Characterization of cellulose produced by Salmonella enterica serovar Typhimurium

Distinct bacterial species belonging to the family of enterobacteriaceae produce cellulose as an exopolysaccharide. Cellulose produced by Salmonella enterica serovar Typhimurium was isolated by various methods. Subsequently, transmission electron microscopy (TEM) studies of the isolated material revealed novel structural details of enterobacterial cellulose.     

The role of the liquid crystalline state in the bundling of Salmonella enterica serovar Typhimurium flagella

Salmonella bacteria owe their motility to the rotation of bundled protein filaments known as flagella. While the method by which flagella impart motility is known, there is a scarcity of data elucidating the critical process of flagellum bundling itself. This work hypothesises the process of flagellum bundling to be an energetically driven phenomenon in which a physical state transition drives the formation of the flagellum bundle at the surface of a Salmonella cell. In vitro analysis of intact flagella, detached and purified from Salmonella enterica serovar Typhimurium cells, with cross-polarised light microscopy demonstrates a transition from an isotropic to mesophasic suspension at physiologically relevant concentrations. We believe the state transition of flagellum suspensions to the liquid crystalline state directs the formation of the flagellum bundle and thus plays a role in Salmonella motility that, until this point, has been sparsely investigated.     

Gold nanorods as a perspective technology platform for SERS analytics

We discuss the application of gold nanorods for forming SERS substrates for chemical and biological sensing. Two approaches are considered: (1) formation of planar arrays on silicon wafers by using suspensions of gold nanorods; and (2) a new approach based on gold nanorod powders that can be easily dissolved in aqueous media. Both SERS platforms are characterized and their SERS enhancement factors are compared.     

A hydrogel-based versatile screening platform for specific biomolecular recognition in a well plate format

Precise determination of biomolecular interactions in high throughput crucially depends on a surface coating technique that allows immobilization of a variety of interaction partners in a non-interacting environment. We present a one-step hydrogel coating system based on isocyanate functional six-arm poly(ethylene oxide)-based star polymers for commercially available 96-well microtiter plates that combines a straightforward and robust coating application with versatile bio-functionalization. This system generates resistance to unspecific protein adsorption and cell adhesion, as demonstrated with fluorescently labeled bovine serum albumin and primary human dermal fibroblasts (HDF), and high specificity for the assessment of biomolecular recognition processes when ligands are immobilized on this surface. One particular advantage is the wide range of biomolecules that can be immobilized and convert the per se inert coating into a specifically interacting surface. We here demonstrate the immobilization and quantification of a broad range of biochemically important ligands, such as peptide sequences GRGDS and GRGDSK-biotin, the broadly applicable coupler molecule biocytin, the protein fibronectin, and the carbohydrates N-acetylglucosamine and N-acetyllactosamine. A simplified protocol for an enzyme-linked immunosorbent assay was established for the detection and quantification of ligands on the coating surface. Cell adhesion on the peptide and protein-modified surfaces was assessed using HDF. All coatings were applied using a one-step preparation technique, including bioactivation, which makes the system suitable for high-throughput screening in a format that is compatible with the most routinely used testing systems.     

SERS as tool for the analysis of DNA-chips in a microfluidic platform

A sequence-specific detection method of DNA is presented combining a solid chip surface for immobilisation of capture DNAs with a microfluidic platform and a readout of the chip based on SERS. The solid chip surface is used for immobilisation of different capture DNAs, where target strands can be hybridised and unbound surfactants can be washed away. For the detection via SERS, short-labelled oligonucleotides are hybridised to the target strands. This technique is combined with a microfluidic platform that enables a fast and automated preparation process. By applying a chip format, the problems of sequence-specific DNA detection in solution phase by means of SERS can be overcome. With this setup, we are able to distinguish between different complementary and non-complementary target sequences in one sample solution.     

Gold nanothorns-macroporous silicon hybrid structure: a simple and ultrasensitive platform for SERS

We synthesised a novel gold-on-porous silicon hybrid material that exhibits a highly sensitive and reproducible surface-enhanced Raman spectroscopy (SERS) response. The material was fabricated simply by reducing gold chloride with hydrofluoric acid on the surface of macro-porous silicon (macro-PSi). The material consists of thorn-shaped gold nanocrystals with characteristic shapes and sizes on the surface of macro-PSi.     

Porous functionalised silica particles: a potential platform for biomolecular screening

Novel, porous, functionalised silica particles have been developed with controlled morphology, which promote covalent attachment of fluorescent dyes which can act as an optical barcode.     

Fluorescent aptasensor for the determination of Salmonella typhimurium based on a graphene oxide platform

We report on an aptamer with high affinity against Salmonella typhimurium (S. typhimurium) and selected from an enriched oligonucleotide pool by a whole-cell SELEX process in a method for the fluorimetric determination of S. typhimurium using a graphene oxide platform. In the absence of target, the fluorescence was fairly weak as result of the FAM-labeled aptamer adjacent to graphene oxide. If, however, the fluorophore is released from the graphene oxide due to the formation of the target/aptamer complexes, fluorescence intensity is substantially increased. Under the optimum conditions, the assay displays a linear response to bacteria in the concentration range from 1?×?10³ to 1?×?10⁸ CFU·mL^?1, with a detection limit of 100 CFU·mL^?1. The method is selective in that fluorescence is not much enhanced in case of other bacteria. This aptasensor displays higher sensitivity and selectivity than others and is believed to possess a large potential with respect to the rapid detection of bacteria.

Microfluidic channel with embedded SERS 2D platform for the aptamer detection of ochratoxin A

A selective aptameric sequence is adsorbed on a two-dimensional nanostructured metallic platform optimized for surface-enhanced Raman spectroscopy (SERS) measurements. Using nanofabrication methods, a metallic nanostructure was prepared by electron-beam lithography onto a glass coverslip surface and embedded within a microfluidic channel made of polydimethylsiloxane, allowing one to monitor in situ SERS fingerprint spectra from the adsorbed molecules on the metallic nanostructures. The gold structure was designed so that its localized surface plasmon resonance matches the excitation wavelength used for the Raman measurement. This optofluidic device is then used to detect the presence of a toxin, namely ochratoxin-A (OTA), in a confined environment, using very small amounts of chemicals, and short data acquisition times, by taking advantage of the optical properties of a SERS platform to magnify the Raman signals of the aptameric monolayer system and avoiding chemical labeling of the aptamer or the OTA target.

Silica encapsulated SERS nanoprobe conjugated to the bacteriophage tailspike protein for targeted detection of Salmonella

Silica-encapsulated Raman-reporter embedded SERS nanoprobes, named nanoaggregate embedded beads (NAEBs), were conjugated to the Salmonella specific tailspike protein (TSP) isolated from the P22 bacteriophage to enable a highly specific and ultrasensitive optical transduction platform. We demonstrate three successful surface conjugation strategies and highlight the detection of a single bacterium using SERS.     

Development of a LIBS assay for the detection of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium from food

Laser-induced breakdown spectroscopy (LIBS) is used for the identification of the presence of hazardous bacteria in food. In this study, our main focus was centered on the identification of S. enterica serovar Typhimurium, a Gram-negative foodborne pathogen, in various liquids such as milk, chicken broth, and brain heart infusion due to the infection being most prevalent in raw meat and dairy products. A Nd:YAG laser of operating wavelength 266 nm was used to obtain the spectra from the artificially inoculated liquid samples. A series of experiments were performed to determine the effectiveness of LIBS to discriminate the bacteria from the background liquids. These results are compared with competing modern molecular methods of detection which include polymerase chain reaction (PCR) and quantitative real-time PCR. In addition to analyzing S. enterica serovar Typhimurium, another common Gram-negative, Escherichia coli, as well as Gram-positive pathogen, Staphlycoccus auerus, were used to determine the specificity of the LIBS technique.     

Development of a protease production platform for structure-based drug design

Structure-based drug design (SBDD) has played an integral role in the development of highly specific, potent protease inhibitors resulting in a number of drugs in clinical trials and on the market. Possessing biochemical assays and structural information on both the target protease and homologous family members helps ensure compound selectivity. We have redesigned the path from clone to protein eliminating many of the traditional bottlenecks associated with protein production to ensure a constant supply to feed many diverse protease drug discovery programs. The process was initiated with the design of a multi-system vector, capable of expression in both eukaryotic and prokaryotic hosts; this vector also facilitated high-throughput cloning, expression and purification. When combined into an expression screen, supplemented with salvage screens for detergent extraction and refolding, a route for protein production was established rapidly. Using this process-orientated approach we have successfully expressed and purified all mechanistic classes of active human and viral proteases for enzymatic assays and crystallization studies. While exploiting recent developments in high-throughput biochemistry, we still employ classical biophysical techniques such as light-scattering and analytical ultracentrifugation, to ensure the highest quality protein enters crystallization trials. We have drawn on examples from our own research programs to illustrate how these strategies have been successfully used in the production of proteases for SBDD.     

High throughput screening platform for new inhibitors of protein biosynthesis

The search for new antibiotics is an important task, which is of interest for both basic research and health care practices. It is essential to elucidate the mechanism of antimicrobial action during the screening for antimicrobial activity and at the same time be able to test thousands of compounds. A robotic screening system for potential antibiotics developed at the Department of Chemistry at Moscow State University has been described that enables the immediate identification of those that inhibit protein biosynthesis.     

Chemical genetics: budding yeast as a platform for drug discovery and mapping of genetic pathways

The budding yeast Saccharomyces cerevisiae is a widely used model organism, and yeast genetic methods are powerful tools for discovery of novel functions of genes. Recent advancements in chemical-genetics and chemical-genomics have opened new avenues for development of clinically relevant drug treatments. Systematic mapping of genetic networks by high-throughput chemical-genetic screens have given extensive insight in connections between genetic pathways. Here, I review some of the recent developments in chemical-genetic techniques in budding yeast.     

High throughput automated chromatin immunoprecipitation as a platform for drug screening and antibody validation

Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein-DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR results only takes one day using HTChIP, as compared to several days up to one week for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. We performed a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell, and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, we believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency, and further facilitate the use of ChIP as a valuable tool for research and discovery.     

Cell-based screening: a high throughput flow cytometry platform for identification of cell-specific targeting molecules

Targeting of drugs and genes to specific cell types is an emerging paradigm in the treatment of many medical conditions. However, targeting structures such as peptides are susceptible to rapid inactivation in vivo. To address this problem, novel targeting molecules can now be rapidly synthesized using a combinatorial approach. Methods to screen the large libraries created in this process are often lacking or compatible only with solution-based screening. This report describes a high-throughput cell-based method utilizing flow cytometry, capable of rapidly screening large libraries of molecules simultaneously for biological functionality and stability. In this method, each library molecule is attached to a microsphere exhibiting a unique set of optical properties, or "fingerprint", conferring modularity and multiplex capability. We investigated the multiplex capability of our flow cytometric method to determine its capacity for high-throughput screening. Current instrumentation in our laboratory allows the screening of at least 75 unique compounds in a single well, a number comparable to available solution-based assays. In state-of-the-art configuration, however, this methodology can support the screening of up to 1875 compounds per well, achieving high-throughput potential in a single multiwell plate. We also investigated the binding capability of targeted microspheres to adherent target cells. These microspheres exhibited a 12-fold increase in binding over control, untargeted microspheres. Competitive inhibition experiments with soluble ligand confirmed the specificity of microsphere binding. Overall, the methodology proposed here is capable of quickly and effectively screening large libraries of targeting molecules using instrumentation readily available to the greater research community.     

Development of a Flow Immunoassay System for the Detection of Salmonella Typhimurium

Abstract

A flow immunoassay system has been developed for the detection of Salmonella typhimurium by immobilization of antibody to Salmonella onto tygon tubing by adsorption and onto polyethylene tubing, by covalent binding. After liquid containing Salmonella cells or Salmonella antigen was allowed to interact with the immobilized. antibody, glucose oxidase labeled antibody to Salmonella was injected to the system. The bound antigen was quantified by electrochemical detection of H₂O₂ produced from the enzymic oxidation of glucose to gluconic acid. In view of the low non-specific binding, sensitivity and reusability, the polyethylene immunoreactor provided a better performance. The system was capable of detecting as low as 10⁵ ? 10⁶ Salmonella cells/ml in less than 10 minutes. The immunoreactor could be regenerated for at least 50 times during one month of the experiment. The detection limit of the tygon immunoreactor was somewhat higher (10⁶ ? 10⁹ cells/ml). Even though the anti-Salmonella antibody was easily immobilized on the tygon tubing, the system was not reusabl and exhibited a very high non-specific binding.     

Development of a liposome-based immunochromatographic strip assay for the detection of Salmonella

Salmonella species are ubiquitous human pathogens which pose a dangerous threat to the elderly and children worldwide. In this study, to develop a more efficient assay procedure for the rapid detection of Salmonella Typhimurium, an immunochromatographic strip assay was developed using immunoliposome (anti-Salmonella IgG-tagged) encapsulated with sulforhodamine B (SRB). The detection sensitivity of the developed immunochromatographic assay was compared with a commercial immunochromatographic test strip using colloidal gold nanoparticles. The liposomes were prepared through a reverse-phase evaporation method by using a lipid and phospholipid mixture and SRB, a fluorescence dye, which was encapsulated in the phospholipid bilayer. Furthermore, the outer surface of the SRB-encapsulated liposome was conjugated with antibody (affinity-purified polyclonal goat anti-Salmonella IgG) to form an immunoliposome (size, 223 nm), used as the analytical reagent in the developed immunoassay. For this strip assay, a plastic-backed nitrocellulose strip was immobilized with two antibody zones. The lower zone of the strip referred to Salmonella antigen capture zone (test line), while the other zone served as a positive control (control line). The lower zone was coated with affinity-purified polyclonal goat anti-Salmonella IgG, while the upper zone was coated with rabbit anti-goat IgG. During capillary migration of the wicking solution (diluted liposome and Salmonella culture, each 50 μl), Salmonella was captured with surface-bound immunoliposomes at the antigen capture zone, while the unbound liposomes migrated upward and bound to another zone. The color density of the antigen capture zone was directly proportional to the amount of S. Typhimurium in the test sample. As a result, the detection limit of the immunochromatographic strip assay developed in this study against S. Typhimurium was found to be 10² CFU/ml, which was significantly higher than the detection limit (10⁷ CFU/ml) of the commercial immunochromatographic test strip assay.