Novel PCR assay for identification of Salmonella enterica serovar Infantis

AIMS: We developed, optimized and tested two novel PCR assays specific for Salmonella enterica subspecies enterica serovar Infantis. METHODS AND RESULTS: The fljB gene was chosen as the target sequence. Primers were designed on a consensus sequence built by sequencing the fljB gene of five genetically unrelated Hungarian S. Infantis strains and using sequence data from the GenBank (http://www.ncbi.nih.gov). Two alternative assays were designed, which share the reverse primer. Both proved to be highly specific to S. Infantis, neither reacted with 42 other nontyphoidal serovariants tested. The detection limit of the assays was determined to be 10(5) CFU ml(-1) from pure culture, and 10(6) CFU g(-1) from artificially spiked chicken faeces samples. CONCLUSIONS: Although the detection limit is rather high to allow for using them for direct detection, the assays may be useful in identification of S. Infantis both for diagnostic and for research purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: The described PCR assays allow for the correct identification of S. Infantis even when traditional serotyping methods fail because lack of expression of flagellar antigens.     

Sensing small neurotransmitter-enzyme interaction with nanoporous gated ion-sensitive field effect transistors

Ion-sensitive field effect transistors with gates having a high density of nanopores were fabricated and employed to sense the neurotransmitter dopamine with high selectivity and detectability at micromolar range. The nanoporous structure of the gates was produced by applying a relatively simple anodizing process, which yielded a porous alumina layer with pores exhibiting a mean diameter ranging from 20 to 35 nm. Gate-source voltages of the transistors demonstrated a pH-dependence that was linear over a wide range and could be understood as changes in surface charges during protonation and deprotonation. The large surface area provided by the pores allowed the physical immobilization of tyrosinase, which is an enzyme that oxidizes dopamine, on the gates of the transistors, and thus, changes the acid-base behavior on their surfaces. Concentration-dependent dopamine interacting with immobilized tyrosinase showed a linear dependence into a physiological range of interest for dopamine concentration in the changes of gate-source voltages. In comparison with previous approaches, a response time relatively fast for detecting dopamine was obtained. Additionally, selectivity assays for other neurotransmitters that are abundantly found in the brain were examined. These results demonstrate that the nanoporous structure of ion-sensitive field effect transistors can easily be used to immobilize specific enzyme that can readily and selectively detect small neurotransmitter molecule based on its acid-base interaction with the receptor. Therefore, it could serve as a technology platform for molecular studies of neurotransmitter-enzyme binding and drugs screening.     

Protein immunosensor using single-wall carbon nanotube forests with electrochemical detection of enzyme labels

Vertically aligned arrays of single-wall carbon nanotubes (SWNT forests) on pyrolytic graphite surfaces were developed for amperometric enzyme-linked immunoassays. Improved fabrication of these SWNT forests utilizing aged nanotube dispersions provided higher nanotube density and conductivity. Biosensor performance enhancement was monitored using nanotube-bound peroxidase enzymes showing a 3.5-fold better sensitivity for H2O2 than when using fresh nanotubes to assemble the forests, and improved detection limits. Absence of improvements by electron mediation for detection of H2O2 suggested very efficient electron exchange between nanotubes and enzymes attached to their ends. Protein immunosensors were made by attaching antibodies to the carboxylated ends of nanotube forests. Utilizing casein/detergent blocking to minimize non-specific binding, a detection limit of 75 pmol mL(-1) (75 nM) was achieved for human serum albumin (HSA) in unmediated sandwich immunosensors using horseradish peroxidase (HRP) labels. Mediation of the immunosensors dramatically lowered the detection limit to 1 pmol mL(-1) (1 nM), providing significantly better performance than alternative methods. In the immunosensor case, the average distance between HRP labels and nanotube ends is presumably too large for efficient direct electron exchange, but this situation can be overcome by electron mediation.     

Comparison of Salmonella enterica serotype Infantis isolates from a veterinary teaching hospital

AIMS: To compare Salmonella enterica serotype Infantis isolates obtained from patients or the environment of a veterinary teaching hospital over a period of 9 years following a nosocomial outbreak to determine whether isolates were epidemiologically related or represented unrelated introductions into the hospital environment. METHODS AND RESULTS: Fifty-six S. Infantis isolates were compared based on their phenotypic (antimicrobial drug [AMD] susceptibility pattern) and genotypic (pulsed-field gel electrophoresis [PFGE] pattern and presence of integrons) characteristics. Epidemiologically unrelated S. Infantis isolates clustered separately from all but two of the hospital isolates, and several isolates from different years and various sources were indistinguishable from each other in cluster analysis of two-enzyme PFGE results. A high percentage of isolates (80.3%) were resistant to at least one AMD, with 67.8% showing resistance to >5 AMD. The majority (74.1%) of isolates tested contained type 1 integrons. CONCLUSION: Results strongly suggest that there was nosocomial transmission of S. Infantis during the initial outbreak, and that contamination arising from this outbreak persisted across years despite rigorous hygiene and biosecurity precautions and may have led to subsequent nosocomial infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Evidence of persistence and transmission of Salmonella clones across years, even in the face of rigorous preventive measures, has important implications for other facilities that have experienced outbreaks of Salmonella infections.     

Fabrication of stable antibody-modified field effect transistors using electrical activation of Schiff base cross-linkages for tumor marker detection

In this paper, we present a method of fabricating a rigid antibody-immobilized surface using electric activation of a glutaraldehyde (GA)-modified aminopropylsilyl surface for stable antibody-modified field effect transistors (FETs). Electric activation of the GA-modified gate surface of the FET reduces Schiff bases, which are easily hydrolyzed and collapsed, formed between GA and 3-aminopropyltriethoxysilane, resulting in preventing the immobilized antibodies from desorbing from the surface. The lack of Raman peaks that could be assigned to a Schiff base after the electrical activation of the GA-modified surface indicated that the electric activation had reduced the Schiff base. The use of the antibody-modified FETs has three advantages for the detection of antigens: increased sensitivity, distinct recognition ability, and improved reproducibility. A tumor marker, alpha-fetoprotein (AFP), was quantitatively detected up to a concentration of 10 ng/mL using the antibody-modified FET. The detection ability of the FET accomplished a cutoff value of hepatic cancer. The quantitative detection of AFP in a solution with contaminating proteins was also demonstrated. This electric activation method is applicable to other antibody-modified FETs.     

Electrochemical behavior of L-cysteine and its detection at carbon nanotube electrode modified with platinum

The electrochemical behavior of L-cysteine (CySH) on platinum (Pt)/carbon nanotube (CNT) electrode was investigated by cyclic voltammetry. CNTs used in this study were grown directly on graphite disk by chemical vapor deposition. Pt was electrochemically deposited on the activated CNT/graphite electrode by electroreduction of Pt(IV) complex ion on the surface of CNTs. Among graphite, CNT/graphite, and Pt/CNT electrodes, improved electrochemical behavior of CySH oxidation was found with Pt/CNT electrode. On the other hand, a sensitive CySH sensor was developed based on Pt/CNT/graphite electrode. A linear calibration curve can be observed in the range of 0.5 microM-0.1 mM. The detection limit of the Pt/CNT electrode is 0.3 microM (signal/nose=3). Effects of pH, scan rate, and interference of other oxidizable amino acids were also investigated and discussed. Additionally, the reproducibility, stability, and applicability of the Pt/CNT electrode were evaluated.     

Development and optimisation of biosensors based on pH-sensitive field effect transistors and cholinesterases for sensitive detection of solanaceous glycoalkaloids

Highly sensitive biosensors based on pH-sensitive field effect transistors and cholinesterases for detection of solanaceous glycoalkaloids have been developed, characterised and optimised. The main analytical characteristics of the biosensors developed have been studied under different conditions and an optimal experimental protocol for glycoalkaloids determination in model solution has been proposed. Using such a biosensor and an enzyme reversible inhibition effect, the total potato glycoalkaloids content can be determined within the range of 0.2-100 microM depending on the type of alkaloid, with lowest detection limits of 0.2 microM for alpha-chaconine, 0.5 microM for alpha-solanine and 1 microM for solanidine. The dynamic ranges for the compounds examined show that such biosensors are suitable for a quantitative detection of glycoalkaloids in real potato samples. High reproducibility, operational and storage stability of the biosensor developed have been shown.     

Single-walled carbon nanotube interactions with HeLa cells

This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma – mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOX™ Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.     

A new type of electrochemical immunosensor based on ion-selective field effect transistors

A new type of an electrochemical sensor based on the use of ion-selective field effect transistors (ISFETs) and conjugates of horse radish peroxidase with specific monoclonal antibodies was developed for the express determination of myoglobine in a solution. For this purpose a simple method of covalent immobilization of myoglobine on the surface of ISFET gate was worked out, an appropriate biochemical approach which allowed potentiometrical registration of the peroxidase activity was used, and an immune chemical analysis was accomplished in competitive way. It was shown that the sensitivity of the analysis with the help of the electrochemical immune sensor corresponds to the demands of medical practice to reveal early stages of myocardial infarction. This sensitivity was significantly higher then that which can be obtained by the traditional ELISA-method. Moreover, overall time of the analysis by the immune sensor was almost one order shorter than this by the ELISA-method. It is concluded that the proposed electrochemical immune sensor based on the ISFETs was very perspective for the express analysis of the level of different types of antigens and antibodies.     

A comparison of three molecular typing methods for the discrimination of Salmonella enterica serovar Infantis

Seventy-six epidemiologically unrelated Salmonella enterica serovar Infantis (S. Infantis) isolates were typed by pulsed-field gel electrophoresis (PFGE), multiple amplification of phage loci typing (MAPLT) and multiple-locus variable-number tandem-repeat analysis (MLVA). PFGE, using the restriction endonuclease XbaI, generated 23 different profiles for the 76 isolates (DI=0.848). MAPLT was undertaken using a combination of 11 primer sets based on bacteriophage sequences and generated 28 different profiles (DI=0.938). By contrast, MLVA only produced nine profiles (DI=0.668) with 13 different primer sets, including the five primer sets routinely used for S. Typhimurium typing. Reducing the number of MAPLT primer sets to four still provided a diversity index of 0.838. All three typing methods revealed two distinct lineages of S. Infantis, with most isolates demonstrating genetic traits of either lineage but not both. The results demonstrate that MAPLT can potentially provide greater discrimination and separation of S. Infantis isolates than both PFGE and MLVA. Furthermore, MAPLT data can be generated much more rapidly and with reduced labour input than PFGE and without the need for expensive PFGE electrophoresis equipment, nor does it require capillary sequencing of PCR fragments to accurately determine PCR fragment lengths as is the case with MLVA.     

Rapid detection of Salmonella in field samples by nested polymerase chain reaction

A simple and universal protocol for the rapid detection of Salmonella spp. in various samples using nested polymerase chain reaction (PCR) was developed. The protocol takes advantage of the rapid purification and concentration of Salmonella by centrifugation onto a layer of 60% sucrose solution. DNA was released by treatment with proteinase K and Triton X-100. Even without pre-enrichment, the detection limit for the nested PCR was 10(5) CFU g-1 of faeces. After pre-enrichment, it was possible to detect Salmonella in faeces where their original concentration was approximately 10(2) CFU g-1 of faeces and in meat samples, it was possible to detect Salmonella in samples where their original concentration was less than 10 cells g-1 of minced meat.     

Detection of DNA and proteins using amorphous silicon ion-sensitive thin-film field effect transistors

Amorphous silicon-based ion-sensitive field-effect transistors (a-Si:H ISFETs) are used for the label-free detection of biological molecules. The covalent immobilization of DNA, followed by DNA hybridization, and of the surface adsorption of oligonucleotides and proteins were detected electronically by the a-Si:H ISFET. The ISFET measurements are performed with an external Ag/AgCl microreference electrode immersed in 100mM phosphate buffer electrolyte with pH 7.0. Threshold voltage shifts in the transfer curve of the ISFETs are observed resulting from successive steps of surface chemical functionalization, covalent DNA attachment to the functionalized surface, surface blocking, and hybridization with a complementary target. The surface sensitivity achieved for DNA oligonucleotides is of the order of 1pmol/cm(2). Point-of-zero charge estimations were made for the functionalized surfaces and for the device surface after DNA immobilization and hybridization. The results show a correlation between the changes in the point-of-zero charge and the shift observed in the threshold voltage of the devices. Electronic detection of adsorbed proteins and DNA is also achieved by monitoring the shifts of the threshold voltage of the ISFETs, with a sensitivity of approximately 50nM.     

Peptide nucleic acid-modified carbon nanotube field-effect transistor for ultra-sensitive real-time detection of DNA hybridization

We have developed a nanosensor array composed of carbon nanotube field-effect transistors (CNTFETs) on SiO₂/Si substrates. Unlike previously reported CNTFETs where the recognition event occurred directly on the CNT, in this case, the reverse surface of the substrate was utilized for the biomolecular functionalization. A self-assembled monolayer (SAM) of peptide nucleic acid (PNA) probes associated with the tumor necrosis factor-α gene (TNF-α) was attached onto the gold electrode on the reverse side of the CNTFET device. A time-dependent conductance increase was monitored upon sequential introduction of wild-type (WT) DNA samples through a microfluidic channel of the poly(dimethylsiloxane) (PDMS) chip. High selectivity of PNA probes only toward the full-complementary WT DNA samples enabled rapid and simple discrimination against single-nucleotide polymorphism (SNP) or non-complementary (NC) DNA. Concentration-dependent measurements indicated a limit-of-detection (LOD) of 6.8 fM WT DNA. Our CNTFET-based biochip is a promising candidate for the development of an integrated, high-throughput, portable device for nucleic acid-based diagnostics.     

Molecular detection of invasive species in heterogeneous mixtures using a microfluidic carbon nanotube platform

Screening methods to prevent introductions of invasive species are critical for the protection of environmental and economic benefits provided by native species and uninvaded ecosystems. Coastal ecosystems worldwide remain vulnerable to damage from aquatic species introductions, particularly via ballast water discharge from ships. Because current ballast management practices are not completely effective, rapid and sensitive screening methods are needed for on-site testing of ships in transit. Here, we describe a detection technology based on a microfluidic chip containing DNA oligonucleotide functionalized carbon nanotubes. We demonstrate the efficacy of the chip using three ballast-transported species either established (Dreissena bugensis) or of potential threat (Eriocheir sinensis and Limnoperna fortuneii) to the Laurentian Great Lakes. With further refinement for on-board application, the technology could lead to real-time ballast water screening to improve ship-specific management and control decisions.     

Sensitive detection of an anthrax biomarker using a glassy carbon electrode with a consecutively immobilized layer of polyaniline/carbon nanotube/peptide

Sensitivity of Anthrax protective antigen (PA) detection has been improved by directly immobilizing a PA-specific peptide onto a multi-wall carbon nanotube (MWCNT). The MWCNT was covalently immobilized onto a polyaniline (PANI) electrode, which was prepared via electropolymerization of the aniline monomer onto a glassy carbon electrode (GCE). Then, the PA-specific peptide was covalently immobilized to the MWCNT layer for measurement. When comparing this technique to that of PA immobilization on an insulting self assembled organic layer, the advantages of the MWCNT are clear. The MWCNT sensor resulted in enhanced electron transfer across the sensing layer. The resulting limit of detection (LOD) was 0.4 pM, a 13-fold improvement over that of our previous self-assembled organic layer was used for immobilization of the same peptide. Neither positive nor negative interferences were observed when a sample containing both 100 pM PA and bovine serum albumin (BSA) was measured, indicating good selectivity of the proposed sensor.     

Electric field effect on (6,0) zigzag single-walled aluminum nitride nanotube

Structural, electronic, and electrical responses of the H-capped (6,0) zigzag single-walled aluminum nitride nanotube was studied under the parallel and transverse electric fields with strengths 0-140?×?10(-4)?a.u. by using density functional calculations. Geometry optimizations were carried out at the B3LYP/6-31G* level of theory using a locally modified version of the GAMESS electronic structure program. The dipole moments, atomic charge variations, and total energy of the (6,0) zigzag AlNNT show increases with increase in the applied external electric field strengths. The length, tip diameters, electronic spatial extent, and molecular volume of the nanotube do not significantly change with increasing electric field strength. The energy gap of the nanotube decreases with increases of the electric field strength and its reactivity is increased. Increase of the ionization potential, electron affinity, chemical potential, electrophilicity, and HOMO and LUMO in the nanotube with increase of the applied parallel electric field strengths shows that the parallel field has a much stronger interaction with the nanotube with respect to the transverse electric field strengths. Analysis of the parameters indicates that the properties of AlNNTs can be controlled by the proper external electric field.     

An algebraic equation for the steady-state response of enzyme-pH electrodes and field effect transistors

The response of enzyme-pH electrodes and field effect transistors (pH-ENFETs) is strongly affected by the pH-buffers present in the test solution, the test solution's pH, and the degree of dissociation of the acidic and/or basic products formed in the enzymic reaction. It is shown that a theoretical model for the sensor's steady-state response, which incorporates alt the above factors, leads to a single transcendental equation which provides the sensor's response. However, when the concentration of the analyte at the face of the enzymic film in contact with the pH sensor (C^o_s) is negligible compared to its concentration at the face in contact with the test solution (C^b_s), the transcendental equation reduces to an algebraic equation. This simple equation is independent of the actual kinetics of the enzymic reaction and the diffusion coefficients of the various species. By controlling (i) the Thiele modulus of the enzymic film and (ii) the concentration of the pH-buffer in the test solution, one can design a sensor so as to have C^o_s ⪡ C^b_s. The aforementioned transcendental equation allows one to compute the required values for these two design variables in order to satisfy the above strong inequality. The response behavior predicted by the algebraic equation is in excellent agreement with the experimental data available on penicillinase—pH and urease—pH sensors     

Structural and functional imaging with carbon nanotube AFM probes

Atomic force microscopy (AFM) has great potential as a tool for structural biology, a field in which there is increasing demand to characterize larger and more complex biomolecular systems. However, the poorly characterized silicon and silicon nitride probe tips currently employed in AFM limit its biological applications. Carbon nanotubes represent ideal AFM tip materials due to their small diameter, high aspect ratio, large Young's modulus, mechanical robustness, well-defined structure, and unique chemical properties. Nanotube probes were first fabricated by manual assembly, but more recent methods based on chemical vapor deposition provide higher resolution probes and are geared towards mass production, including recent developments that enable quantitative preparation of individual single-walled carbon nanotube tips [J. Phys. Chem. B 105 (2001) 743]. The high-resolution imaging capabilities of these nanotube AFM probes have been demonstrated on gold nanoparticles and well-characterized biomolecules such as IgG and GroES. Using the nanotube probes, new biological structures have been investigated in the areas of amyloid-beta protein aggregation and chromatin remodeling, and new biotechnologies have been developed such as AFM-based haplotyping. In addition to measuring topography, chemically functionalized AFM probes can measure the spatial arrangement of chemical functional groups in a sample. However, standard silicon and silicon nitride tips, once functionalized, do not yield sufficient resolution to allow combined structural and functional imaging of biomolecules. The unique end-group chemistry of carbon nanotubes, which can be arbitrarily modified by established chemical methods, has been exploited for chemical force microscopy, allowing single-molecule measurements with well-defined functionalized tips.     

Investigation of class 1 integrons and virulence genes in the emergent Salmonella serovar Infantis in Turkey

International Microbiology - The emerging situation of Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) in Turkey was investigated in terms of virulence genes and mobile genetic...