Near-infrared spectroscopy of H3O+⋯Xn (X = Ar, N2, and CO, n = 1-3).

Near-infrared (NIR) spectra of H3O+⋯Xn (X = Ar, N2, and CO, n = 1-3) in the first overtone region of OH-stretching vibrations (4800-7000 cm-1) were measured. Not only OH-stretching overtones but also several combination bands are major features in this region, and assignments of these observed bands are not obvious at a glance. High-precision anharmonic vibrational simulations based on the discrete variable representation approach were performed. The simulated spectra show good agreement with the observed ones and provide firm assignments of the observed bands, except in the case of X = CO, in which higher order vibrational mode couplings seem significant. This agreement demonstrates that the present system can be a benchmark for high precision anharmonic vibrational computations of NIR spectra. Band broadening in the observed spectra becomes remarkable with an increase of the interaction with the solvent molecule (X). The origin of the band broadening is explored by rare gas tagging experiments and anharmonic vibrational simulations of hot bands.

Enhancement of Fluorescence-Based Sandwich Immunoassay Using Multilayered Microplates Modified with Plasma-Polymerized Films.

A functional modification of the surface of a 96-well microplate coupled with a thin layer deposition technique is demonstrated for enhanced fluorescence-based sandwich immunoassays. The plasma polymerization technique enabling the deposition of organic thin films was employed for the modification of the well surface of a microplate. A silver layer and a plasma-polymerized film were consecutively deposited on the microplate as a metal mirror and the optical interference layer, respectively. When Cy3-labeled antibody was applied to the wells of the resulting multilayered microplate without any immobilization step, greatly enhanced fluorescence was observed compared with that obtained with the unmodified one. The same effect could be also exhibited for an immunoassay targeting antigen directly adsorbed on the multilayered microplate. Furthermore, a sandwich immunoassay for the detection of interleukin 2 (IL-2) was performed with the multilayered microplates, resulting in specific and 88-fold-enhanced fluorescence detection.

A sniffer-camera for imaging of ethanol vaporization from wine: the effect of wine glass shape.

A two-dimensional imaging system (Sniffer-camera) for visualizing the concentration distribution of ethanol vapor emitting from wine in a wine glass has been developed. This system provides image information of ethanol vapor concentration using chemiluminescence (CL) from an enzyme-immobilized mesh. This system measures ethanol vapor concentration as CL intensities from luminol reactions induced by alcohol oxidase and a horseradish peroxidase (HRP)-luminol-hydrogen peroxide system. Conversion of ethanol distribution and concentration to two-dimensional CL was conducted using an enzyme-immobilized mesh containing an alcohol oxidase, horseradish peroxidase, and luminol solution. The temporal changes in CL were detected using an electron multiplier (EM)-CCD camera and analyzed. We selected three types of glasses-a wine glass, a cocktail glass, and a straight glass-to determine the differences in ethanol emission caused by the shape effects of the glass. The emission measurements of ethanol vapor from wine in each glass were successfully visualized, with pixel intensity reflecting ethanol concentration. Of note, a characteristic ring shape attributed to high alcohol concentration appeared near the rim of the wine glass containing 13 °C wine. Thus, the alcohol concentration in the center of the wine glass was comparatively lower. The Sniffer-camera was demonstrated to be sufficiently useful for non-destructive ethanol measurement for the assessment of food characteristics.

Biomedical soft contact-lens sensor for in situ ocular biomonitoring of tear contents.

A soft contact-lens biosensor (SCL-biosensor) for novel non-invasive biomonitoring of tear fluids was fabricated and tested. Wearing a biosensor on eye enabled the in situ monitoring of tear contents. The biosensor has an enzyme immobilized electrode on the surface of a polydimethyl siloxane (PDMS) contact lens. The SCL-biosensor was fabricated using microfabrication techniques for functional polymers (PDMS and 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer). In investigation of in vitro characterization, the SCL-biosensor showed excellent relationship between the output current and glucose concentration from 0.03 to 5.0 mmol·L(-1), with a correlation coefficient of 0.994. The calibration range covered the reported tear glucose concentrations (0.14 mmol·L(-1)). Based on the result, ocular biomonitoring with the SCL-biosensor was carried out. The SCL-biosensor well worked both in the static state and the dynamic state. The tear glucose level of rabbit was estimated to 0.12 mmol·L(-1) at first and then the tear turnover was successfully calculated to be 29.6 ± 8.42% min(-1). The result indicated that SCL-biosensor is useful for advanced biomonitoring on eye.

Fluorescence immunoassay using an optical fiber for determination of Dermatophagoides farinae (Der f1).

Immunoassay methods are generally used for measuring of allergenic substances. However, they need special facilities, skilled handling, and time-consuming procedure. In this work, a fiber-optic immunoassay system which could measure allergen by fluorescent intensities of immune complexes formed by allergens and fluorescently labeled antibodies was established. Immune complexes absorbed on the optical fiber probe surface, and excitation light was injected into the probe, then evanescent field is created in the proximity of the probe. The fluorophores were excited by the evanescent light, and fluorescence was detected by a photo diode. The target allergen detected by our system was Der f1 derived from Dermatophagoides farinae that is one of the house dust mite and major source of inhaled allergens. The fluorophore used labeling on detecting antibody was cyanine 5. The system enabled to detect and quantitatively determine of Der f1. The measurement range was from 0.24 to 250 ng/ml, and the result competes with ELISA. The measurement time was 16 min/sample. The immunoassay system was applied to measurement of Der f1 from actual dust samples. Calculated values of Der f1 showed good correlations between the fiber-optic fluoroimmunoassay and ELISA.

A soft and flexible biosensor using a phospholipid polymer for continuous glucose monitoring.

A flexible biosensor using a phospholipid polymer to immobilization of glucose oxidase (GOD) was fabricated and tested. At first, an enzyme membrane formed by immobilizing GOD onto a porous polytetrafluoroethylene (PTFE) membrane using the phospholipid polymer (2-methacryloyloxyethyl phosphorylcholine (MPC) copolymerized with 2-ethylhexylmethacrylate (EHMA):PMEH) was evaluated. According to the result of amperometric measurement, average density of GOD to be immobilized was optimized to 38.9 units cm(-2). Temperature and pH dependences were also investigated. Then, a flexible glucose sensor was fabricated by immobilizing GOD onto a flexible hydrogen peroxide electrode using PMEH. The flexible glucose sensor showed a linear relationship between output currents and glucose concentration in 0.05-1.00 mmol L(-1), with a correlation coefficient of 0.999. The calibration range covered the normal tear glucose level of 0.14-0.23 mmol L(-1). This indicates that the flexible biosensor is considered to be useful for monitoring of glucose in tear fluids.

Improved performance Air bio-battery based on efficient oxygen supply with a gas/liquid highly-porous diaphragm cell.

Performance of a glucose-driven bio-battery was improved by enhancing electrode characteristics and oxygen supply efficiency to a cathode. The bio-battery generates electric power from glucose through three enzymatic reactions using glucose dehydrogenase, diaphorase and bilirubin oxidase. A flexible and thin Pt electrode was employed instead of a glassy carbon (GC) electrode on which enzymes, a coenzyme, and mediators were immobilized by layer-by-layer method. The maximum current and power densities of the constructed bio-battery were 257 ±â€¯22 µA/cm2 and 86 ±â€¯3 µW/cm2, respectively, in 5 mM glucose solution. In addition, a newly designed compact gas/liquid diaphragm cell, which allowed to reduce the internal resistance by shortening the anode-cathode distance and enhance oxygen supply to a cathode using a highly-porous cotton mesh diaphragm, was implemented to the bio-battery to develop a high-performance Air bio-battery. As a result, improved Air bio-battery showed the maximum current and power densities of 451 ±â€¯27 µA/cm2 and 162 ±â€¯7 µW/cm2, which was 3.6-fold improvement from the previous GC electrode-based bio-battery. In addition, continuous operation for 210 min revealed high stability of power generation as it decreased by 3.3% at the end of operation. Additional supply of oxygen to a cathode exhibited proportional increase of the power density to the oxygen concentration, which demonstrates a promising potential of Air bio-battery for a high-performance and continuous powering device.

Anti-HIV and vitamin C-synergized radical scavenging activity of cacao husk lignin fractions.

Cacao husk lignin fractions, prepared by acid precipitation and 50% ethanol precipitation showed unexpectedly higher anti-human immunodeficiency virus (HIV) activity, as compared with the corresponding fractions from the cacao mass, amounting to the level comparable with that of popular anti-HIV compounds. The cacao husk lignin fractions also showed anti-influenza virus activity, but did not show antibacterial activity. The cacao husk lignin fractions synergistically enhanced the superoxide anion and hydroxyl radical scavenging activity of vitamin C. The cacao husk lignin fractions stimulated nitric oxide generation by mouse macrophage-like cells, to a level higher than that attained by lipopolysaccharide (LPS). The present study suggests the functionality of cacao husk lignin fractions as complementary alternative medicine.

Improving the efficiency of evolutionary de novo peptide design: strategies for probing configuration and parameter settings.

Evolutionary molecular design based on genetic algorithms (GAs) has been demonstrated to be a flexible and efficient optimization approach with potential for locating global optima. Its efficacy and efficiency are largely dependent on the operations and control parameters of the GAs. Accordingly, we have explored new operations and probed good parameter setting through simulations. The findings have been evaluated in a helical peptide design according to "Parameter setting by analogy" strategy; highly helical peptides have been successfully obtained with a population of only 16 peptides and 5 iterative cycles. The results indicate that new operations such as multi-step crossover-mutation are able to improve the explorative efficiency and to reduce the sensitivity to crossover and mutation rates (CR-MR). The efficiency of the peptide design has been furthermore improved by setting the GAs at the good CR-MR setting determined through simulation. These results suggest that probing the operations and parameter settings through simulation in combination with "Parameter setting by analogy" strategy provides an effective framework for improving the efficiency of the approach. Consequently, we conclude that this framework will be useful for contributing to practical peptide design, and gaining a better understanding of evolutionary molecular design.

[Comparison of DNA sequencing analysis with phenotyping test for identification of yeasts isolated from foods].

The identification of 20 strains of yeasts isolated from foods by means of DNA sequence analysis with two kinds of universal primers for the rDNA region was examined, and the results were compared with those of the conventional phenotyping test using API 20C AUX. In the analysis of the 26S region, all 20 yeast strains tested were identified at the species level. In the ITS1 region, 16 strains were also classified at the species level. In addition, all results of DNA sequence analysis were consistent with those of the phenotyping test at the genus level. Furthermore, DNA sequence analysis was able to identify causative yeasts observed in two suspect foods, though phenotyping tests alone failed to identify them.

[Isolation of thermostable direct hemolysin producing Vibrio parahaemolyticus from food using screening by PCR in food-borne outbreaks].

The producibility of thermostable direct hemolysin (TDH) is the most important pathogenic factor in Vibrio parahaemolyticus. TDH (+) V. parahaemolyticus is usually isolated from patients having V. parahaemolyticus food-borne disease. TDH (+) V. parahaemolyticus is, however, very difficult to isolate from food and environmental samples. In the 5 years from 2000 to 2004 in Tokyo, V. parahaemolyticus was isolated from food samples related to 67 of 227 V parahaemolyticus food-borne outbreaks. In these outbreaks, TDH (+) strains were also tried to isolate using PCR as the screening methods. TDH (+) V. parahaemolyticus strains were able to isolate from enrichment broth in which toxR and tdh genes become positive in PCR. TDH (+) strains of the same serotype with patients were able to be isolated from 23 food samples related to 11 outbreaks (16.4%); 3 outbreaks in 2000, 2 in 2001, 2 in 2002, 1 in 2003, and 3 in 2004. The serotypes of V. parahaemolyticus isolated from food were O3 : K6 (10 samples), O3 : K5 (6 samples), O1 : K25 (4 samples), O3 : K29 (2 samples), O4 : K 8 (1 sample), and O4 : K11 (1 sample). The isolation rate of the TDH (+) strain from enrichment broth differed with samples. In several samples TDH (+) strains were isolated easily only by examining 3 colonies, hence no TDH (+) strains were isolated in spite of the examination of 250 colonies. No correlation was seen between the number of V. parahaemolyticus and the isolation rate of TDH (+) strains in food samples. Screening using PCR is very effective method for isolating TDH (+) V. parahaemolyticus from food samples.

Characteristics and modeling of Escherichia coli growth in pouched food.

Characteristics of the growth kinetics of Escherichia coli cells in pouched mashed potatoes under various conditions were studied with a mathematical model. Bacterial cells were inoculated in sterile mashed potatoes and then sealed in vinyl pouches, in which a very small amount of air was included. The growth curves of cells in the pouched mashed potatoes at constant temperature (12-34 degrees C) were sigmoidal with time on a semi-logarithmic plot and were successfully described with a new logistic model recently developed by us. The rate constant of growth showed a highly linear relationship to the temperature with the square-root model, and the lag period was longer at lower temperatures. The growth curve in glass tubes containing a large volume of air was similar to that in pouches, showing that the rate of growth was not affected by the volume of the surrounding air. The growth curves in pouched mashed potatoes were very similar to those in nutrient broth or on the surface of nutrient agar, which we previously reported. These results suggested that the growth kinetics of the bacterial cells under various conditions of rich nutrition might be almost identical, and can be described with a simple growth model like ours.

Model comparison for Escherichia coli growth in pouched food.

We recently studied the growth characteristics of Escherichia coli cells in pouched mashed potatoes (Fujikawa et al., J. Food Hyg. Soc. Japan, 47, 95-98 (2006)). Using those experimental data, in the present study, we compared a logistic model newly developed by us with the modified Gompertz and the Baranyi models, which are used as growth models worldwide. Bacterial growth curves at constant temperatures in the range of 12 to 34 degrees C were successfully described with the new logistic model, as well as with the other models. The Baranyi gave the least error in cell number and our model gave the least error in the rate constant and the lag period. For dynamic temperature, our model successfully predicted the bacterial growth, whereas the Baranyi model considerably overestimated it. Also, there was a discrepancy between the growth curves described with the differential equations of the Baranyi model and those obtained with DMfit, a software program for Baranyi model fitting. These results indicate that the new logistic model can be used to predict bacterial growth in pouched food.

Novel method for estimating viable Salmonella cell counts using real-time PCR.

A novel method for estimating viable Salmonella Enteritidis cell counts with 5'-nuclease real-time PCR was developed in this study. Our method was based on the increase kinetics of the target DNA region (invA) of the microorganism growing in a food/clinical sample in a culture medium during incubation. The index of increase in the target DNA region studied here was threshold cycle, CT. A test Salmonella strain was grown in buffered peptone water at the optimal temperature (39 degrees C). As Salmonella cells were grown, the value of CT decreased with time, generating a downward sigmoidal curve. The slope of the curve was constant at various initial cell concentrations. With higher initial cell concentration, the CT value evaluated from the slope at a given time was lower. With this relationship, a novel method for estimating the initial viable cell concentration of a sample was developed. Dead Salmonella cells or bacteria other than the target cell caused deviation in the CT curve. Incubation in a selective media suppressed the deviation caused by other bacterial cells. We think that this method could be applied to many other microorganisms cultivable in a suitable medium.

[Development of a predictive program for microbial growth under various temperature conditions].

A predictive program for microbial growth under various temperature conditions was developed with a mathematical model. The model was a new logistic model recently developed by us. The program predicts Escherichia coli growth in broth, Staphylococcus aureus growth and its enterotoxin production in milk, and Vibrio parahaemolyticus growth in broth at various temperature patterns. The program, which was built with Microsoft Excel (Visual Basic Application), is user-friendly; users can easily input the temperature history of a test food and obtain the prediction instantly on the computer screen. The predicted growth and toxin production can be important indices to determine whether a food is microbiologically safe or not. This program should be a useful tool to confirm the microbial safety of commercial foods.

[Development of effective detection method for Coxiella burnetii in mayonnaise by real-time PCR and investigation of C. burnetii contamination in commercial mayonnaise in Tokyo].

A PCR method for the effective detection of Coxiella burnetii in commercially available mayonnaise was developed. Sample preparations were isolated from 50 g portions of each mayonnaise product by four successive extraction steps in phosphate buffer with 2.0 M NaCl. These extracts were then centrifuged at 20,000 x g for 60 min. DNA was isolated from the solution containing the precipitate with a commercial kit, and amplified quantitatively using real-time PCR that targeted the com1 region of C. burnetii. The recoveries of C. burnetii from 2 kinds of commercial mayonnaise specimens, with a baseline control of 1 x 10(7) particles of the Nine Mile phase II strain, were 85.0 +/- 6.0% and 72.0 +/- 0.4%, respectively. The determination limit of this method was 500 C. burnetii particles per 50 g of mayonnaise. The DNA specimens isolated from 50 different commercial mayonnaise samples sold in Tokyo using this method were amplified using both nested PCR and real-time PCR. No contamination by C. burnetii was detected in any of the mayonnaise samples.