Application of a fluorescence stereomicroscope as an in-line detection unit for electrophoretic separation of bacteria

Electrophoretic separation of bacteria as well as potential applications of electrophoresis in microbiological medical analysis has gained the interest of several research groups. The undesired effects such as aggregation and/or adhesion of the cells have a strong effect on the separation process. For a better understanding of the phenomena we decided to apply a fluorescence stereomicroscope as an in-line detection unit for observation of electrophoresis of microorganisms. The microscope was connected to the capillary electrophoretic system which allowed observing the moving bacteria through a 10 cm wide window created in the capillary that was kept in a special holder. The experiments showed that the stained bacteria adhered to the inner capillary surface thus creating a green fluorescent layer. The short (2 mm) polymeric monolithic bed, the pore size of which disabled bacteria cells to pass through, was also photosynthesized in the centre of the window and served to preconcentrate the bacterial suspension.     

Enantioselective analysis of ofloxacin enantiomers by partial-filling capillary electrophoresis with bacteria as chiral selectors

The enantiomeric separation of ofloxacin enantiomers (OFLX) was achieved by using capillary electrophoresis partial-filled with Escherichia coli, Pseudomonas aeruginosa (Gram-negative), and Staphylococcus aureus (Gram-positive) as chiral selectors. Experimental parameters, including the concentration of background electrolyte, applied voltage, length of the filled bacteria plug, and pH of the buffer, were intensively investigated. Baseline separation of OFLX could be achieved within 7 min by using E. coli and P. aeruginosa as chiral selectors under the following conditions: electrophoretic buffer composed of 10 mM phosphate buffer at pH 7.4, applied voltage at 15 kV, and the bacteria (6.0 × 10(8) cells/mL) were injected into the capillary by gravity with injection height of 17.5 cm for 180 s (E. coli), 300 s (P. aeruginosa), and 300 s (S. aureus), respectively. E. coli and P. aeruginosa had better chiral selectivity for OFLX than S. aureus, which was in good agreement with OFLX having better antimicrobial activity on Gram-negative rather than Gram-positive bacteria. A novel method was developed for the enantioselective separation of enantiomers using bacteria as chiral selectors, which provides a new approach for antimicrobials enantioselective analysis, chiral pharmacodynamics, and chiral pharmacokinetics studies.     

利用高度结构对称的共聚试剂制备高比表面积的硼亲和整体柱

硼亲和整体柱是一种能对顺式二羟基化合物进行选择性分离的重要色谱介质。然而,整体柱微结构在制备过程中难以控制。本文以分子结构高度对称的三聚氰胺和三(2,3-环氧基丙基)异氰酸酯(TEPIC)作为共聚试剂,利用“团队硼亲和”原理,采用原位聚合的方法制备了一种新型硼亲和整体柱。“团队硼亲和”的作用使该硼亲和整体柱对顺式二羟基化合物的结合pH降低至中性范围。结构高度对称的共聚试剂有效地提高了整体柱材料的比表面积,使其达80.3 m²/g,显著高于文献中报道的其它硼亲和整体柱。     

利用高度结构对称的共聚试剂制备高比表面积的硼亲和整体柱

硼亲和整体柱是一种能对顺式二羟基化合物进行选择性分离的重要色谱介质。然而,整体柱微结构在制备过程中难以控制。本文以分子结构高度对称的三聚氰胺和三(2,3-环氧基丙基)异氰酸酯(TEPIC)作为共聚试剂,利用“团队硼亲和”原理,采用原位聚合的方法制备了一种新型硼亲和整体柱。“团队硼亲和”的作用使该硼亲和整体柱对顺式二羟基化合物的结合pH降低至中性范围。结构高度对称的共聚试剂有效地提高了整体柱材料的比表面积,使其达80.3 m²/g,显著高于文献中报道的其它硼亲和整体柱。     

Optical imaging of bacterial infection in living mice using a fluorescent near-infrared molecular probe

An optical imaging probe was synthesized by attaching a near-infrared carbocyanine fluorophore to an affinity group containing two zinc(II) dipicolylamine (Zn-DPA) units. The probe has a strong and selective affinity for the surfaces of bacteria, and it was used to image infections of Gram-positive S. aureus and Gram-negative E. coli bacteria in living nude mice. After intravenous injection, the probe selectively accumulates at the sites of localized bacterial infections in the thigh muscles of the mice.     

Inherent antibacterial activity of a peptide-based beta-hairpin hydrogel

Among several important considerations for implantation of a biomaterial, a main concern is the introduction of infection. We have designed a hydrogel scaffold from the self-assembling peptide, MAX1, for tissue regeneration applications whose surface exhibits inherent antibacterial activity. In experiments where MAX1 gels are challenged with bacterial solutions ranging in concentrations from 2 x 10(3) colony forming units (CFUs)/dm2 to 2 x 10(9) CFUs/dm2, gel surfaces exhibit broad-spectrum antibacterial activity. Results show that the hydrogel surface is active against Gram-positive (Staphylococcus epidermidis, Staphylococcus aureus, and Streptococcus pyogenes) and Gram-negative (Klebsiella pneumoniae and Escherichia coli) bacteria, all prevalent in hospital settings. Live-dead assays employing laser scanning confocal microscopy show that bacteria are killed when they engage the surface. In addition, the surface of MAX1 hydrogels was shown to cause inner and outer membrane disruption in experiments that monitor the release of beta-galactosidase from the cytoplasm of lactose permease-deficient E. coli ML-35. These data suggest a mechanism of antibacterial action that involves membrane disruption that leads to cell death upon cellular contact with the gel surface. Although the hydrogel surface exhibits bactericidal activity, co-culture experiments indicate hydrogel surfaces show selective toxicity to bacterial versus mammalian cells. Additionally, gel surfaces are nonhemolytic toward human erythrocytes, which maintain healthy morphologies when in contact with the surface. These material attributes make MAX1 gels attractive candidates for use in tissue regeneration, even in nonsterile environments.     

金纳米粒子修饰聚合物固相微萃取整体柱的制备及其性能研究

通过在毛细管聚合物整体柱表面修饰金纳米粒子,制备了一种可选择性捕获含巯基化合物的固相微萃取整体柱.首先制备聚(甲基丙烯酸缩水甘油酯-乙二醇二甲基丙烯酸酯)整体柱,在其表面化学修饰半胱胺;通过半胱胺上的巯基基团将金纳米粒子固定在整体柱的孔表面.以巯基类化合物为探针,评价了固相微萃取整体柱的萃取性能.结果表明,由于Au对巯...     

基于正电荷和光热协同效应的新型半导体聚合物纳米抗菌材料

由于抗生素的不当使用和细菌多药耐药的出现, 迫切需要开发新的抗菌剂. 本文制备了具有光热转换性能的正电荷半导体高分子材料及具有协同抗菌活性的半导体聚合物纳米粒子(SP-PPh₃ NPs). SP-PPh₃ NPs的光热转化效率为43.8%. 带正电荷的SP-PPh₃ NPs可以附着在细菌上, 有助于将热量有效传递给细菌. 在热和正电荷的协同作用下, SP-PPh₃ NPs对革兰氏阴性大肠杆菌(E. coli)和革兰氏阳性金黄色葡萄球菌(S. aureus)均具有抗菌活性, 其对二者的体外抑菌率分别为99.9%和98.6%. 此外, SP-PPh₃ NPs具有良好的生物相容性, 对小鼠的主要器官几乎无副作用. 对细菌感染的小鼠皮肤伤口用SP-PPh₃ NPs治疗12 d后, 伤口可以很好地愈合.     

Recent advances in the control of morphology and surface chemistry of porous polymer-based monolithic stationary phases and their application in CEC

This review focuses on developments in the field of polymer-based monolithic columns for CEC published in the literature since the beginning of the year 2005. The possibility of in-situ preparation as well as easy control over their porous properties and surface chemistries clearly make monolithic separation media an attractive alternative to capillary columns packed with particles. Different variables such as polymerization conditions, morphology, and surface chemistry are shown to directly affect performance of monolithic capillary columns in terms of efficiency, analysis time, and retention.     

Using flow to switch the valency of bacterial capture on engineered surfaces containing immobilized nanoparticles

Toward an understanding of nanoparticle-bacterial interactions and the development of sensors and other substrates for controlled bacterial adhesion, this article describes the influence of flow on the initial stages of bacterial capture (Staphylococcus aureus) on surfaces containing cationic nanoparticles. A PEG (poly(ethylene glycol)) brush on the surface around the nanoparticles sterically repels the bacteria. Variations in ionic strength tune the Debye length from 1 to 4 nm, increasing the strength and range of the nanoparticle attractions toward the bacteria. At relatively high ionic strengths (physiological conditions), bacterial capture requires several nanoparticle-bacterial contacts, termed "multivalent capture". At low ionic strength and gentle wall shear rates (on the order of 10 s(-1)), individual bacteria can be captured and held by single surface-immobilized nanoparticles. Increasing the flow rate to 50 s(-1) causes a shift from monovalent to divalent capture. A comparison of experimental capture efficiencies with statistically determined capture probabilities reveals the initial area of bacteria-surface interaction, here about 50 nm in diameter for a Debye length κ(-1) of 4 nm. Additionally, for κ(-1) = 4 nm, the net per nanoparticle binding energies are strong but highly shear-sensitive, as is the case for biological ligand-receptor interactions. Although these results have been obtained for a specific system, they represent a regime of behavior that could be achieved with different bacteria and different materials, presenting an opportunity for further tuning of selective interactions. These finding suggest the use of surface elements to manipulate individual bacteria and nonfouling designs with precise but finite bacterial interactions.     

Miniaturized bead-beating device to automate full DNA sample preparation processes for gram-positive bacteria

We have developed a miniaturized bead-beating device to automate nucleic acids extraction from Gram-positive bacteria for molecular diagnostics. The microfluidic device was fabricated by sandwiching a monolithic flexible polydimethylsiloxane (PDMS) membrane between two glass wafers (i.e., glass-PDMS-glass), which acted as an actuator for bead collision via its pneumatic vibration without additional lysis equipment. The Gram-positive bacteria, S. aureus and methicillin-resistant S. aureus, were captured on surface-modified glass beads from 1 mL of initial sample solution and in situ lyzed by bead-beating operation. Then, 10 μL or 20 μL of bacterial DNA solution was eluted and amplified successfully by real-time PCR. It was found that liquid volume fraction played a crucial role in determining the cell lysis efficiency in a confined chamber by facilitating membrane deflection and bead motion. The miniaturized bead-beating operation disrupted most of S. aureus within 3 min, which turned out to be as efficient as the conventional benchtop vortexing machine or the enzyme-based lysis technique. The effective cell concentration was significantly enhanced with the reduction of initial sample volume by 50 or 100 times. Combination of such analyte enrichment and in situ bead-beating lysis provided an excellent PCR detection sensitivity amounting to ca. 46 CFU even for the Gram-positive bacteria. The proposed bead-beating microdevice is potentially useful as a nucleic acid extraction method toward a PCR-based sample-to-answer system.     

一种聚甲基丙烯酸酯类亲水作用毛细管电色谱整体柱的制备与评价

以2-羟基乙基甲基丙烯酸酯(HEMA)为单体,乙二醇二甲基丙烯酸酯(EDMA)为交联剂,制备了亲水分离模式的聚HEMA-co-EDMA新型毛细管电色谱(CEC)整体柱。考察了整体柱的结构特征及其CEC性能,研究了极性物质的保留行为,并对其可能的保留机理进行了探讨。所制备的整体柱稳定性好,固定相表面带有极性羟基功能团,不仅能提供亲水相互作用位点,且能吸附流动相中的阴离子产生阳极电渗流(EOF)。在流动相中乙腈含量较高(＞62%,体积分数)的条件下,整体柱表现出典型的亲水作用,实现了对核苷、碱基和苯胺类带有碱性的强极性化合物的高效分离,并成功分离了苯酚类、苯甲酸类等中性或酸性的极性化合物。     

Structural and biological behaviors of some nonionic Schiff-base amphiphiles and their Cu(II) and Fe(III) metal complexes

Novel series of nonionic Schiff bases was synthesized and characterized using microelemental analysis, FTIR and (1)H NMR spectra. These Schiff bases and their complexes with Cu and Fe have been evaluated for their antibacterial activity against bacterial species such as Staphylococcus aureus, Pseudomonas aureus, Candida albi, Bacillus subtilis and Escherichia coli and their fungicidal activity against Aspcrgillus niger and Aspcrgillus flavus. The results of the biocidal activities showed high potent action of the synthesized Schiff bases towards both bacteria and fungi. Furthermore, complexation of these Schiff bases by Cu(II) and Fe(III) show the metal complexes to be more antibacterial and antifungal than the Schiff bases. The results were correlated to the surface activity and the transition metal type. The mode of action of these complexes was discussed.     

Capillary electrochromatography of peptides on a neutral porous monolith with annular electroosmotic flow generation

A new kind of monolithic capillary column was prepared for capillary electrochromatography (CEC) with a positively charged polymer layer on the inner wall of a fused-silica capillary and a neutral monolithic packing as the bulk stationary phase. The fused-silica capillary was first silanized with 3-glycidoxypropyltrimethoxysilane (GPTMS). Polyethyleneimine (PEI) was then covalently bonded to the GPTMS coating to form an annular positively charged polymer layer for the generation of electroosmotic flow (EOF). A neutral bulk monolithic stationary phase was then prepared by in situ copolymerization of vinylbenzyl chloride (VBC) and ethylene glycol dimethacrylate in the presence of 1-propanol and formamide as porogens. Benzyl chloride functionalities on the monolith were subsequently hydrolyzed to benzyl alcohol groups. Effects of pH on the EOF mobility of the column were measured to monitor the completion of reactions. Using a column with this design, we expected general problems in CEC such as irreversible adsorption and electrostatic interaction between stationary phase and analytes to be reduced. A peptide mixture was successfully separated in counter-directional mode CEC. Comparison of peptide separations in isocratic monolithic CEC, gradient HPLC and capillary zone electrophoresis (CZE) indicated that the separation in CEC is governed by a dual mechanism that involves a complex interplay between selective chromatographic retention and differential electrophoretic migration.     

Detection of Escherichia coli O157:H7 bacteria by a combination of immunofluorescent staining and capillary electrophoresis

As the number of incidents of bacterial infections continues to rise around the globe, simpler, faster, and more sensitive diagnostic techniques are required to improve the safety of the food supply and to screen for potential bacterial infections in humans. We present here direct and indirect approaches for the detection of bacteria, which are based upon a combination of immunofluorescent staining and capillary electrophoresis. In the direct approach, Escherichia coli O157:H7 bacteria stained with fluorescein-tagged specific antibodies are detected by CE, while in the indirect approach fluorescein-tagged specific antibodies to E. coli are first captured by E. coli O157:H7 bacteria and then released and detected by CE. We have identified suitable bacteria staining and CE protocols, which involved a 10 mM Tris-borate-EDTA (TBE) buffer, 0.25 micro g antibody/1 million bacteria, and capillaries dynamically coated with poly-N-hydroxyethylacrylamide (polyDuramide). We have also successfully detected the presence of E. coli O157:H7 in contaminated meat. The total time required for analysis was 6-8 h, which is less than that realized in most commercial assays presently available.     

Rapid identification of Escherichia coli and Helicobacter pylori in biological samples by capillary zone electrophoresis

The differences in surface charge of different bacteria can be exploited for their separation by capillary electrophoresis. However, this method of separation of microorganisms is beset with various drawbacks such as adhesion of bacteria to the fused silica surface or cluster formation. To overcome these phenomena we investigated the addition of poly(ethylene oxide) as a focusing agent to the running buffer and used calcium and myoinositol hexakisphosphate as specific ions that interact with the bacterial surface, changing its electrical properties and electrophoretic mobilities. In the present work, we applied CZE to identification of E. coli in infected urine (direct injection) from patients with urinary tract infections and to identification of Helicobacter pylori, which is a gram-negative bacillus responsible for one of the most common infections found in humans worldwide. Helicobacter pylori colonize the stomach and are responsible for severe diseases of the gastric tract, ranging from chronic gastric ulcer to gastric cancer.     

Rapid detection of Staphylococcus aureus by a combination of monoclonal antibody-coated latex and capillary electrophoresis

The rapid detection of pathogenic bacteria is extremely important in biotechnology and clinical diagnosis. CE has been utilized in the field of bacterial analysis for many years, but to some extent, simultaneous separation and identification of certain microbes from complex samples by CE coupled with UV detector is still a challenge. In this paper, we propose a new strategy for rapid separation and identification of Staphylococcus aureus (S. aureus) in bacterial mixtures by means of specific mAb-coated latex coupled with CZE. An appropriate set of conditions that selectively isolated S. aureus from the microorganisms Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were established. S. aureus could be differentiated from the others by unique peaks in the electropherograms. The validity was also confirmed by LIF with antibodies specific to both the latex and the microbial cells. The LOD is as low as 9.0 x 10(5) colony forming unit/mL. We have also utilized this technology to identify S. aureus in a stool sample coming from a healthy volunteer spiked successfully with S. aureus. This CZE-UV technique can be applied to rapid diagnosis of enteritis caused by S. aureus or other bacterial control-related fields needing rapid identification of target pathogens from microbial mixtures. In theory, this method is suitable for the detection of any bacterium as long as corresponding bacterium-specific antibody-coated latex is available.     

Polymethacrylate monolithic columns for capillary liquid chromatography

In recent years, continuous separation media have attracted considerable attention because of the advantages they offer over packed columns. This research resulted in two useful monolithic material types, the first based on modified silica gel and the second on organic polymers. This work attempts to review advances in the development, characterization, and applications of monolithic columns based on synthetic polymers in capillary chromatography, with the main focus on monolithic beds prepared from methacrylate-ester based monomers. The polymerization conditions used in the production of polymethacrylate monolithic capillary columns are surveyed, with attention being paid to the concentrations of monomers, porogen solvents, and polymerization initiators as the system variables used to control the porous and hydrodynamic properties of the monolithic media. The simplicity of their preparation as well as the possibilities of controlling of their porous properties and surface chemistries are the main benefits of the polymer monolithic capillary columns in comparison to capillary columns packed with particulate materials. The application areas considered in this review concern mainly separations in reversed-phase chromatography, ion-exchange chromatography, hydrophobic and hydrophilic interaction modes; enzyme immobilization and sample preparation in the capillary chromatography format are also addressed.     

Robust and Self-healable Antibiofilm Multilayer Coatings

The infection induced by implantation of biomedical materials may result from the biofilm formation after bacteria attachment.Hence, the antibiofilm surface coating represents a novel technique to improve the antibacterial activity of biomedical materials. The traditional antibiofilm surface coatings exhibited some disadvantages and provided a limited service life. In this work, we used polyethyleneimine grafted 3-maleimidopropionic acid(PEIM) and poly(acrylic acid) grafted 2-furfurylamine(PAAF) to achieve robust and self-healable crosslinked multilayer coatings, employing Layer-by-Layer(LbL) self-assembly technique and Diels-Alder reaction. Then, thiol-terminated poly((3-acrylamidopropyl)trimethylammonium chloride)(PAMPTMA-SH) was grafted onto the crosslinked multilayer coating by thiol-ene click reaction to form a novel multilayer coating(PEIM/PAAF)_(10)-PAMPTMA. We found that this coating showed robust and self-healable activity, and significantly inhibited the bacterial growth and biofilm formation after infection with Escherichia coli(E. coli) and Staphylococcus aureus(S. aureus) by in vitro and in vivo assays for 120 h. In addition, the multilayer coating did not induce significant hemolysis or affect the cell viability of red blood cells. In vivo studies also showed that(PEIM/PAAF)_(10)-PAMPTMA coating efficiently blocked the infiltration of inflammatory cells and gene expression in the mouse skin challenged with E. coli or S. aureus. Taken together, these results showed that the prepared multilayer coating exhibited strong antibiofilm activity and provided a new strategy for the application of highly efficient antibiofilm surface coating of biomedical materials.     

应用于全氟辛烷磺酸萃取富集的全氟癸基修饰毛细管硅胶整体柱的制备及应用

利用溶胶-凝胶法,经过烷氧基硅烷的水解、硅羟基的缩聚、凝胶化、陈化、中孔制备、干燥和表面修饰等步骤制备了全氟癸基修饰的毛细管硅胶整体柱。采用该整体柱对全氟辛烷磺酸(PFOS)进行萃取富集,考察其富集特性和效率,并与传统的C18毛细管硅胶整体柱进行对比。结果表明,全氟癸基修饰毛细管硅胶整体柱(15 cm×75 μm)对PFOS具有更高的吸附量和更好的富集选择性,其平均吸附量可以达到75 ng;样品中PFOS的质量浓度为0.25 mg/L时,富集倍数平均可以达到29倍。此全氟癸基修饰毛细管硅胶整体柱对PFOS具有良好的萃取富集性能,可用于水质中痕量PFOS的萃取富集。