Detection of Salmonella Enterica in Egg Yolk by PCR on a Microfluidic Disc Device Using Immunomagnetic Beads.

Salmonella enterica is a pathogenic bacterium that causes foodborne illness. One of the vehicle foods of S. enterica are chicken eggs. Efficient collection of the bacterium is necessary to detect it specifically. We developed a method to detect S. enterica by PCR on a microfluidic disc device using a fluorescent probe. Salmonella enterica cells were isolated in the microchambers on the device, followed by thermal lysis and PCR targeting with the invA gene, a gene specific to S. enterica, were observed by measurement of the fluorescent signal that resulted from gene amplification. However, the developed method was unable to discriminate viable cells from dead cells. Consequently, in this study, magnetic beads modified with anti-Salmonella antibody were utilized to detect viable Salmonella cells from egg yolk prior to PCR on the device. While using the antibody-modified beads, egg yolk components, which inhibit PCR, were removed. The collected cells were subsequently detected by PCR of the invA gene on a microfluidic disc device. This method enabled the detection of viable cells without the inhibition of PCR by any egg component. S. enterica was detected at 5.0×104 cells mL-1 or at a higher concentration of egg yolk within 6 h including the sampling time.

Rapid Detection of Salmonella enterica in Food Using a Compact Disc-Shaped Device.

Rapid detection of food-borne pathogens is essential to public health and the food industry. Although the conventional culture method is highly sensitive, it takes at least a few days to detect food-borne pathogens. Even though polymerase chain reaction (PCR) can detect food-borne pathogens in a few hours, it is more expensive and unsatisfactorily sensitive relative to the culture method. We have developed a method to rapidly detect Salmonella enterica by using a compact disc (CD)-shaped device that can reduce reagent consumption in conventional PCR. The detection method, which combines culture and PCR, is more rapid than the conventional culture method and is more sensitive and cheaper than PCR. In this study, we also examined a sample preparation method that involved collecting bacterial cells from food. The bacteria collected from chicken meat spiked with S. enterica were mixed with PCR reagents, and PCR was performed on the device. At a low concentration of S. enterica, the collected S. enterica was cultured before PCR for sensitive detection. After cultivation for 4 h, S. enterica at 1.7 × 104 colony-forming units (CFUs)·g-1 was detected within 8 h, which included the time needed for sample preparation and detection. Furthermore, the detection of 30 CFUs·g-1 of S. enterica was possible within 12 h including 8 h for cultivation.

Study on Electrochemical Insulin Sensing Utilizing a DNA Aptamer-Immobilized Gold Electrode.

We investigated an insulin-sensing method by utilizing an insulin-binding aptamer IGA3, which forms an anti-parallel G-quadruplex with folded single strands. Spectroscopic observation indicates that some anti-parallel G-quadruplex bind hemin and show peroxidase activity. In this study, the peroxidase activity of IGA3 with hemin was confirmed by spectrophotometric measurements, i.e., the activity was three-times higher than hemin itself. IGA3 was then immobilized onto a gold electrode to determine its electrochemical activity. The peroxidase activity of the immobilized IGA3-hemin complex was determined by cyclic voltammetry, and a cathodic peak current of the electrode showed a dependence on the concentration of H2O2. The cathodic peak current of the IGA3-hemin complex decreased by binding it to insulin, and this decrease depended on the concentration of insulin.

Fluorescence Sensing of the Interaction between Biomembranes with Different Lipid Composition and Endocrine Disrupting Chemicals.

Fluorescence sensing of the interaction between biomembranes with different lipid composition and endocrine disrupting chemicals (EDCs) was carried out by using a liposome-encapsulating fluorescence dye (carboxyfluorescein (CF)-liposome). We detected a significant increase in fluorescence intensity in CF-liposome solutions due to the leakage of fluorescence caused by the interaction of EDCs with the biomembranes of liposomes. The temporal increases in fluorescent were significantly different among the lipid compositions of CF-liposome and the EDCs. Results were considered by summarizing the interactions in radar charts and by showing the pattern of interaction of each EDC. Each chart showed a dissimilar pattern reflecting the complexity of the biomembrane-EDC interaction. The results indicate that this fluorescence sensing could be useful to evaluate the interaction.

Detection of expressed gene in isolated single cells in microchambers by a novel hot cell-direct RT-PCR method.

In order to be able to detect the expression of a gene in individual cells, the ability to isolate and lyse a single cell and to perform reverse transcription polymerase chain reaction (RT-PCR) in one device is important. As is common, when performing cell lysis and RT-PCR in the same reaction chamber, it is necessary to add the reagent for RT-PCR after cell lysis. In this study, we propose an original formula for cell lysis and RT-PCR in the same reaction chamber without the addition of reagent by only a heat process, which we termed hot cell-direct RT-PCR. Hot cell-direct RT-PCR was enabled by using Tth DNA polymerase, which is a thermostable polymerase and has high reverse transcription activity in the presence of manganese ions. Direct detection of RT-PCR products was performed by detecting fluorescence with the use of a double-dye fluorescent probe. We attempted to detect the mRNA of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in isolated Jurkat cells on a microfluidic device, which we had already developed for single cell isolation. After cell isolation and successive hot cell-direct RT-PCR on the device, fluorescent signals from RT-PCR products for a single cell were detected and differentiated from the chamber containing no cells. A highly positive linear relationship (r = 0.9933) was observed between the number of chambers containing cell(s) and those containing RT-PCR products from 10 to 400 cells µL(-1). Thus it was possible to use the novel hot cell-direct RT-PCR method to detect the expressed gene in isolated cells.

Use of a novel microfluidic disk in the analysis of single-cell viability and the application to Jurkat cells.

Jurkat cells were trapped in the microchambers of a novel disk-shaped cell separation device and stained with Cellstain. Approximately 90% of the cells were living. Single cells were isolated with a branching microchannel after rotation at 4500rpm for 30s, demonstrating that a living single cell could be trapped in the microchambers.

Compact disk (CD)-shaped device for single cell isolation and PCR of a specific gene in the isolated cell.

For immediate discrimination among isolated cells we propose a novel device and technique for isolation of cells and sequential detection of specific gene(s) within them by polymerase chain reaction (PCR). In this study, we isolated Salmonella enterica cells and detected the Salmonella-specific invA gene from isolated cells by PCR on a compact disk (CD)-shaped device. This device enabled liquid flow by centrifugal force without a micro pump, and was fabricated from silicon wafer and glass to avoid evaporation of a small amount of reagent. One device has 24 microchannels, and 313 microchambers integrated on each microchannel. One microliter of PCR mixture containing cells was separated into microchambers on the device at 5000 rpm for 30 s. Each microchamber contained approximately 1.5 nL PCR mixture. A Poisson distribution of S. enterica cells was observed for different densities of cell suspension. At 200 cells µL(-1) of S. enterica or less, isolated single cells could be determined on the device by amplification of DNA of the invA gene; at 400 cells µL(-1), chambers containing no, one, two, or three cells could be determined on the device. Selective detection of S. enterica was achieved by PCR from a mixture of S. enterica and Escherichia coli on the CD-shaped device.

A photoimmobilizable sulfobetaine-based polymer for a nonbiofouling surface.

A novel photoreactive polymer containing sulfobetaine polar groups was prepared by copolymerization of two kinds of methacrylic acids with sulfobetaine and azidoaniline. The polymer was photoimmobilized on polyester and polystyrene surfaces. Its effects on surface modification were investigated from its interactions with water, proteins and cells. Polymer immobilization altered both of the plain surfaces to becoming hydrophilic in a similar range of static contact angles (12.5±1.6° on polyester and 14.7±2.2° on polystyrene). This suggests that the surfaces were covered with sulfobetaine polar groups. Micropattern immobilization was carried out on both polymers using a photomask. The formed pattern was identical to the photomask, showing that the polymer was formed in response to ultraviolet irradiation. Measurements using atomic force microscopy showed that the polymer was formed at a thickness of 550nm, demonstrating that the polymer was cross-linked with itself and with the substrate molecules. Measurements using time-of-flight secondary ion mass spectrometry detected an abundance of sulfur-containing ions in the patterned polymer, confirming that sulfobetaine had been immobilized. Protein adsorption and mammalian cell adhesiveness were reduced markedly on the immobilized regions. The reduction of cell adhesiveness was concentration-dependent for the immobilized polymer on polyester surfaces. In conclusion, a novel sulfobetaine-containing polymer was immobilized photoreactively on conventional polymer surfaces and significantly reduced interactions with proteins and mammalian cells.

Degradation of liposome cluster caused by the interaction with endocrine disrupting chemicals (EDCs).

The interaction between endocrine disrupting chemicals (EDCs) and liposome clusters was investigated using UV-vis spectroscopy and observed by microscope. Since liposome clusters are composed of small unilamellar liposomes and membrane proteins, they are regarded as a model of plasma membranes. The size of each cluster was 10microm in diameter. UV-vis spectroscopy of liposome clusters showed an apparent absorption at 300nm which was affected by the interaction with EDCs. Four EDCs caused an alteration in absorption in a concentration-dependence manner between 0.1 and 10ppm (i.e. 0.1-10mg/l). Microscopic observation showed that the effect was caused by the degradation of liposome clusters while degradation was induced by the interaction; liposome was broken down by EDCs. This report suggests that the liposome cluster can be applied for universal detection of EDCs based on the interaction between a plasma membrane model and EDCs.

6-Chloro-N,N-diethyl-1,3,5-triazine-2,4-diamine (CAT) sensor based on biomimetic recognition utilizing a molecularly imprinted artificial receptor.

We aimed to develop a 6-chloro-N,N-diethyl-1,3,5-triazine-2,4-diamine (CAT)-sensing system based on a biomimetic receptor of a molecularly imprinted polymer for CAT and electrochemical determination of CAT. A molecularly imprinted polymer for CAT was prepared by the polymerization of methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylate (EDMA) as a cross-linker with a template molecule (CAT) in dimethyl formamide (DMF). The polymer prepared with the ratio of these monomers (CAT:MAA:EDMA = 1:7.5:20) showed the most selective rebinding to CAT, and the obtained polymer was recognized as a CAT-imprinted polymer (CAT-MIP). The effect of the specific imprinting sites of CAT-MIP was demonstrated by Scatchard analysis. In an aqueous solution of CAT, CAT-MIP showed the maximum binding of CAT in a 0.05 M phosphate buffer (PB), pH 5.0. The binding amount of CAT to CAT-MIP was 24% more than atrazine and 72% more than propazine. The CAT-sensing system was composed of a column of CAT-MIP particles and a voltammetry analyzer. The reductive current of CAT depended on the concentration of CAT up to 30 microM with the system.

Difference in Surface Properties between Insoluble Monolayer and Adsorbed Film from Kinetics of Water Evaporation and BAM Image.

The evaporation rate of water molecules across three kinds of interfaces (air/water interface (1), air/surfactant solution interface (2), and air/water interface covered by insoluble monolayer (3)) was examined using a remodeled thermogravimetric balance. There was no difference in both the evaporation rate and the activation energy for the first two interfaces for three types of surfactant solutions below and above the critical micelle concentration (cmc). This means that the molecular surface area from the Gibbs surface excess has nothing to do with the evaporation rate. In the third case, the insoluble monolayer of 1-heptadecanol decreased the evaporation rate and increased the activation energy, indicating a clear difference between an insoluble monolayer and an adsorbed film of soluble surfactant. This difference was substantiated by BAM images, too. The images of three surfactant solution interfaces were similar to that of just the water surface, while distinct structures of molecular assemblies were observed for the insoluble monolayer. The concentration profile of water molecules in an air/liquid interfacial region was derived by Fix's second law. The profile indicates that a definite layer just beneath the air/liquid interface of the surfactant solution is made mostly of water molecules and that the layer thickness is a few times the root-mean-square displacement %@mt;sys@%%@rl;;@%2%@ital@%Dt%@rsf@%%@rlx@%%@mx@% of the water molecules. The thickness was found to be more than a few nanometers, as estimated from several relaxation times derived from the other kinetics than evaporation of amphiphilic molecules in aqueous systems and a maximum evaporation rate of purified water.

Atrazine sensor based on molecularly imprinted polymer-modified gold electrode.

Molecularly imprinted polymers (MIP) have been elucidated to work as artificial receptors. In our present study, a MIP was applied as a molecular recognition element to a chemical sensor. We have constructed an atrazine sensor based on a MIP layer selective for atrazine and its electrochemical reduction on gold electrode. The atrazine sensor was fabricated by directly polymerizing the atrazine-imprinted polymer composed from methacrylic acid and ethylene glycol dimethacrylate onto the surface of a gold electrode. By introducing LiCl into the MIP, atrazine was reduced below -800 mV vs Ag/AgCl reference electrode, at pH 3. The cathodic current of atrazine depended on the concentration of atrazine at the range of 1-10 microM. The sensor exhibited a selective response to atrazine. A nonimprinted polymer-modified electrode did not show selective response to atrazine, thus implying that the imprinted polymer acts as recognition element of atrazine sensor.

Potentiometric phosphate-sensing system utilizing phosphate-binding protein.

A phosphate-detection system has been developed which uses phosphate-binding protein (PBP) from Escherichia coli. PBP was immobilized on a sheet of nitrocellulose membrane by cross-linking and the membrane potential of the immobilized PBP was measured. The response time of the system to phosphate was 5 min. The response was selective to phosphate among other anions. Under optimum conditions 0.1-1.5 mmol L(-1) phosphate can be determined with this system.