Continuous production of glucose oxidase with Aspergillus sp. under ultrasound waves

Glucose oxidase (β-d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4, GOD) was continuously released from Aspergillus sp. under mild ultrasound waves (20 kHz, 15 W). However, GOD was not released from the cells under normal conditions because of their thick wall. GOD production under ultrasound waves was optimum at pH 7.5 and 30°C and decreased with increasing ultrasonic frequency. Ultrasonic cavitation accelerated GOD release from the cells. Microscopic observation and determination of ATP and nucleic acids in the broth revealed that the mycelia were not broken during a 5 h reaction under ultrasound waves (15 W). About 10% of GOD produced in cells was released during the reaction.     

Single nucleotide polymorphism typing on DNA array with hydrophobic surface fabricated by plasma-polymerization technique

A DNA array has been fabricated on glass substrates, which enables high-throughput analysis of single-base mismatches. In this work, microfabrication-compatible plasma-polymerization (PP) method was used for immobilizing probe DNAs to study the hybridization behavior by changing surface properties. The immobilization matrix consisting of 35 A of PP layer, applied additionally on the streptavidin absorbed hexamethyldisiloxane (HMDS)-PP layer, was constructed on the substrates to anchor biotinylated DNA probes onto the surface. The hydrophobic immobilization matrix was considered to enhance hybridization accuracy and efficiency, compared with its hydrophilic acetonitrile-PP layers. The oligonucleotide arrays fabricated on HMDS-PP surface were shown to be effective in detection of single nucleotide polymorphisms (SNPs) of ApoE gene.     

In vitro selection of DNA aptamers which bind to cholic acid

DNA aptamers which bind to cholic acid have been identified by in vitro selection from a pool of approximately 9x10(14) DNA molecules. After 13 rounds of selection, 19 clones with 95-100 nucleotide length were sequenced. Deletion-mutant experiments and computational sequence analysis suggested that all clones contained cholic acid binding sequences which could fold into three-way junction structures. By comparing the sequences involved in the predicted three-way junction structure of these 19 clones, it was determined that the nucleotide sequences and lengths of three stem and loop regions have no similarity. The most conserved structure seems to have three base pairs flanking the junction of the three stems and they may form a hydrophobic cavity in which they interact with cholic acid.     

A fluorescence polarization assay using oligonucleotide probes for the rapid detection of verotoxin-producing Escherichia coli

A hybridization assay using fluorescence polarization was combined with the asymmetric polymerase chain reaction (PCR) in a method for the detection of the verotoxin type 2 gene of verotoxin-producing Escherichia coli. Six oligonucleotide probes labeled with FITC were designed and evaluated. One of these gave a detection limit of 10(3) colony forming units per assay, and assay results could be obtained within 5 min after PCR. It appears that the detection limit was restricted mainly by the extent and fidelity of PCR amplification, rather than by the sensitivity of the fluorescence polarization technique, indicating that good probe design facilitates the rapid detection of the PCR product. The fluorescence polarization assay, in conjunction with DNA amplification by PCR, is a powerful and widely applicable method for the rapid and sensitive detection of oligonucleotide sequences.     

Effect of incident angle of light on sensitivity and detection limit for layers of antibody with surface plasmon resonance spectroscopy

The effect of the incident angle of light on sensitivity and the detection limit for surface-plasmon resonance spectroscopy were examined. The sensitivities and the detection limit were experimentally measured using an antibody as a modeled analyte in the incident angles of a light region of 66-76 degrees. The results showed that the sensitivity of a smaller incident angle was higher than that of a larger one. For instance, the sensitivity of a 66 degree incident angle was three times higher than that of a 76 degree incident angle. The detection limit with a 66 degree incident angle was one-tenth of that with a 76 degree incident angle. These sensitivities and detection limits were compared with those of a commercially produced surface-plasmon resonance instrument. This comparison demonstrated that a wavelength resolution of the order of less than 10(-2) nm was necessary to obtain satisfactory sensitivities and detection limits. In addition, the refractive index and thickness of the antibody layer formed on a sensor surface was proposed by experimental results and theoretical calculation.     

Increasing the sensitivity of piezoelectric odour sensors based on molecularly imprinted polymers

The molecular imprinting technique has been used to create sensors with a predetermined selectivity for molecules in the gas phase. Based on previous results, piezoelectric quartz crystals were first coated with a layer of nylon, to provide increased sensitivity, and then a layer of a polymer imprinted with 2-methylisoborneol (MIB) was applied to endow the device with selectivity. The response of the 'imprinted' sensor was significantly higher than the response of the 'non-imprinted' sensor at MIB concentrations above 10 ppb. Our new sensor is thus 20 times more sensitive than our previous device. At concentrations greater than 10 ppb, the response of the 'non-imprinted' sensor to geosmin, another odorant often accompanying MIB, was significantly higher than the response of the 'imprinted' sensor. A number of other odorants were examined and, as in our previous work, the responses of the 'non-imprinted' sensors were found to be to greater than the responses of the 'imprinted' sensors.     

Antioxidants from carbon dioxide fixing Chlorella sorokiniana

Chlorella sorokiniana H-84, which has toleranceto high temperatures and high concentrations ofCO₂, has been isolated from a hot spring inJapan. Large-scale culturing of C. sorokinianawas carried out in air containing 10% CO₂.Analysis of the biomass shows that protein, carbohydrate and lipids comprised 68.5, 11.9 and10.0% of dry matter, respectively. The totalcarotenoids comprised 0.69% dry matter. The luteinand -carotene contents were 4300 and 600 gg^-1 dry weight, respectively. The-tocopherol content was 112 g g^-1 dry weight. These carotenoids and -tocopherolare known to possess radical scavenging activity.Two fractions with radical scavenging activity wereisolated from the aqueous extract of C.sorokiniana H-84. The extract showed several singlepeaks by reversed phase HPLC analysis and two of themhad molecular weights of 710 and 1286, respectively.     

Electron transfer mediator micro-biosensor fabrication by organic plasma process

We propose a new strategy for constructing a mediator-type biosensor as a Bio-MicroElectroMechanical Systems (BioMEMS) application. A vinylferrocene plasma-polymerized film (PPF) was deposited directly onto the surface of an electrode under dry conditions. The resulting redox film was extremely thin, adhered well onto a substrate (electrode), and had a highly crosslinked network structure. This technique, capable of polymeric deposition of any kind of monomer, can also serve the purpose of anti-fouling coating, or layer-to-layer interface creation. With a subsequent plasma process, additional polymeric layer of hydrophilic acetonitrile was superimposed onto the existing vinylferrocene-PPF surface to offer crucial features such that the wettability could be adjusted for a better electron transfer, and amino functional groups could be attached to immobilize a large amount of enzyme. Based upon this scheme, the device fabrication could be designed in a manner that the whole procedure was made up of dry wafer-handling processes, which is compatible with mass production. A prototype device was fabricated to have an array of needle-shaped amperometric micro-biosensors. The resultant thin polymer layer carried a large number of the mediator molecules, accomplishing a lower overpotential (+410 mV) and a rapid response time (<5s). Stressing the advantages of the plasma polymerization process together with some additional features accomplished in our device fabrication, we would discuss new possibilities in the field of BioMEMS.     

Electric field control of enzyme membrane activity.

Lipase was immobilized in liquid crystal-collagen membrane. No difference in the optimum pH between the lipase-liquid crystal-collagen membrane and native lipase was observed. However, the immobilized lipase shows flat-pH profiles. The activity of the lipase membrane on a platinum electrode used for cathode increased with increase in terminal voltage and then returned to the initial activity with decrease of terminal voltage.