A novel pathogen detection system based on high‐resolution CE‐SSCP using a triblock copolymer matrix

Although CE‐SSCP analysis combined with 16S ribosomal RNA gene‐specific PCR has enormous potential as a simple and versatile pathogen detection technique, low resolution of CE‐SSCP causes the limited application. Among the experimental conditions affecting the resolution, the polymer matrix is considered to be most critical to improve the resolution of CE‐SSCP analysis. However, due to the peak broadening caused by the interaction between hydrophobic moiety of polymer matrices and DNA, conventional polymer matrices are not ideal for CE‐SSCP analysis. A poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) (PEO‐PPO‐PEO) triblock copolymer, with dynamic coating ability and a propensity to form micelles to minimize exposure of hydrophobic PPO block to DNA, can be an alternative matrix. In this study, we examined the resolution of CE‐SSCP analysis using the PEO‐PPO‐PEO triblock copolymer as the polymer matrix and four same‐sized DNA fragments of similar sequence content. Among 48 commercially available PEO‐PPO‐PEO triblock copolymers, three were selected due to their transparency in the operable range of viscosity and PEO₁₃₇PPO₄₃PEO₁₃₇ exhibited the most effective separation. Significant improvement in resolution allowed discrimination of the similar sequences, thus greatly facilitated CE‐SSCP analysis compared to the conventional polymer matrix. The results indicate that PEO‐PPO‐PEO triblock copolymer may serve as an ideal matrix for high‐resolution CE‐SSCP analysis.     

A new single‐step quantitative pathogen detection system: Template‐tagging followed by multiplex asymmetric PCR using common primers and CE‐SSCP

Rapid diagnosis of bacterial infection is important for patient management and appropriate therapy during the early phase of bacteria‐induced disease. Among the existing techniques for identifying microbial, CE‐SSCP combined with 16S ribosomal RNA gene‐specific PCR has the benefits of excellent sensitivity, resolution, and reproducibility. However, even though CE‐SSCP can separate PCR products with high‐resolution, multiplex detection and quantification are complicated by primer‐dimer formation and non‐specific amplification. Here, we describe a novel technique for multiplex detection and quantification of pathogens by template‐tagging followed by multiplex asymmetric PCR and subsequent CE‐SSCP. More specifically, we reverse transcribed 16S ribosomal RNAs from seven septicemia‐inducing pathogens, tagged the templates with common end sequences, and amplified them using common primers. The resulting amplicons could be successfully separated by CE‐SSCP and quantified by comparison to an internal standard. This method yielded results that illustrate the potential of this system for diagnosing infectious disease.     

Micellar ordered structure effects on high‐resolution CE‐SSCP using Pluronic triblock copolymer blends

Pluronic F108 block copolymers have shown a great promise to achieve the desirable high resolution in the conformation‐sensitive separation of ssDNA using CE‐SSCP. However, fundamental understanding of the structures and properties of Pluronic matrix affecting the resolution is still limited. Unlike conventional gel‐forming homopolymers, Pluronic F108 block copolymers are amphiphilic macromolecules consisting of poly(ethylene oxide)‐b‐poly(propylene oxide)‐b‐poly(ethylene oxide) triblock copolymers, which are capable of forming a highly ordered micellar structure in aqueous solution. In this study, we have performed a series of experiments by blending different types of Pluronic polymers to control the formation of micelles and to study the correlation between separation and rheological characteristics of Pluronic gels affecting the resolution of CE‐SSCP. Our experiments have been specifically designed to elucidate how the micellar structure affects the resolution of CE‐SSCP upon altering the size uniformity and constituent homogeneity of the micelles. Our results suggest that uniformly sized micelle packing is the primary structural feature of Pluronic gel matrix for the high‐resolution separation, while the size and constituent of the micelle themselves need to be considered as secondary factors.     

Rapid and sensitive detection of lower respiratory tract infections by stuffer‐free multiplex ligation‐dependent probe amplification

Lower respiratory tract infection is one of the most common infectious diseases. However, conventional methods for detecting infectious pathogens are time‐consuming, and generally have a limited impact on early therapeutic decisions. We previously reported a rapid and sensitive method for detecting such pathogens using stuffer‐free multiplex ligation‐dependent probe amplification coupled with high‐resolution CE‐SSCP. In this study, we report an application of this method to the detection of respiratory pathogens. As originally configured, this method was capable of simultaneously detecting seven bacterial species responsible for lower respiratory tract infections, but its detection limit and assay time were insufficient to provide useful information for early therapeutic decisions. To improve sensitivity and shorten assay time, we added a target‐specific preamplification step, improving the detection limit from 50 pg of genomic DNA to 500 fg. We further decreased time requirements by optimizing the hybridization step, enabling the entire assay to be completed within 7 h while maintaining the same detection limit. Taken together, these improvements enable the rapid detection of infectious doses of pathogens (i.e. a few dozen cells), establishing the strong potential of the refined method, particularly for aiding early treatment decisions.     

High‐resolution pluronic‐filled microchip CE‐SSCP analysis system via channel width control

Although the resolution of CE‐SSCP has been significantly improved by using a poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) (PEO‐PPO‐PEO; Pluronic^®) triblock copolymer as a separation medium, CE‐SSCP on a microchip format is not widely applicable because their resolution is limited by short channel length. Therefore, a strategy to improve the resolution in channels of limited lengths is highly required for enabling microchip‐based CE‐SSCP. In this study, we developed a high‐resolution CE‐SSCP microchip system by controlling the width of the pluronic‐filled channel. We tested four different channel widths of 180, 240, 300, and 400 μm, and found that 300 μm showed the highest resolution in the separation of two pathogen specific markers. Potential applications of our method in various genetic analyses were also shown by using SNP markers for spinal muscular atrophy.     

Highly multiplex and sensitive SNP genotyping method using a three‐color fluorescence‐labeled ligase detection reaction coupled with conformation‐sensitive CE

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost‐effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation‐dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error‐prone hybridization‐based detection, the multiplex assay process is complicated because of the size‐based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high‐resolution CE‐SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar‐sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three‐color fluorescence‐labeled probes.     

A multiplex single nucleotide polymorphism genotyping method using ligase‐based mismatch discrimination and CE‐SSCP

Accuracy, simplicity, and cost‐effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase‐based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray‐based detection, the design process and multiplex assay procedure are complicated because of the DNA size‐dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition‐optimized ligation and high‐resolution CE‐based SSCP. With this high‐resolution CE‐SSCP system, we are able to use similar‐sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single‐base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.     

Synthesis,characterization, effect of architecture on crystallization,and spherulitic growth of poly(L‐lactide)‐b‐poly(ethylene oxide) copolymers with different branch arms

Well‐defined poly(L ‐lactide)‐b‐poly(ethylene oxide) (PLLA‐b‐PEO) copolymers with different branch arms were synthesized via the controlled ring‐opening polymerization of L ‐lactide followed by a coupling reaction with carboxyl‐terminated poly(ethylene oxide) (PEO); these copolymers included both star‐shaped copolymers having four arms (4sPLLA‐b‐PEO) and six arms (6sPLLA‐b‐PEO) and linear analogues having one arm (LPLLA‐b‐PEO) and two arms (2LPLLA‐b‐PEO). The maximal melting point, cold‐crystallization temperature, and degree of crystallinity (X_c) of the poly(L ‐lactide) (PLLA) block within PLLA‐b‐PEO decreased as the branch arm number increased, whereas X_c of the PEO block within the copolymers inversely increased. This was mainly attributed to the relatively decreasing arm length ratio of PLLA to PEO, which resulted in various PLLA crystallization effects restricting the PEO block. These results indicated that both the PLLA and PEO blocks within the block copolymers mutually influenced each other, and the crystallization of both the PLLA and PEO blocks within the PLLA‐b‐PEO copolymers could be adjusted through both the branch arm number and the arm length of each block. Moreover, the spherulitic growth rate (G) decreased as the branch arm number increased: G_{6sPLLA‐b‐PEO} < G_{4sPLLA‐b‐PEO} < G_{2LPLLA‐b‐PEO} < G_{LPLLA‐b‐PEO}. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 2034–2044, 2006     

Toughening of a propylene‐b‐(ethylene‐co‐propylene) copolymer by a plastomer

Several blends, covering the entire range of compositions, of a metallocenic ethylene‐1‐octene copolymer (CEO) with a multiphasic block copolymer, propylene‐b‐(ethylene‐co‐propylene) (CPE) [composed of semicrystalline isotactic polypropylene (iPP) and amorphous ethylene‐co‐propylene segments], have been prepared and analyzed by differential scanning calorimetry, X‐ray diffraction, optical microscopy, stress‐strain and microhardness measurements, and dynamic mechanical thermal analysis. The results show that for high CEO contents, the crystallization of the iPP component is inhibited and slowed down in such a way that it crystallizes at much lower temperatures, simultaneously with the crystallization of the CEO crystals. The mechanical results suggest very clearly the toughening effect of CEO as its content increases in the blends, although it is accompanied by a decrease in stiffness. The analysis of the viscoelastic relaxations displays, first, the glass transition of the amorphous blocks of CPE appearing at around 223 K, which is responsible for the initial toughening of the plain CPE copolymer in relation to iPP homopolymer. Moreover, the additional toughening due to the addition of CEO in the blends is explained by the presence of the β relaxation of CEO that appears at about 223 K. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 1869–1880, 2002     

Fabrication of Poly(L‐lactide)‐block‐Poly(ethylene glycol)‐block‐Poly(L‐lactide) Triblock Copolymer Thin Films with Nanochannels: An AFM Study

This paper aims to report the fabrication of biodegradable thin films with micro‐domains of cylindrical nanochannels through the solvent‐induced microphase separation of poly(L ‐lactide)‐block‐poly(ethylene glycol)‐block‐poly(L ‐lactide) (PLA‐b‐PEG‐b‐PLA) triblock copolymers with different block ratios. In our experimental scope, an increase in each of the block lengths of the PLA and PEG blocks led to both a variation in the average number density (146 to 32 per 100 µm²) and the size of the micro‐domains (140 to 427 nm). Analyses by atomic force microscopy (AFM) and fluorescence microscopy indicated that the hydrophilic PEG nanochannels were dispersed in the PLA matrix of the PLA‐b‐PEG‐b‐PLA films. We demonstrated that the micro‐domain morphology could be controlled not only by the block length of PEG, but also by the solvent evaporation conditions.

     

Water‐soluble poly(ethylene oxide)‐block‐poly(p‐phenylene vinylene) copolymer: Synthesis and characterization

Water‐soluble and photoluminescent block copolymers [poly(ethylene oxide)‐block‐poly(p‐phenylene vinylene) (PEO‐b‐PPV)] were synthesized, in two steps, by the addition of α‐halo‐α′‐alkylsulfinyl‐p‐xylene from activated poly(ethylene oxide) (PEO) chains in tetrahydrofuran at 25 °C. This copolymerization, which was derived from the Vanderzande poly(p‐phenylene vinylene) (PPV) synthesis, led to partly converted PEO‐b‐PPV block copolymers mixed with unreacted PEO chains. The yield, length, and composition of these added sequences depended on the experimental conditions, namely, the order of reagent addition, the nature of the monomers, and the addition of an extra base. The addition of lithium tert‐butoxide increased the length of the PPV precursor sequence and reduced spontaneous conversion. The conversion into PPV could be achieved in a second step by a thermal treatment. A spectral analysis of the reactive medium and the composition of the resulting polymers revealed new evidence for an anionic mechanism of the copolymerization process under our experimental conditions. Moreover, the photoluminescence yields were strongly dependant on the conjugation length and on the solvent, with a maximum (70%) in tetrahydrofuran and a minimum (<1%) in water. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 4337–4350, 2005     

Dual‐enzyme,co‐immobilized capillary microreactor combined with substrate recycling for high‐sensitive glutamate determination based on CE

A new method for high‐sensitive determination of glutamate was developed and evaluated based on CE by using dual‐enzyme co‐immobilized capillary microreactor combined with substrate recycling. The capillary microreactor was prepared by covalently co‐immobilizing glutamate dehydrogenase (GDH) and glutamic pyruvic transaminase (GPT) on the inner surface of a capillary and was characterized by SEM, ultraviolet‐visible spectroscopy, and fluorescence spectroscopy. The GDH‐GPT co‐immobilized capillary microreactor showed great stability and reproducibility. The apparent K_m for glutamate with GDH‐GPT coupled reaction was determined to be 0.61±0.06 mM but 2.56±0.24 mM when only GDH was immobilized. Glutamate determination was based on on‐column monitoring UV absorption at 340 nm of the reaction product reduced nicotinamide adenine dinucleotide, of which peak area was directly related to the glutamate concentration. The response of the present co‐immobilized GDH‐GPT assay for glutamate is greatly enhanced over single enzyme system, and a 15.7‐fold improvement in sensitivity was obtained. The detection limit of the proposed method is 0.15 μM glutamate (S/N=3). Selectivity for glutamate is good over most of the 20 amino acids. Finally, this method was successfully applied to determine the glutamate content in rat plasma and serum samples.     

Precise characterization method of antibody‐conjugated magnetic nanoparticles for pathogen detection using stuffer‐free multiplex ligation‐dependent probe amplification

Antibody‐conjugated magnetic nanoparticles (Ab‐MNPs) have potential in pathogen detection because they allow target cells to be easily separated from complex sample matrices. However, the sensitivity and specificity of pathogen capture by Ab‐MNPs generally vary according to the types of MNPs, antibodies, and sample matrices, as well as preparation methods, including immobilization. Therefore, achieving a reproducible analysis utilizing Ab‐MNPs as a pathogen detection method requires accurate characterization of Ab‐MNP capture ability and standardization of all handling processes. In this study, we used high‐resolution CE‐single strand conformational polymorphism coupled with a stuffer‐free multiplex ligation‐dependent probe amplification system to characterize Ab‐MNPs. The capture ability of Ab‐MNPs targeting Salmonella enteritidis and nine pathogens, including S. enteritidis, was analyzed in phosphate buffer and milk. The effect of storage conditions on the stability of Ab‐MNPs was also assessed. The results showed that the stuffer‐free multiplex ligation‐dependent probe amplification system has the potential to serve as a standard characterization method for Ab‐MNPs. Moreover, the precise characterization of Ab‐MNPs facilitated robust pathogen detection in various applications.     

New super‐tough poly(butylene terephthalate) materials based on compatibilized blends with metallocenic poly(ethylene‐octene) copolymer

New super‐tough poly(butylene terephthalate) (PBT)/poly(ethylene‐octene) copolymer (PEO) blends containing 2 wt% poly(ethylene‐co‐glycidyl methacrylate) (EGMA) as a compatibilizer were obtained by extrusion and injection molding. The blends comprised of an amorphous PBT‐rich phase with some miscibilized EGMA, a pure PEO amorphous phase, and a crystalline PBT phase that was not influenced by the presence of either PEO or EGMA. The blends showed a fine particle size up to 20 wt% PEO content. Super‐tough blends were obtained with PEO contents equal to or higher than 10%. The maximum toughness was very high (above 710 J/m) and was attained with 20% PEO without chemical modification of the commercial components used. Copyright © 2003 John Wiley & Sons, Ltd.     

Effect of PEO‐PPO‐PEO copolymer on the mechanical and thermal properties and morphological behavior of biodegradable poly (L‐lactic acid) (PLLA) and poly (butylene succinate‐co‐L‐lactate) (PBSL) blends

A blend of two biodegradable and semi‐crystalline polymers, poly (L‐lactic acid) (PLLA; 70 wt%) and poly (butylene succinate‐co‐L‐lactate) (PBSL; 30 wt%), was prepared in the presence of various polyethylene oxide‐polypropylene oxide‐polyethylene oxide (PEO‐PPO‐PEO) triblock copolymer contents (0.5, 1, 2 wt%). Mechanical, thermal properties, and Fourier transform infrared (FTIR) analysis of the blends were investigated. It was found that the addition of copolymer to PLLA/PBSL improved the fracture toughness of the blends as shown by mode I fracture energies. It was supported by morphological analysis where the brittle deformation behavior of PLLA changed to ductile deformation with the presence of elongated fibril structure in the blend with copolymer system. The glass transition temperature (T_g), melting temperature (T_m) of PLLA, and PBSL shift‐closed together indicated that some compatibility exists in the blends. In short, PEO‐PPO‐PEO could be used as compatibilizer to improve the toughness and compatibility of the PLLA/PBSL blends. Copyright © 2010 John Wiley & Sons, Ltd.     

Biodegradable electrospun mat: Novel block copolymer of poly (p‐dioxanone‐co‐L‐lactide)‐block‐poly(ethylene glycol)

Ultrafine fibers of a laboratory‐synthesized new biodegradable poly(p‐dioxanone‐co‐L ‐lactide)‐block‐poly(ethylene glycol) copolymer were electrospun from solution and collected as a nonwoven mat. The structure and morphology of the electrospun membrane were investigated by scanning electron microscopy, differential scanning calorimetry (DSC), wide‐angle X‐ray diffraction (WAXD), and a mercury porosimeter. Solutions of the copolymer, ranging in the lactide fraction from 60 to 80 mol % in copolymer composition, were readily electrospun at room temperature from solutions up to 20 wt % in methylene chloride. We demonstrate the ability to control the fiber diameter of the copolymer as a function of solution concentration with dimethylformamide as a cosolvent. DSC and WAXD results showed the relatively poor crystallinity of the electrospun copolymer fiber. Electrospun copolymer membrane was applied for the hydrolytic degradation in phosphate buffer solution (pH = 7.5) at 37 °C. Preliminary results of the hydrolytic degradation demonstrated the degradation rate of the electrospun membrane was slower than that of the corresponding copolymers of cast film. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 1955–1964, 2003     

Graphene‐doped electrochemical copolymer coating of 2,2‐bithiophene and 3‐methylthiophene for the highly effective solid‐phase microextraction of volatile benzene homologues

We report the electrochemical fabrication of a poly(2,2‐bithiophene‐co‐3‐methylthiophene)‐graphene composite coating and its application in the headspace solid‐phase microextraction and gas chromatography determination of benzenes (i.e., bromobenzene, 4‐bromotoluene, 2‐nitrotoluene, 3‐nitrotoluene and 1,2,4‐trichlorobenzene). The coating was uniform and showed cauliflower‐like microstructure. It had high thermal stability (up to 375°C) and could be used for at least 180 times of solid‐phase microextraction without a decrease in extraction performance. Furthermore, it presented high extraction capacity for the benzenes due to the hydrophobic effect and π–π interaction between the analytes and the coating. Under optimized extraction conditions, good linearity (correlation coefficients higher than 0.9946), wide linear range (0.01–50 μg/L), and low limits of detection (5.25–12.5 ng/L) were achieved for these analytes. The relative standard deviation was lower than 5.7% for five successive measurements with one fiber, and the relative standard deviation for fiber‐to‐fiber was 4.9–6.8% (n = 5). The solid‐phase microextraction and gas chromatography method was successfully applied for the determination of three real samples, and the recoveries for standards added were 89.6–106% for nail polish, 85.8–110% for hair dye, and 90–106.2% for correction fluid, respectively.     

Development of variance‐filtered instrumental transfer methods for high‐resolution NMR spectroscopy

The introduction of a variance‐filter to both direct standardization (DS) and piece‐wise direct standardization (PDS) instrumental transfer methods for the analysis of NMR spectral data is described. The variance‐filter modification allows for the identification of regions in the NMR spectra that are not adequately represented by the limited number of transfer calibration samples used during the calculation of the instrument‐to‐instrument transfer matrix. For these spectral frequencies, the corresponding portion of the transfer matrix is replaced by identity (or scaled identity) prior to the secondary instrumental data sets being transferred to the target instrument response. The spectral matching performance of the variance‐filtered instrumental transfer method as applied to high‐resolution ¹H NMR spectra is presented along with possible uses and limitations. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of a capillary‐assembled microfluidic system for separation of cephalosporins

This paper demonstrates a simple and easy setting up of a fused‐silica capillary‐assembled microfluidic system (μCE). This system incorporates a split‐flow pressure injection of the sample into a microfluidic system made from PDMS and a short (～20 cm) length of fused‐silica capillary as a separation unit. The on‐capillary detection was carried out by fiber optic spectrometry. A mixture of six cephalosporin antibiotics was separated in the μCE system and the obtained results were compared to those achievable by conventional CE. The six components could be separated within 8.5 min with the number of theoretical plates around 10 000.