suggest that it is possible to quantify the saltiness enhancement effect using a taste sensor with lipid/polymer membranes.

This paper reports the improvement of a bitterness sensor based on a lipid polymer membrane consisting of phosphoric acid di-n-decyl ester (PADE) as a lipid and bis(1-butylpentyl) adipate (BBPA) and tributyl o-acetylcitrate (TBAC) as plasticizers. Although the commercialized bitterness sensor (BT0) has high sensitivity and selectivity to the bitterness of medicines, the sensor response gradually decreases to almost zero after two years at room temperature and humidity in a laboratory. To reveal the reason for the deterioration of the response, we investigated sensor membranes by measuring the membrane potential, contact angle, and adsorption amount, as well as by performing gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS). We found that the change in the surface charge density caused by the hydrolysis of TBAC led to the deterioration of the response. The acidic environment generated by PADE promoted TBAC hydrolysis. Finally, we succeeded in fabricating a new membrane for sensing the bitterness of medicines with higher durability and sensitivity by adjusting the proportions of the lipid and plasticizers.

Objective taste evaluation has been much in demand in the food, beverage and pharmaceutical industries. A taste-sensing system, which is an electronic tongue with “global selectivity,” is one of the methods used for objective taste evaluation. A taste sensor electrode responds to only one of the basic tastes (saltiness, sourness, sweetness, bitterness and umami) as a change in membrane potential caused by interactions with tastants. Sweet substances are compounds with diverse chemical structures and sizes. Since the taste-sensing system is a potentiometric measurement system using a change in membrane potential, three types of sweetness sensors are required, one for sweeteners with each type of electric charge (uncharged, positively charged and negatively charged). A sweetness sensor for uncharged sweeteners has been developed. Therefore, negatively charged sweeteners, such as saccharine sodium and acesulfame potassium, were chosen as the target substances in this study. We investigated the responses of various sensor membranes using a lipid and nine kinds of plasticizers to each basic taste sample. Furthermore, not only the selectivity of the membranes but also the concentration dependence of their response to sweeteners was investigated. As a result, one of the developed sensors showed responses of over 20 mV to 5 mM saccharine sodium and 10 mM acesulfame potassium in CPA value measurement (CPA: change in membrane potential caused by adsorption). On the other hand, the sensor also showed nearly zero responses to other basic taste samples. In addition, saltiness was the only interfering taste, and the responses to target substances in relative value measurement were over 140 mV. The developed sweetness sensor had high selectivity and concentration-dependent responses. Hence, we concluded that the sensor is suitable for use as a sweetness sensor for high-potency sweeteners with a negative electric charge.

High-potency sweeteners are applied to low-calorie diets and bitterness-masking ingredients in pharmaceutical products. We have studied taste sensors with lipid polymer membranes based on potentiometric measurement system for high-potency sweeteners. However, the sensor also responds to astringency substances because of hydrophobic characteristics of the lipid polymer membrane. In this study, we developed a new taste sensor using a lipid polymer membrane for saccharin sodium and acesulfame potassium as negatively charged high-potency sweeteners. We optimized the quantities and types of lipids and plasticizers for the fabrication of the sensor with high selectivity and sensitivity. We succeeded in the fabrication of the new sensor, the output of which could be suppressed under −5 mV for astringency substances. Moreover, the sensor has a good sensitivity and selectivity for saccharin sodium and acesulfame potassium.

The detection and recognition of metabolically derived aldehydes, which have been identified as important products of oxidative stress and biomarkers of cancers; are considered as an effective approach for early cancer detection as well as health status monitoring. Quartz crystal microbalance (QCM) sensor arrays based on molecularly imprinted sol-gel (MISG) materials were developed in this work for highly sensitive detection and highly selective recognition of typical aldehyde vapors including hexanal (HAL); nonanal (NAL) and bezaldehyde (BAL). The MISGs were prepared by a sol-gel procedure using two matrix precursors: tetraethyl orthosilicate (TEOS) and tetrabutoxytitanium (TBOT). Aminopropyltriethoxysilane (APT); diethylaminopropyltrimethoxysilane (EAP) and trimethoxy-phenylsilane (TMP) were added as functional monomers to adjust the imprinting effect of the matrix. Hexanoic acid (HA); nonanoic acid (NA) and benzoic acid (BA) were used as psuedotemplates in view of their analogous structure to the target molecules as well as the strong hydrogen-bonding interaction with the matrix. Totally 13 types of MISGs with different components were prepared and coated on QCM electrodes by spin coating. Their sensing characters towards the three aldehyde vapors with different concentrations were investigated qualitatively. The results demonstrated that the response of individual sensors to each target strongly depended on the matrix precursors; functional monomers and template molecules. An optimization of the 13 MISG materials was carried out based on statistical analysis such as principle component analysis (PCA); multivariate analysis of covariance (MANCOVA) and hierarchical cluster analysis (HCA). The optimized sensor array consisting of five channels showed a high discrimination ability on the aldehyde vapors; which was confirmed by quantitative comparison with a randomly selected array. It was suggested that both the molecularly imprinting (MIP) effect and the matrix effect contributed to the sensitivity and selectivity of the optimized sensor array. The developed MISGs were expected to be promising materials for the detection and recognition of volatile aldehydes contained in exhaled breath or human body odor.

Taste evaluation technology has been developed by several methods, such as sensory tests, electronic tongues and a taste sensor based on lipid/polymer membranes. In particular, the taste sensor can individually quantify five basic tastes without multivariate analysis. However, it has proven difficult to develop a sweetness sensor, because sweeteners are classified into three types according to the electric charges in an aqueous solution; that is, no charge, negative charge and positive charge. Using membrane potential measurements, the taste-sensing system needs three types of sensor membrane for each electric charge type of sweetener. Since the commercially available sweetness sensor was only intended for uncharged sweeteners, a sweetness sensor for positively charged high-potency sweeteners such as aspartame was developed in this study. Using a lipid and plasticizers, we fabricated various lipid/polymer membranes for the sweetness sensor to identify the suitable components of the sensor membranes. As a result, one of the developed sensors showed responses of more than 20 mV to 10 mM aspartame and less than 5 mV to any other taste. The responses of the sensor depended on the concentration of aspartame. These results suggested that the developed sweetness sensor had high sensitivity to and high selectivity for aspartame.

A taste sensor using a lipid/polymer membrane, i.e., an electronic tongue with global selectivity, has been developed for objective evaluation of the taste of foods and beverages. Moreover, the taste sensor has been also contributing to safety of foods, e.g., the sensor membrane with strong hydrophobicity was used to detect sodium dodecyl sulfate (SDS), a negatively charged surfactant, which was generally added into the pesticide because of its strongly melting effect. An immersion process in monosodium glutamate (MSG) solution, called “MSG preconditioning” was needed to obtain the change in membrane electric potential caused by adsorption (CPA) for sensor membrane before measurement. However, what happened to sensor membrane during MSG preconditioning is unclear. In this paper, we examined the relationship between the CPA value and the period of MSG preconditioning. The amount of adsorbed SDS and MSG was measured to figure out whether the CPA value is related to the amount of adsorption. As a result, with the precondition process progressed, the CPA values showed concentration dependence on SDS concentration, and increased to a peak by preconditioning for one day then decreased to a stable state after that. The amount of adsorbed SDS depended on the SDS concentration but did not change with the increasing of preconditioning time. In conclusion, we revealed that the most suitable time of MSG preconditioning for the membrane for SDS was one day. The CPA value was affected by both the surface charge density and the amount of absorption.

Recently, highly functional biosensors have been developed in preparation for possible large-scale terrorist attacks using chemical warfare agents. Practically applicable sensors are required to have various abilities, such as high portability and operability, the capability of performing rapid and continuous measurement, as well as high sensitivity and selectivity. We developed the detection method of capsaicinoids, the main component of some lachrymators, using a surface plasmon resonance (SPR) immunosensor as an on-site detection sensor. Homovanillic acid, which has a vanillyl group similar to capsaicinoids such as capsaicin and dihydrocapsaicin, was bound to Concholepas concholepas hemocyanin (CCH) for use as an immunogen to generate polyclonal antibodies. An indirect competitive assay was carried out to detect capsaicinoids using SPR sensor chips on which different capsaicin analogues were immobilized. For the sensor chip on which 4-hydroxy-3-methoxybenzylamine hydrochloride was immobilized, a detection limit of 150 ppb was achieved. We found that the incubation time was not required and the detection can be completed in five minutes.

This paper reports the development of membrane sensors based on an artificial lipid and plasticizers with high selectivity and sensitivity to drug bitterness by using bis(1-butylpentyl) adipate (BBPA), bis(2-ethylhexyl) sebacate (BEHS), phosphoric acid tris(2-ethylhexyl) ester (PTEH), and tributyl o-acetylcitrate (TBAC) as a plasticizer and phosphoric acid di-n-decyl ester (PADE) as an artificial lipid to optimize surface hydrophobicity of the sensors. In addition, a sensor with highly correlated bitterness sensory score was developed by blending BBPA and TBAC to detect the bitterness suppression effect of sucrose, and other bitter-masking materials. Therefore, this sensor can be used to evaluate the bitterness of various drug formulations with high accuracy. Copyright © 2009 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc.

A surface plasmon resonance (SPR) immunosensor for detection of 2,4-dinitrotoluene (2,4-DNT), which is a signature compound of 2,4,6-trinitrotoluene-related explosives, was developed by using a novel oligo (ethylene glycol) (OEG)-based sensor surface. A rabbit polyclonal antibody against 2,4-DNT (anti-DNPh-KLH-400 antibody) was prepared, and the avidity for 2,4-DNT and recognition capability were investigated by indirect competitive ELISA. The sensor surface was fabricated by immobilizing a 2,4-DNT analog onto an OEG-based self-assembled monolayer formed on a gold surface via an OEG linker. The fabricated surface was characterized by Fourier-transform infrared-refractive absorption spectrometry (FTIR-RAS). The immunosensing of 2,4-DNT is based on the indirect competitive principle, in which the immunoreaction between the anti-DNPh-KLH-400 antibody and 2,4-DNT on the sensor surface was inhibited in the presence of free 2,4-DNT in solution. The limit of detection for the immunosensor, calculated as three times the standard deviation of a blank value, was 20 pg mL(-1), and the linear dynamic range was found to be between 1 and 100 ng mL(-1). Additionally, the fabricated OEG-based surface effectively prevented non-specific adsorption of proteins, and the specific response to anti-DNPh-KLH-400 antibody was maintained for more than 30 measurement cycles.

Taste sensor responds to different taste qualities by unique patterns of output signals. The sensor uses lipid membranes as recognition element of tastes. Food constituents affect the membrane potential of the sensor mainly with shielding effect and/or adsorption. However suitable concentration of lipid in membrane has not been studied up to now. In this paper, we changed the concentration of lipid dioctyl phosphate (C) and trioctylmethyl ammonium chloride (T) in membranes, and investigated the response of the sensor to taste substances. Dilution of lipid improved the detection limit of the sensor to adsorptive taste substances, whereas the detection limit to non-adsorptive taste substances did not remarkably changed.

Environmental pollution of soil, river water, and underground water by explosive–related compounds is a serious problem in the areas of buried landmines and/or landmine factories (Hilmi et al., 1999). These compounds are suspected to have human toxicity, mutagenicity, and carcinogenicity (Kennel et al., 2000; Sayama et al., 1998; Tchounwou et al., 2001; Banerjee et al., 1999). Therefore, there is a pressing need to determine the concentrations and distributions of these compounds in the environment. The main component of landmines is 2,4,6–trinitrotoluene (TNT). TNT released into the environment is decomposed and converted to dinitrotoluenes (DNTs) and aminodinitrotoluenes (ADNTs), among other products, by microorganisms, light, and heat. These compounds have been measured by using HPLC or other instrumental analytical devices (Borch and Gerlach, 2004; Ahmad and Roberts; 1995). These instrumental analyses, however, require tedious pre–treatments such as extraction from samples (Halasz et al., 2002). On the other hand, detection methods using an antigen–antibody reaction, such as enzyme–linked immunosorbent assay (ELISA) and fluorescent and chemiluminescent immunosensors, are more useful and are able to detect ppb levels of explosive compounds (Goldman et al., 2003; Green et al., 2002; Wilson et al., 2003). We have been engaged in development of an antibody with high avidity to explosive–related compounds. In recent years, we have focused on the development of highly sensitive surface plasmon resonance (SPR) sensing methods for the detection of TNT using an antigen– antibody reaction (Sakai et al., 2003; Shankaran et al., 2004; Shankaran et al., 2005a; Shankaran et al., 2005b; Matsumoto et al., 2005; Shankaran et al., 2006). According to the reports of Jenkins et al. (2000, 2001), the concentrations of 2,4–DNT and other decomposed nitroaromatic compounds around and over buried landmines are higher than the concentration of TNT. In the present study, a polyclonal antibody against 2,4–DNT was raised in a rabbit, and the antibody was applied to the detection of 2,4–DNT using ELISA. The aim was to prepare anti–DNPh–KLH antibody from a rabbit and to clarify the characteristics for the detection of 2,4–DNT based on the principle of indirect competitive ELISA. In the course of the detection of free 2,4–DNT, we recognized that the combination of the structure of the coating antigen–protein, free 2,4–DNT, and the anti– DNPh–KLH antibody are an important factor in sensitivity using an indirect competitive immunoassay. Therefore, the effect of the structures of the coating antigen–protein conjugates on the sensitivity of the detection of free 2,4–DNT was investigated in detail. Preparation of Anti–dinitrotoluene Polyclonal Antibody and Effect of the Hapten Spacer Length in Coating Antigen on Immunoassay Sensitivity

Simple, automated and cost-effective sensor systems intended for detection of low-molecular-weight (LMW) fragrant compounds are indispensable at automated, bottle-filling production lines of food and beverage industries. Here, we report our investigations on the development of a highly regenerable and storageable surface plasmon resonance (SPR)-based biosensor for detection of trace amounts of benzaldehyde (BZ), a characteristic fragrant compound of peach products. The sensing surface was fabricated by self-assembling of long-chain polyethyleneglycol(PEG)-dialkanethiols on thin Au-films and by followed covalent-binding of a BZ analog on the self-assembly of PEG-dialkanethiols. The all-chemical based BZ-bound PEG-monolayer chip shows specific binding affinity toward anti-BZ antibody (BZ-Ab). The principle of indirect competitive immunoassay, promising highly sensitive and consistent detection of small molecular analytes, has been applied, wherein immunospecific binding of BZ-Ab onto the BZ-bound sensor surface is inhibited by analyte BZ present in test sample. The immunosensor exhibited excellent sensitivity for BZ detection over a wide concentration range of 0.1–80 ng/ml (ppb). The BZ-bound monolayer surface was highly regenerable and storageable for repeated use of a same sensor chip. The assay reproducibility was proved through sequential multiple analysis of the same sensor chip for more than 50 regeneration cycles. Sensitivity was enhanced further by an add-on strategy involving anti-(rabbit IgG)-functionalized Au nanoparticles (nAu-Ab), with which the sensor-signal is enhanced by about 15–18 times and the amount of BZ-Ab antibody required for an analysis could be reduced to one-tenth. With the enhancement technique, detection of as low as 7 pg/ml (ppt) BZ directly from beverage samples has been demonstrated.

This paper describes the development and comparison of two immunoassays for the detection of 2,4,6-trinitrotoluene (TNT) based on competitive inhibition. Two polyclonal antibodies were used in the immunoassay; one was prepared from 2,4,6-trinitrophenol–bovine serum albumin conjugate (anti-TNP-BSA Ab, goat IgG) and another from 2,4,6-trinitrophenyl–keyhole limpet hemocyanine conjugate (anti-TNPh-KLH Ab, rabbit IgG). A solid-phase immunoassay that consisted of physically immobilized TNP-BSA conjugate was allowed to interact with these antibodies and the resonance angle changes due to the biomolecular interactions were monitored by surface plasmon resonance (SPR). The quantification of TNT was based on the change in the resonance angle shift for binding of antibodies with a TNP-BSA conjugate, which occurred in the presence of TNT in solution. Both antibodies showed a high degree of affinity for TNT and varying degrees of cross-reactivity to the related nitroaromatic derivative compounds, such as 2,4-dinitrotoluene (DNT), 1,3-dinitrobenzene (DNB), 2-amino-4,6-dintitrotoluene (2A-4,6-DNT), 4-amino-2,6-dinitrotoluene (4A-2,6-DNT) and TNP. The anti-TNPh-KLH Ab prepared in our laboratory showed a detection limit of 0.006 ng/ml (6 ppt), which was comparable to or even better than the detection limit (0.095 ng/ml or 95 ppt) of a commercially available anti-TNP-BSA Ab for detection of TNT. A pepsin solution prepared with a glycine–HCl buffer (pH 2.0) was used for regeneration of the sensor surface. The duration for a single immunocycle was ∼19 and ∼13 min for anti-TNP-BSA Ab and anti-TNPh-KLH Ab immunosystems, respectively. The proposed immunoassay is simple and possesses good analytical characteristics for quantification of TNT.

A simple and highly sensitive surface-plasmon-resonance (SPR) immunoassay has been demonstrated for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT). The immunoreaction between a home-made polyclonal anti-2,4,6-trinitrophenyl-keyhole-limpet-hemocyanine antibody (P-TNPh-KLH Ab) with a physically immobilized TNPh-ovalbumin (TNPh-OVA) conjugate was utilized for TNT quantification based on the principle of indirect competitive immunoreaction. The performance of the P-TNPh-KLH Ab was compared with three commercially-available antibodies and was observed that the home-made antibody exhibited the highest sensitivity to TNT (1 ppt) among the other antibodies with a response time of 2 min.

A taste sensor using lipid-polymer membranes has been developed to evaluate the taste of foods, beverages and medicines. The response of the taste sensor, measured as a change in the membrane potential caused by adsorption (CPA), corresponds to the aftertaste felt by humans. The relationships between the CPA value and the amount of adsorbed taste substances, quinine and iso-α acid (bitterness), and tannic acid (astringency), have been studied so far. However, that of epigallocatechin gallate (EGCg) has not been clarified, although EGCg is abundantly present in green tea as one of its astringent substances. This study aimed at clarifying the response of the taste sensor to EGCg and its relationship with the amount of EGCg adsorbed onto lipid-polymer membranes. The lipid concentration dependence of the CPA value was similar to that of the amount of adsorbed EGCg, indicating a high correlation between the CPA value and the amount of adsorbed EGCg. The CPA value increased with increasing amount of adsorbed EGCg, however, the CPA value showed a tendency of leveling off when the amount of adsorbed EGCg further increased.

A surface plasmon resonance (SPR) immunosensor based on a competitive immunoreaction for the determination of trinitrophenol (TNP) is described. A goat anti-mouse IgG (1st antibody), which recognizes an Fc moiety of an antibody, was immobilized on a gold film of an SPR sensor chip by physical adsorption. A TNP solution containing a fixed concentration of a mouse anti-TNP monoclonal antibody (2nd antibody) and a TNP-keyhole limpet hemocyanin (KLH) conjugate was incubated in one-pot and introduced into the sensor chip. The TNP-KLH conjugate competes with TNP for binding with the 2nd antibody. The resulting complex of the 2nd antibody with the TNP-KLH conjugate was bound to the 1st antibody, which is immobilized on the sensor chip. The SPR sensor signal based on resonance angle shift is dependent on the concentration of TNP in the incubation solution in the range from 25 ppt to 25 ppb, and the coefficient of variation of the SPR signals for the 25 ppb TNP solution was determined to be 13% (n = 4). The experimental results for the adsorption constant of the 1st antibody on the sensor chip and the binding constant of the 1st antibody complex with the 2nd antibody are discussed, together with theoretical considerations.

A surface plasmon resonance (SPR) immunosensor for the determination of 2,4,6-trinitrotoluene (TNT) has been developed based on the principle of indirect competitive immunoreaction. 2,4,6-trinitrophenol-bovine serum albumin (TNP-BSA) conjugate was immobilized onto a SPR gold chip by means of simple physical adsorption. Binding of anti-TNP antibody with TNP-BSA conjugate was detected based on an increase in resonance angle due to antigen-antibody interaction. Preincubation of anti-TNP antibody with TNT suppresses its interaction with immobilized TNP-BSA conjugate, which leads to a decrease in resonance angle shift. Following the dependence of the resonance angle shift, concentration of TNT was detected. Pepsin solution was used for the regeneration of the sensing surface. The response time for TNT measurement is about 22 min. The immunosensor showed excellent sensitivity to TNT in a wide concentration range from 60 ppt to 1000 ppb with good selectivity, stability, and reproducibility. The proposed system is promising for future application for the on-site detection of landmines.

We have examined the sensing characteristics of a surface plasmon resonance (SPR) immunoassay for the detection of 2,4,6-trinitrotoluene (TNT) using an immunoreaction between 2,4,6-trinitrophenol-ovalbumin (TNP-OVA) conjugate and anti-2,4,6-trinitrophenol antibody (anti-TNP antibody). TNP-OVA conjugate was attached to a SPR-gold sensing surface by means of physical immobilization, which undergoes binding interaction with anti-TNP antibody. Both the immobilization and binding processes were studied from a change in the SPR-resonance angle. The quantification of TNT is based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-OVA conjugate and anti-TNP antibody was inhibited in the presence of free TNT in solution. The decrease in the resonance angle shift is proportional to an increase in concentration of TNT used for incubation. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range from 0.09 to 1000 ng/ml with good stability and reproducibility. The immunosensor developed could detect TNT as low as 0.09 ng/ml, within a response time of approximately 22 min. The sensor surface was regenerated by a brief flow of pepsin solution, which disrupts the antigen-antibody complex without destroying the conjugate biofilm. Cross-reactivity of the SPR sensor to some structurally related nitroaromatic derivative and the detection of TNT in the presence of these nitroaromatic compounds were investigated. The cross-reactivity of the SPR sensor to 2,4-dinitrotoluene (2,4-DNT), 1,3-dinitrobenzene (1,3-DNB), 2-amino-4,6-dinitrotoluene (2A-4,6-DNT) and 4-amino-2,6-dinitrotoluene (4A-2,6-DNT) were very low (or =1.1%). The analytical characteristics of the proposed immunosensor are highly promising for the development of new field-portable sensors for on-site detection of landmines.

This paper reports a new measurement method to detect ppb levels of noncharged organic substances using lipid/polymer membrane sensors. Noncharged organic substances have large influences on the adsorption of positively-charged lipids to negatively-charged membranes. Organic solvents (trichloroethylene) and endocrine disrupting chemicals (di-2-ethylhexylphthalate) were detected by utilizing the sensor output, which is the change of membrane potential caused by interactions among the lipid membrane, noncharged organic substances, and lipids in solution. This new potentiometric method has a possibility of detection of a trace amount of noncharged toxic substances.

Taste qualities were studied using a method for detecting the change in interactions between an electrode surface and taste substances in aqueous solution. The present method to detect chemicals was categorized into impedance spectroscopy in electrochemistry. Taste substances affect the electrochemical impedance by adsorption to the electrode surface. Dependences of the impedance on the electrode potential showed different patterns for five basic taste substances with different taste qualities. Furthermore, the response profiles obtained for capsaicin, piperine and allyl isothiocyanate, which are natural pungent-taste substances and have strong hydrophobic groups, were discriminated from those for bitter taste substances using a new method detecting the change of electrode impedance by addition of the mediators of chemical interactions to the taste substances. Hydrophobic substances, which were difficult to discriminate using the electrochemical method, could be discriminated by a proposed method; hence, it was possible to detect wide range of chemical substances from hydrophilic electrolytes to various hydrophobic non-electrolytes.

The monitoring of salivary cortisol as a key biomarker of an individual's stress response has been increasingly focused on. This paper describes the development of a novel cortisol immuno-assay method based on an indirect competitive method using a commercially available surface plasmon resonance instrument. The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog. A detection limit of 38 ppt range with a measurement range of 10 ppt-100 ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8 min from the sample injection. We experimentally compared our immuno-assay with a commercialized salivary cortisol enzyme-linked immunosorbent assay (ELISA) kit using human saliva samples. It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R = 0.96). Our findings indicate the potential utility of the cortisol immuno-assay for measurements of human salivary cortisol levels.

Phospholipids, such as phosphatidic acid, suppress bitter taste without affecting other taste qualities. In the present study, we detected and quantified this suppression effect with an electronic tongue whose transducer is composed of several kinds of lipid/polymer membranes with different characteristics. We measured a phospholipid cocktail and various kinds of taste substances with five basic taste qualities. The responses to quinine hydrochloride and L-tryptophan, which have a bitter taste, were reduced as the phospholipid concentration was increased, and the responses to the other taste substances were not affected by the phospholipids, as with the human sensation test. Furthermore, the change of bitter intensity caused by phospholipid was quantified by principal component analysis and the tau scale, which expresses the relationship between taste intensity and taste substance concentration. The results are compared with those of the human sensory test and discussed.

A multichannel taste sensor using lipid/polymer membranes has higher sensitivities to electrolytes such as sour and salty substances than nonelectrolytes such as sweet substances. The purpose of this study is to improve sensitivity to sweet taste substances. The response to sweetness is assumed to have a mechanism different from electrolytic taste substances due to its nonelectrolyte. We could make large improvement of the response to sweetness by mixing positively and negatively charged lipid/polymer membranes and utilizing their hydrophobic property.

This paper describes an application of taste sensor for detection of a trace amount of organic substances. This sensor consists of multichannel sensors with lipid membranes which are modeled on living organisms. One of characteristics of this sensor is a global selectivity. We discovered that organic substances have large influences on the adsorption of a plus charged lipid (additive in a solution) to a lipid membrane made by minus charged lipid. Using this method, some of organic substances, such as oil, Trichioroethylene and Di-2-ethylhexylphthalate, could be detected about 100ppb within five minutes. In conclusion, the taste sensor has a possibility for real-time monitoring of water quality using this new methods.

It has been noticed recently that there is a possibility of elution of chemical substances from packages of foods. The chemicals act as harmful substances such as environmental hormones for biological bodies at very low concentration. It is therefore necessary to develop a sensing device, which can detect such substances easily and quickly. The purpose of the present study is high sensitive measurement of environmental hormones using electrode polarity controlling method. The responding ability of the polarity-controlled sensor covers various chemicals from electrolytes to nonelectrolytes and from hydrophilic to hydrophobic substances. As a result, we could sensitively detect bisphenol-A at about 50ppb and dibutyl phthalate at about 3ppb, and the sensor could distinguish such chemicals from ordinary taste substances.

A chloride ion-selective solvent polymeric membrane electrode (Cl-ISE) using quaternary ammonium salts for determination of the ethanol concentration during sake fermentation is described. The ethanol response by a Cl-ISE is based on the change in the transfer activity coefficient of Cl- from water to ethanol. The logarithm value of the transfer activity coefficient increases linearly with increasing ethanol concentration. The Cl-ISE using a mixture of o-nitro-phenyloctyl ether and n-tetradecylalcohol as plasticizers and tetradodecylammonium salts as sensing materials showed good linearities for ethanol (−2.39 mV/%) and for Cl- (−53.6 mV/decade). Furthermore, the interference from co-existent components in sake mash with the response of the Cl-ISE was improved in comparison with that using other plasticizers and sensing materials. The Cl-ISE was found to show good accuracy in the measurement of ethanol in sake mash.

The present study investigates adsorption of taste substances to lipid membranes for the multichannel taste sensor. To clarify the mechanism of adsorption of taste substances to a lipid membrane, we measured adsorption quantities of taste substances, such as amino acids and quinine-HCl, with fluorometry, and the surface of the lipid membrane was analyzed with ESCA (Electron spectroscopy for chemical analysis). Furthermore, we measured potential responses of a lipid membrane and pH of monosodium glutamate and lipid solutions. The results showed that amino acids adsorb to the lipid membrane, but no specific adsorption site exists on the membrane. The adsorption occurred by weak interaction between a phosphoric acid group of lipid molecules and an amino group of amino acids. Quinine-HCl adsorbed to membrane with strong electrostatic coupling and weak hydrophobic effects to lipid molecules in the membrane.

We have developed a multichannel taste sensor with lipid membranes which can detect and quantify the basic taste substances in aqueous solution. The aim of the present study is to increase selectivity to adsorptive taste substances (bitter, umami and astringent taste substances) for quantification of taste by improving measuring methods. High selectivity to adsorptive taste substances is obtained by CPA measurement algorithm (CPA: Change of membrane Potential caused by Adsorption). High repeatability is also obtained by developing a cleaning technique of taste sensor.

Impedance changes in phospholipid LB (Langmuir-Blodgett) and protein-mixed phospholipid LB films by several taste substances were measured. The resistance of the phospholipid LB film increased by bitter and sweet substances, whereas the resistance decreased for salty, sour and umami substances. For sweetness the larger change was observed in the protein-mixed phospholipid LB film, and hence discrimination between sweetness and bitterness was possible.

Interactions between a lipid-protein membrane and taste substances were investigated using SPR (surface plasmon resonance). The used membrane was the LB (Langmuir-Blodgett) multi-layered film composed of DHP (dihexadecyl phosphate) and the sugar-binding protein ConA. The LB membrane including ConA showed larger responses to almost all the taste substances, such as quinine, caffeine, sucrose and NaCl than the LB membrane without ConA. Suggestion was also made on the interactions of astringent and pungent substances with the charged lipid LB membrane.

We have developed a multichannel taste sensor with artifical lipid membranes and have applied it to quantification of taste of green tea. We used multiple regression analysis and found high correlations of outputs of the taste sensor with the results of sensory test (taste, flavor and color) and chemical analyses (amino acids and tannin that are main taste substances in green tea). It is concluded that the taste sensor has a potential for quantification of taste of green tea. The taste sensor responds not only to amino acids and tannin, but also to many other taste substances, and hence it contains much more taste information than conventional chemical analyses.

The taste of Japanese sake was investigated using a taste sensor with eight kinds of lipid membranes and an enzymatic glucose sensor. The electric-potential pattern constructed of eight outputs from the taste sensor has information on the taste quality and intensity. A standard solution for sake measurement was synthesized. Therefore, chemical meanings were assigned to two perpendicular axes transformed from the nine variables of outputs from the two sensors; commercial brands of sake were scattered on this plane. The taste of sake was discussed objectively.

The titrable acidity of and the ethanol concentration in sake mash were measured using a multichannel taste sensor system. A positively charged membrane responded to ethanol and the electric response of the membrane showed good agreement with those determined by gas chromatography in the measurement of sake mash. It was considered that the responses were induced by a change in the activity coefficient of anions. The titrable acidity of sake mash was measured using an electrode with a negatively charged membrane, because the electric response of the membrane showed a strong correlation with the titrable acidity of sake. The result showed good agreement with the value determind by titration, and the agreement was slightly increased by multiple regression together with the electric response of the positively charged membrane. Therefore, it is concluded that this method is useful for determination of the ethanol concentration in and the titrable acidity of sake mash. The results show that the taste sensor can be used for monitoring of those values in sake fermentation.

Lipid membranes are useful materials to transform information about taste substances into electric signals. A lipid monolayer membrane for a taste sensor has been prepared by adsorbing lipid materials to the hydrophobic surface of a polymer membrane. The lipid-modified membranes respond to such electrolytic taste substances as HCl (sourness) or NaCl (saltiness) with large response magnitudes and such non-electrolytic substances as caffeine (bitterness) or sugar (sweetness) with high sensitivities. Thus the present membrane has a high ability to detect taste.

A taste sensor that uses lipid/polymer membranes can evaluate aftertastes felt by humans using Change in membrane Potential caused by Adsorption (CPA) measurements. The sensor membrane for evaluating bitterness, which is caused by acidic bitter substances such as iso-alpha acid contained in beer, needs an immersion process in monosodium glutamate (MSG) solution, called "MSG preconditioning". However, what happens to the lipid/polymer membrane during MSG preconditioning is not clear. Therefore, we carried out three experiments to investigate the changes in the lipid/polymer membrane caused by the MSG preconditioning, i.e., measurements of the taste sensor, measurements of the amount of the bitterness substance adsorbed onto the membrane and measurements of the contact angle of the membrane surface. The CPA values increased as the preconditioning process progressed, and became stable after 3 d of preconditioning. The response potentials to the reference solution showed the same tendency of the CPA value change during the preconditioning period. The contact angle of the lipid/polymer membrane surface decreased after 7 d of MSG preconditioning; in short, the surface of the lipid/polymer membrane became hydrophilic during MSG preconditioning. The amount of adsorbed iso-alpha acid was increased until 5 d preconditioning, and then it decreased. In this study, we revealed that the CPA values increased with the progress of MSG preconditioning in spite of the decrease of the amount of iso-alpha acid adsorbed onto the lipid/polymer membrane, and it was indicated that the CPA values increase because the sensor sensitivity was improved by the MSG preconditioning.

A taste sensor with a multichannel electrode was developed by using lipid membranes as a transducer of taste substances. The sensor can detect the taste in a similar manner to the human gustatory sensation by response patterns of electric potential to taste substances. The sensitivity, reproducibility and durability were superior to those of humans. The same taste as that elicited by some commercial aqueous drink was reproduced by making aqueous solution mixed from four kinds of basic taste substances, the concentrations of which were determined so that the electric-potential pattern of this mixed solution could agree well with the pattern by the drink. Different brands of beer were easily distinguished by the electric-potential patterns.

A series of n-alkanols from ethanol to tridecanol interacted with a negatively charged lipid membrane composed of dioleyl phosphate, which exhibited a

Benzaldehyde (Bz) is a typical fragrant compound for peach-flavored beverages. In the food and beverage industries there is great demand for a sensitive and easy detection system of Bz in order to ensure product quality control and to avoid contamination. For the noncompetitive detection of Bz, we applied an open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) utilizing an antigen-dependent reassociation of antibody variable region fragments, VH and VL. We cloned the VH and VL genes of an anti-Bz monoclonal antibody, and the fragments were individually expressed and purified as a bacterial alkaline phosphatase (BAP)-conjugated form for VH and as a thioredoxine (Trx)-fused form for VL, respectively. Using these VH and VL fragments, we successfully constructed the OS-ELISA system for Bz detection. The Bz-induced formation of a trimolecular complex composed of VH-BAP/Bz/Trx-VL was readily detected by a dose-dependent increase in the BAP activity of the VH-fusion protein.

In this chapter, we describe an “electronic dog nose” based on a surface plasmon resonance (SPR) immunosensor for highly sensitive detection of aromatic nitro compounds leaking from landmines or explosives. To measure explosives on an SPR immunosensor, the vapors of the target substances are sampled using a preconcentrator. Here, we explain the principle of SPR immunosensors, production of antibodies, method for the measurement of trinitrotoluene (TNT), and sampling processes of aromatic nitro compounds.

The taste sensor was widely used in distinguishing various taste substances. However, the taste sensor has poorer sensitivity to uncharged molecules such as caffeine, a bitter substance, than to charged taste substances. In the present study, we discussed the sensitivity of caffeine detection using a taste sensor with lipid/polymer membranes that were formed with different length of methyl group of lipid, namely, tetra-n-ctylammonium bromide (R8), tetrakis-(decyl)-ammonium bromide (R10), tetradodecylammonium bromide (TDAB; R12), and tetrahexadecylammonium bromide (R16). As a result, we observed that the electric responses of the lipid membranes to caffeine were associated with the length of alkyl chain of a lipid and an optimum concentration of the lipids in membranes was also observed to enhance the sensitivity of caffeine with taste sensor.

We have developed a taste sensor consisting of a multichannel electrode with transducers composed of lipid membranes immobilized with a polymer, i.e., eight different kinds of lipid analogs mixed with poly(vinyl chloride) and plasticizer. This multichannel sensor responds to five basic taste substances in five different ways; the taste patterns composed of eight electric-potential responses could be clearly distinguished from each other for the five basic taste substances. Moreover, similar but distinguishable patterns were obtained for substances eliciting a similar taste in humans. The reproducibility is noticeably improved from those of lipid membranes which were previously investigated for application in a taste sensor. The present results imply that the multichannel lipid membrane device has the ability to sense tastes like a human being.

We report our investigations on the development of a novel self-assembled monolayer (SAM)-based flow-through surface-plasmon-resonance (SPR) immunosensor for highly specific and rapid detection of benzaldehyde (BZ). The functional sensing surface was immobilized with a BZ derivative on the SAM of polyethyleneglycol-alkanethiols. Both the self-assembling of alkanethiols and the immobilization of BZ were controlled by the analysis of SPR resonance angle measurements. The BZ-bound SAM surface shows high specificity for immunoaffinity reaction with anti-BZ antibody (BZ-Ab). The quantification of benzaldehyde is based on the principle of indirect competitive immunoassay, in which the immunoreaction between the BZ derivative on sensor surface and anti-BZ antibody was inhibited in the presence of free BZ in solution. The decrease in SPR angle is proportional to the increase in the concentration of BZ in test samples. The SPR immunosensor exhibited excellent sensitivity for the detection of BZ in the concentration range from 0.1-80 ppb (ng ml-1). Detection limit of the present sensor is superior compared to various high-performance SPR and QCM sensors. Similarly, HPLC and GC techniques reported a low-detection-limit of only 10 ppb BZ, despite a set of sample pretreatments. In addition, enhancement of the SPR sensor signal and the sensitivity were investigated by introducing a single-step add-on strategy involving either Au nanoparticles or a simple biotinavidin coupling reaction, with which the detection limit has been improved dramatically.

This paper describes the fabrication and sensing characteristics of a self-assembled monolayer (SAM)-based surface plasmon resonance (SPR) immunosensor for detection of benzaldehyde (BZ). The functional sensing surface was fabricated by the immobilization of a benzaldehyde-ovalbumin conjugate (BZ-OVA) on Au-thiolate SAMs containing carboxyl end groups. Covalent binding of BZ-OVA on SAM was found to be dependent on the composition of the base SAM, and it is improved very much with the use of a mixed monolayer strategy. Based on SPR angle measurements, the functional sensor surface is established as a compact monolayer of BZ-OVA bound on the mixed SAM. The BZ-OVA-bound sensor surface undergoes immunoaffinity binding with anti-benzaldehyde antibody (BZ-Ab) selectively. An indirect inhibition immunoassay principle has been applied, in which analyte benzaldehyde solution was incubated with an optimal concentration of BZ-Ab for 5 min and injected over the sensor chip. Analyte benzaldehyde undergoes immunoreaction with BZ-Ab and makes it inactive for binding to BZ-OVA on the sensor chip. As a result, the SPR angle response decreases with an increase in the concentration of benzaldehyde. The fabricated immunosensor demonstrates a low detection limit (LDL) of 50 ppt (pg mL(-1)) with a response time of 5 min. Antibodies bound to the sensor chip during an immunoassay could be detached by a brief exposure to acidic pepsin. With this surface regeneration, reusability of the same sensor chip for as many as 30 determination cycles has been established. Sensitivity has been enhanced further with the application of an additional single-step multi-sandwich immunoassay step, in which the BZ-Ab bound to the sensor chip was treated with a mixture of biotin-labeled secondary antibody, streptavidin and biotin-bovine serum albumin (Bio-BSA) conjugate. With this approach, the SPR sensor signal increased by ca. 12 times and the low detection limit improved to 5 ppt with a total response time of no more than ca. 10 min. Figure A single-step multi-sandwich immunoassay step increases SPR sensor signal by ca. 12 times affording a low detection limit for benzaldehyde of 5 ppt.