Selective adsorption of bacterial cells onto zeolites.

Zeolites adsorb microbial cells on their surfaces and selective adsorption for specific microorganisms was seen with certain zeolites. Tests for the adsorption ability of zeolites were conducted using various established microbial cell lines. Specific cell lines were shown to selectively absorb to certain zeolites, species to species. In order to understand the selectivity of adsorption, we tested adsorption under various pH conditions and determined the zeta-potentials of zeolites and cells. The adsorption of some cell lines depended on the pH, and some microorganisms were preferentially adsorbed at acidic pH. The values of zeta-potentials were used for calculating the electric double layer interaction energy between zeolites and microbial cells. There was a correlation between the experimental adsorption results and the interaction energy. Moreover, we evaluated the surface hydrophobicity of bacterial cells by using the microbial adherence to hydrocarbon (MATH) assay. In addition, we also applied this method for zeolites to quantify relative surface hydrophobicity. As a result, we found a correlation between the adsorption results and the hydrophobicity of bacterial cells and zeolites. These results suggested that adsorption could be explained mainly by electric double layer interactions and hydrophobic interactions. Finally, by using the zeolites Na-BEA and H-Y, we succeeded in clearly separating three representative microbes from a mixture of Escherichia coli, Bacillus subtilis and Staphylococcus aureus. Zeolites could adsorb each of the bacterial cell species with high selectivity even from a mixed suspension. Zeolites can therefore be used as effective carrier materials to provide an easy, rapid and accurate method for cell separation.

Easy detection of multiple Alexandrium species using DNA chromatography chip.

In this study, the Kaneka DNA chromatography chip (KDCC) for the Alexandrium species was successfully developed for simultaneous detection of five Alexandrium species. This method utilizes a DNA-DNA hybridization technology. In the PCR process, specifically designed tagged-primers are used, i.e. a forward primer consisting of a tag domain, which can conjugate with gold nanocolloids on the chip, and a primer domain, which can anneal/amplify the target sequence. However, the reverse primer consists of a tag domain, which can hybridize to the solid-phased capture probe on the chip, and a primer domain, which can anneal/amplify the target sequence. As a result, a red line that originates from gold nanocolloids appears as a positive signal on the chip, and the amplicon is detected visually by the naked eye. This technique is simple, because it is possible to visually detect the target species soon after (<5min) the application of 2µL of PCR amplicon and 65µL of development buffer to the sample pad of the chip. Further, this technique is relatively inexpensive and does not require expensive laboratory equipment, such as real-time Q-PCR machines or DNA microarray detectors, but a thermal cycler. Regarding the detection limit of KDCC for the five Alexandrium species, it varied among species and it was <0.1-10pg and equivalent to 5-500 copies of rRNA genes, indicating that the technique is sensitive enough for practical use to detect several cells of the target species from 1L of seawater. The detection sensitivity of KDCC was also evaluated with two different techniques, i.e. a multiplex-PCR and a digital DNA hybridization by digital DNA chip analyzer (DDCA), using natural plankton assemblages. There was no significant difference in the detection sensitivity among the three techniques, suggesting KDCC can be readily used to monitor the HAB species.

Heterogeneous nucleation of protein crystals on fluorinated layered silicate.

Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface.

Use of layer silicate for protein crystallization: effects of Micromica and chlorite powders in hanging drops.

Two kinds of layer silicate powder, Micromica and chlorite, were used to aid protein crystallization by the addition to hanging drops. Using appropriate crystallization buffers, Micromica powder facilitated crystal growth speed for most proteins tested in this study. Furthermore, the addition of Micromica powder to hanging drops allowed the successful crystallization of lysozyme, catalase, concanavalin A, and trypsin even at low protein concentrations and under buffer conditions that otherwise would not generate protein crystals. Except for threonine synthase and apoferritin, the presence of chlorite delayed crystallization but induced the formation of large crystals. X-ray analysis of thaumatin crystals generated by our novel procedure gave better quality data than did that of crystals obtained by a conventional hanging drop method. Our results suggest that the speed of crystal growth and the quality of the corresponding X-ray data may be inversely related, at least for the formation of thaumatin crystals. The effect of Micromica and chlorite powders and the application of layer silicate powder for protein crystallization are discussed.