Optimal Design for U-bent Fiber-optic LSPR Sensor Probes

The refractive index (RI) sensitivity of a localized surface plasmon resonance (LSPR)-based fiber-optic probes is dependent on surface coverage of gold nanoparticles (GNP), fiber core diameter, and probe geometry. For U-bent LSPR fiber-optic probes, which demonstrated an order higher absorption sensitivity over straight probes, bend diameter and probe length may also have a significant influence on the sensitivity. This study on U-bent fiber-optic LSPR probes is aimed at optimizing these parameters to obtain highest possible RI sensitivity. RI sensitivity increases linearly as a function of surface coverage of GNP in the range of 2–22 %. U-bent fiber-optic probes made of 200-, 400-, and 600-μm fiber core diameter show optimum bend diameter value as ～1.4 mm. In addition, RI sensitivity is almost the same irrespective of fiber core diameter demonstrating flexibility in choice of the fiber and ease in optical coupling. The length of the probe preceding and succeeding the bend region has significantly less influence on RI sensitivity allowing miniaturization of these probes. In addition to these experimental studies, we present a theoretical analysis to understand the relative contribution of evanescent wave absorbance of GNP and refractive losses in the fiber due to GNP, towards the RI sensitivity.     

Proving the antimicrobial spectrum of an amphoteric surfactant-sol-gel coating: a food-borne pathogen study

An antimicrobial coating was evaluated in this work for its antimicrobial efficacy against common food-borne pathogens. Dodecyl-di(aminoethyl)-glycine, an organic disinfectant, was immobilized in a silicon oxide matrix to generate thin films over surfaces by means of the sol-gel process. Tetraethoxysilane was used as the polymeric precursor. No alteration of optical transparency on the covered surfaces was observed. Topographic images obtained with atomic force microscopy showed a homogeneous film with no additional roughness added by the polymer to the surface. The attenuated total reflectance-Fourier transform infrared spectral data showed the presence of dodecyl-di(aminoethyl)-glycine in the silicon oxide network after a normal cleaning procedure. The antimicrobial efficacy test was performed by exposing coated slides to suspensions of common food-borne pathogens: Escherichia coli, Staphyloccocus aureus, E. coli O157:H7, Salmonella typhi, S. cholerasuiss, Listeria innocua and L. monocytogenes. The coating activity was not only bacteriostatic but also bactericidal. The percent reduction of viable microorganism exposure over 24 h to the coated surface ranged between 99.5%, for the more resistant gram-positive bacteria, and over 99.999%, for most gram-negative bacteria. The silicon matrix itself did not account for any reduction of viable microbial, even more an increase was observed.     

The Effect of Annealing Temperature on the Plasma Edge in Reflectance Spectra of Al/Al2O3 Composites Synthesized by Thermal Oxidation of Aluminum Thin Films

In this study, the structural and optical properties of aluminum oxide thin films were investigated. Aluminum oxide thin films were prepared on silicon and glass substrate by DC magnetron sputtering of aluminum targets with subsequent thermal oxidation of the aluminum-deposited thin films. Important result obtained included the presence of a plasma edge for the individual aluminum atoms. In addition, the temperatures that resulted in the highest concentration of surface plasmons were determined. On other hand, the relationship between the plasma edge and the optical energy gap was investigated.

     

Surface Plasmon Resonance-Based Fiber-Optic Hydrogen Gas Sensor Utilizing Indium–Tin Oxide (ITO) Thin Films

We report experimental study on an indium?Ctin oxide (ITO)-coated surface plasmon resonance-based fiber-optic hydrogen gas sensor operating at room temperature. The sensor works on intensity modulation interrogation. Indium?Ctin oxide (In₂O₃?+?SnO₂) films were grown on unclad core of the fiber by thermal evaporation technique. The surface plasmon resonance (SPR) spectra for 100?% nitrogen gas as well as for a mixture of 4?% hydrogen gas and 96?% nitrogen gas were obtained. In the case of mixture of hydrogen and nitrogen gases, a sharp dip in the SPR spectrum was observed implying that the hydrogen gas changes the dielectric properties of ITO. The performance of the sensor has been studied for different percentages of tin oxide in indium oxide and for different thicknesses of ITO film. Both the parameters have been optimized for the best performance of the sensor.     

O-10THE ACCURACY OF CERVICAL CYTOLOGY: A COMPARISON BETWEEN THE THINPREP IMAGING SYSTEM AND CONVENTIONAL METHODS

Douglass Hanly Moir is a large Australian laboratory which has recently introduced the ThinPrep Imaging System (TPI) for reading ThinPrep slides, which is still performed using a split-sample technique. The Imager is a computerized system which identifies 22 fields for the cytologist to review using automated light microscopy. We compared the accuracy of TPI and conventional cytology (CC) during normal laboratory operation. The ThinPrep sample was prepared after taking a conventional Pap smear. TPI and CC reading was done without knowledge of the result of the other reading. The final cytology report issued to the referring doctor reflected the more severe of these two results. Histology results for all cases in which TPI and CC cytology results showed more than minimal disagreement were sought from the NSW Pap Test Register. Of 55 164 split sample pairs, 3.1% of CC of slides and 1.8% of TPI slides were unsatisfactory. There were 1758 women for whom there was more than minimal discrepancy between TPI and CC cytology results. TPI gave the more severe result in 1193 of the 1758 cases. In cases where only one of each pair of discrepant cytology results was CIN1 or higher grade, TPI detected 133 cases of high-grade histology among 380 biopsies (35%), whereas CC detected 62 cases among 210 biopsies (29.5%). A repeat analysis based on reading of histology by one pathologist blinded to initial Pap smear result showed a similar result. Reading times were measured over 5 months for both TPI and CC for twenty cytologists who read both types of smears. On average, they read 13.3 TPI slides per hour and 6.1 CC slides per hour. This study provides evidence that cervical cytology read using the TPI detects more histological high-grade disease than does CC. Further evidence shows that reading times are significantly reduced for cytologists using the TPI.     

Innovative metal oxide-based substrates for DNA microarrays

In the present report, we propose a novel approach to synthesize DNA microarrays that employs immobilization of the nucleic acid molecules onto zinc and iron oxide surfaces through their phosphate backbone. Oxide films were prepared by the sol–gel technique and the resulting surfaces were characterized especially with respect to surface chemistry and morphological features by both X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). ZnO films annealed at T ? 300 °C show the most promising surface features to be employed for DNA microarray preparation, i.e. high density of binding sites (hydroxyl groups), smooth and homogeneous surfaces, high optical transmittance in the visible spectral range suitable for detection using fluorescence, and easy handling during preparation procedures. The analysis of nucleic acid retention on the oxide layers was performed by the scanning of dye-labelled DNA previously printed on the substrate using the DNA microarray robotic arm. Clearly visible spots with regular shape were revealed above the background noise indicating that anchoring of the DNA on the treated surface is efficient and does not interfere with hybridization processes. The use of suitably engineered zinc oxide film makes the immobilization strategy ideal for facile, efficient, and cost-effective manufacturing of DNA microarrays.     

Assessment of metagenomic assembly using simulated next generation sequencing data

Due to the complexity of the protocols and a limited knowledge of the nature of microbial communities, simulating metagenomic sequences plays an important role in testing the performance of existing tools and data analysis methods with metagenomic data. We developed metagenomic read simulators with platform-specific (Sanger, pyrosequencing, Illumina) base-error models, and simulated metagenomes of differing community complexities. We first evaluated the effect of rigorous quality control on Illumina data. Although quality filtering removed a large proportion of the data, it greatly improved the accuracy and contig lengths of resulting assemblies. We then compared the quality-trimmed Illumina assemblies to those from Sanger and pyrosequencing. For the simple community (10 genomes) all sequencing technologies assembled a similar amount and accurately represented the expected functional composition. For the more complex community (100 genomes) Illumina produced the best assemblies and more correctly resembled the expected functional composition. For the most complex community (400 genomes) there was very little assembly of reads from any sequencing technology. However, due to the longer read length the Sanger reads still represented the overall functional composition reasonably well. We further examined the effect of scaffolding of contigs using paired-end Illumina reads. It dramatically increased contig lengths of the simple community and yielded minor improvements to the more complex communities. Although the increase in contig length was accompanied by increased chimericity, it resulted in more complete genes and a better characterization of the functional repertoire. The metagenomic simulators developed for this research are freely available.     

BIPES,a cost-effective high-throughput method for assessing microbial diversity

Pyrosequencing of 16S rRNA (16S) variable tags has become the most popular method for assessing microbial diversity, but the method remains costly for the evaluation of large numbers of environmental samples with high sequencing depths. We developed a barcoded Illumina paired-end (PE) sequencing (BIPES) method that sequences each 16S V6 tag from both ends on the Illumina HiSeq 2000, and the PE reads are then overlapped to obtain the V6 tag. The average accuracy of Illumina single-end (SE) reads was only 97.9%, which decreased from ∼99.9% at the start of the read to less than 85% at the end of the read; nevertheless, overlapping of the PE reads significantly increased the sequencing accuracy to 99.65% by verifying the 3′ end of each SE in which the sequencing quality was degraded. After the removal of tags with two or more mismatches within the medial 40–70 bases of the reads and of tags with any primer errors, the overall base sequencing accuracy of the BIPES reads was further increased to 99.93%. The BIPES reads reflected the amounts of the various tags in the initial template, but long tags and high GC tags were underestimated. The BIPES method yields 20–50 times more 16S V6 tags than does pyrosequencing in a single-flow cell run, and each of the BIPES reads costs less than 1/40 of a pyrosequencing read. As a laborsaving and cost-effective method, BIPES can be routinely used to analyze the microbial ecology of both environmental and human microbiomes.     

Antimicrobial activity on glass materials subject to disinfectant xerogel coating

The antimicrobial compound dodecyl-di(aminoethyl)-glycine was immobilized in a silicon oxide xerogel matrix and used for glass surface coating. Coated glasses were tested for surface antimicrobial activity. The utilization of tetraethoxysilane (TEOS) as a silicon oxide polymer precursor, using the dip-coating process, allowed for the generation of transparent thin films over glass surfaces. Different concentrations of the antimicrobial compound were used to generate the coatings. The presence of dodecyl-di(aminoethyl)-glycine on coated and uncoated slides was analyzed by FT-IR spectra. Coated glass slides were exposed to suspensions of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus for 24 h. Surface contamination was evaluated by the microbial plate count technique. When antimicrobial-coated glasses were compared with antimicrobial-free coated glasses, the former showed greater than 99% reduction of colony-forming units (cfu) for E. coli and P. aeruginosa, when 1% of antimicrobial was present in the coating solution. The same percentage of reduction for S. aureus was achieved when 1.5% of the antimicrobial was present in the coating solution. In a direct inhibition test on agar plates, no inhibitory zone was observed, indicating that the antimicrobial did not diffuse into the media.G.J. Copello and S. Teves are equally responsible for this work.In memorian of Prof. Benjamin Frydman.     

A visual exploration apparatus for infant monkeys

An apparatus was constructed to study visual exploration in infant rhesus macaques. It consisted of an enclosed two-chamber box with a peephole at each end. The floor was made of stainless steel bars, and the walls and top were made of Plexiglas covered with Masonite. The peepholes were recessed in alcoves. An infrared photobeam crossed the alcoves in front of each peephole so that whenever the monkey looked out its head broke the photobeam. Slides of complex scenes were projected on back-lit frosted plexiglas screens. The monkey's position in the box was monitored by its resistance across the floor bars. Whenever the monkey went from one side of the box to the other, a new slide was projected on the side just entered. The session progressed until either 40 slides had been displayed on each side or 30 minutes had elapsed. The primary behavioral measures taken were session length, number of slides displayed, time spent looking, number of looks, and time spent looking at the first slide on each side. Serveral other performance measures were derived from these basic measures: time looked/slide, number of looks/slide, and average length of look (time looked/number of looks). The monkeys readily performed in this apparatus, looking out through the peepholes for an average of about 14% of each session with attentional episodes of just under 3 seconds. This apparatus has proven useful for automatically measuring visual exploration behavior in infant monkeys and can be readily adapted for use in many types of studies.     

The role of conditioning film formation in Pseudomonas aeruginosa PAO1 adhesion to inert surfaces in aquatic environments

Bacterial initial adhesion to inert surfaces in aquatic environments is highly dependent on the surface properties of the substratum, which can be altered significantly by the formation of conditioning films. In this study, the impact of conditioning films formed with extracellular polymeric substances (EPS) on bacterial adhesion was investigated. Adhesion of wild type Pseudomonas aeruginosa PAO1 to slides coated with model EPS components (alginate, humic substances, and bovine serum albumin (BSA)) was examined. Surface roughness of conditioning film coated slides was evaluated by atomic force microscopy (AFM), and its effect on the bacterial initial adhesion was not significant. X-ray photoelectron spectroscopy (XPS) studies were performed to determine the elemental surface compositions of bacterial cells and substrates. Results showed that bacterial adhesion to bare slides and slides coated with alginate and humic substances increased as ionic strength increased. Conversely, BSA coating enhanced bacterial adhesion at low ionic strength but hindered adhesion at higher ionic strength. It was concluded that forces other than hydrophobic and electrostatic interactions were involved in controlling bacterial adhesion to BSA coated surfaces. A steric model for polymer brushes that considers the combined influence of steric effects and DLVO interaction forces was shown to adequately describe the observed bacterial adhesion behaviors.     

Long-read pyrosequencing using pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer.

Pyrosequencing, a nonelectrophoretic DNA sequencing method that uses a luciferase-based enzymatic system to monitor DNA synthesis in real time, has so far been limited to sequencing of short stretches of DNA. To increase the signal-to-noise ratio in pyrosequencing the natural dATP was replaced by dATPalphaS (M. Ronaghi et al., 1996, Anal. Biochem. 242, 84-89). The applied dATPalphaS was a mixture of two isomers (Sp and Rp). We show here that by the introduction of pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer in pyrosequencing substantial longer reads could be obtained. The pure Sp-isomer allowed lower nucleotide concentration to be used and improved the possibility to read through poly(T) regions. In general, a doubling of the read length could be obtained by the use of pure Sp-isomer. Pyrosequencing data for 50 to 100 bases could be generated on different types of template. The longer read will enable numerous new applications, such as identification and typing of medically important microorganisms as well as resequencing of DNA fragments for mutation screening and clone checking.     

The effects of surface configuration on the retentive properties of parallel-sided dowels cemented with a composite luting agent

Factors which have effect on the retention of any dowel include the shape, length, diameter, and surface configuration of the dowel, the cementing medium, precision fitness and material, etc. The shape and surface configuration are the most important among all factors. Recent developments of the etching technique for cast metal has improved the bonding strength between base metal alloys and composite cement which offers a great opportunity for better retention of the dowel if its surface has been etched. In the past, because it has often been ignored that the bonding at the cement-dentin interface is weaker than that between the metal and cement, the true impact of surface configuration and cement thickness on the retention of dowels has never really been observed. In this study, plastic transparent rods were substituted for natural teeth to avoid anatomical variation and circular horizontal grooves were placed on the channel walls to strengthen the retention between the cement and the plastic rods. A total of 140 dowel samples were cast in Rexillium III and were divided into seven groups. The surfaces of the samples were treated differently by smoothing, sandblasting, chemical etching, electrolytic etching and serrating, and fixed into the channels of the plastic rods by cementation with composite resin. Immediately thereafter, the samples were submerged in 37 degrees C water for 7 days. Instron crosshead speed 1 mm/min was used in measuring the tensile forces required for extracting the dowels from the channels. Data were analyzed with the ANOVA method. Among the three factors known to have an effect on the retention being considered in this study, the circular horizontal groove proved to be the most influential, and surface configuration came next, while cement thickness was the least influential. Among the different surface configurations, the serrated dowels showed the best retention and were followed in order by etched and smooth or sandblasted dowels.     

生物素化ATP硫酸化酶的表达、固定化与应用

现代大规模焦测序技术的产生是DNA测序技术的一次革命,其关键技术之一是得到高活性的、固定于磁性微球表面的ATP硫酸化酶．生物素化的ATP硫酸化酶可以通过生物素与亲和素之间的特异结合特性固定在包被亲和素的磁性微球表面,但是利用化学修饰法将ATP硫酸化酶进行生物素化修饰很可能会影响酶的活性．利用融合表达策略,将大肠杆菌生物素酰基载体蛋白C端87个氨基酸肽段(BCCP87)与ATP硫酸化酶在大肠杆菌内融合表达,经SDS-PAGE和Western blot分析,表达的融合蛋白分子质量约为64 ku,并且能够在大肠杆菌内被生物素化．生物素化的ATP硫酸化酶能够与亲和素包被的磁珠结合,固定后的ATP硫酸化酶具有活性,并且能够用于定量检测焦磷酸盐(PPi)和焦测序,为今后建立高通量大规模焦测序系统提供了一个有效的工具酶．     

Bionanofabrication by Near-Field Optical Methods

Near-field optical methods offer unique potential in nanofabrication, because they provide the capacity to initiate highly selective chemical transformations with nanometer scale precision. The basic principles behind scanning near-field photolithography (SNP), in which a scanning near-field optical microscope coupled to a UV laser is used to initiate surface chemical reactions, are described. The fundamental principles underlying the patterning of self-assembled monolayers by SNP are described, and the resolution limits and the basic principles that enable routine achievement of sub-50 nm resolution are discussed. Illustrations are provided of the application of SNP to the patterning of protein molecules on gold surfaces. The patterning of molecular adsorbates on oxide surfaces, including the fabrication of highly miniaturized arrays of DNA on silicon dioxide, is also described. It is argued that SNP holds great promise for the organization of biomolecules on nanometer length scales.     

Quantifying microbial communities with 454 pyrosequencing: does read abundance count?

Pyrosequencing technologies have revolutionized how we describe and compare complex microbial communities. In 454 pyrosequencing data sets, the abundance of reads pertaining to taxa or phylotypes is commonly interpreted as a measure of genic or taxon abundance, useful for quantitative comparisons of community similarity. Potentially systematic biases inherent in sample processing, amplification and sequencing, however, may alter read abundance and reduce the utility of quantitative metrics. Here, we examine the relationship between read abundance and biological abundance in a sample of house dust spiked with known quantities and identities of fungi along a dilution gradient. Our results show one order of magnitude differences in read abundance among species. Precision of quantification within species along the dilution gradient varied from R(2) of 0.96-0.54. Read-quality based processing stringency profoundly affected the abundance of one species containing long homopolymers in a read orientation-biased manner. Order-level composition of background environmental fungal communities determined from pyrosequencing data was comparable with that derived from cloning and Sanger sequencing and was not biased by read orientation. We conclude that read abundance is approximately quantitative within species, but between-species comparisons can be biased by innate sequence structure. Our results showed a trade off between sequence quality stringency and quantification. Careful consideration of sequence processing methods and community analyses are warranted when testing hypotheses using read abundance data.     

Surface composition and morphology of starch,amylose, and amylopectin films

The surfaces of solution-cast films of starch, amylose, and amylopectin were examined with scanning electron microscopy (SEM), atomic force microscopy (AFM), electron spectroscopy for chemical analysis (ESCA), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The surface topography visualized by SEM showed that amylopectin films were very smooth whereas amylose and starch films were rougher. It appears that crystallinity or phase separation in the bulk of the film affects the surface topography. AFM showed that the outmost surfaces of all films were covered with small protrusions, 15-35 nm wide and 1-4 nm high. Studies with ESCA revealed the presence of 3-8% nitrogen on the surfaces. ToF-SIMS indicated that the nitrogen originates from protein because ionic fragments from amino acids and the peptide backbone were found. Extracts from the top surface layer of the starch film showed protein bands in gel electrophoresis (SDS-PAGE) around 60 kDa, which is in the same molecular weight range as the biosynthesizing enzyme GBSS I present in starch granules. The proteins apparently phase separated during film formation and migrated to the surface, resulting in an extensive enrichment of proteins in the film surface, where about 8% of the protein is present in the top 0.01% of the film. We believe that the protrusions observed with AFM could be one or a few proteins aggregated side by side.     

Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform

Background

16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform

Methodology/Principal Findings

The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5–1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management.

Conclusions

Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.     

Maximization of acetic acid production in partial acidogenesis of swine wastewater.

Swine wastewater was biologically treated to produce short-chain volatile organic acids (VOAs) in laboratory-scale continuously stirred tank reactors. The maximum production rates of acetic and butyric acids associated with simultaneous changes in pH and hydraulic retention time (HRT) were investigated, in which the degree of acidification of swine wastewater to the short-chain VOAs was <25% of influent chemical oxygen demand (COD) concentration. A constant inoculum system was used to minimize the experimental error due to the use of inconsistent inoculum. The inoculum system was operated with synthetic wastewater at 6000 mg soluble chemical oxygen demand per liter (pH 6.0) and 35 degrees C at 0.5 day hydraulic retention time. Response surface methodology was applied successfully to determine the optimum physiological condition for which the maximum rate of acetic acid production occurred, which was pH 5.90 and 0.88 day hydraulic retention time at 35 degrees C. The partial acidification process to manage swine waste should be operated in the optimum condition for acetic acid production because the optimum operating condition for butyric acid production approached the washout point.