Increasing the sensitivity of Listeria monocytogenes assays: evaluation using ELISA and amperometric detection

An immunosensor for the detection of Listeria monocytogenes was developed. ELISA and amperometric studies were run in parallel to develop a more sensitive and rapid assay for the bacterium. Conditions for the immunosensor were primarily characterised using ELISA. A direct sandwich assay was employed and the affinities of two polyclonal (goat and rabbit) and one monoclonal (mouse) anti-L. monocytogenes antibodies were compared using this format. Owing to low sensitivity being obtained with all antibodies, biotin-avidin amplification and an indirect sandwich assay were employed. The system was then transferred to screen-printed electrodes (SPEs), the primary antibody being immobilised by cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the mode of detection being amperometric. Various parameters (limit of detection, working range, incubation time, cross-reactivity) of the systems were characterised. The effect of direct incubation in milk is also discussed. The final immunosensor had a working range of 1 x 10(6)-1 x 10(3) cells ml-1 and a detection limit of 9 x 10(2) cells ml-1. The assay took about 3.5 h to complete.     

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of anatoxins in cyanobacteria and drinking water

Anatoxin-a (AN) and homoanatoxin-a (HMAN) are potent neurotoxins produced by a number of cyanobacterial species. A new, sensitive liquid chromatography/multiple tandem mass spectrometry (LC/MS(n)) method has been developed for the determination of these neurotoxins. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer in positive ion mode. The [M+H](+) ions at m/z 166 (anatoxin-a) and m/z 180 (homoanatoxin-a) were used as the precursor ions for multiple MS experiments. MS(2)bond;MS(4) spectra displayed major fragment ions at m/z 149 (AN), 163 (HMAN), assigned to [Mbond;NH(3)+H](+); m/z 131 (AN), 145 (HMAN), assigned to [Mbond;NH(3)bond;H(2)O+H](+), and m/z 91 [C(7)H(7)](+). Although the chromatographic separation of these neurotoxins is problematic, reversed-phase LC, using a C(18) Luna column, proved successful. Calibration data for anatoxin-a using spiked water samples (10 mL) in LC/MS(n) modes were: LC/MS (25-1000 microg/L), r(2) = 0.998; LC/MS(2) (5-1000(microg/L), r(2) = 0.9993; LC/MS(3) (2.5-1000 microg/L), r(2) = 0.9997. Reproducibility data (% RSD, N = 3) for each LC/MS(n) mode ranged between 2.0 at 500 microg/L and 7.0 at 10 microg/L. The detection limit (S/N = 3) for AN was better than 0.03 ng (on-column) for LC/MS(3) which corresponded to 0.6 microg/L.     

The influence of strontium substitution in fluorapatite glasses and glass-ceramics

Strontium is often substituted for calcium in order to confer radio-opacity in glasses used for dental cements, biocomposites and bioglass-ceramics. The present paper investigates the influence of substituting strontium for calcium in a glass of the following composition: 4.5SiO₂3Al₂O₃1.5P₂O₅3CaO2CaF₂, having a Ca:P ratio of 1.67 corresponding to calcium fluorapatite (Ca₅(PO₄)₃F). The glasses were characterized by magic angle spinning nuclear magnetic resonance (MAS-NMR), by differential scanning calorimetry (DSC) and X-ray powder diffraction (XRD). The ²⁹Si, ²⁷Al and ³¹P NMR spectra for the glasses with different strontium contents were identical. The ¹⁹F spectra indicated the presence of F-Ca(n) and Al-F-Ca(n) species in the calcium glasses and in the strontium glasses F-Sr(n) and Al-F-Sr(n). It can be concluded that strontium substitutes for calcium with little change in the glass structure as a result of their similar charge to size ratio. The low strontium glasses bulk nucleated to a calcium apatite phase. Intermediate strontium content glasses surface nucleated to a mixed calcium-strontium apatite and the fully strontium substituted glass to strontium fluorapatite.     

Silicone-protein surfactants; stability of water-in-silicone oil emulsions

Small scale water-in-silicone oil emulsions were readily prepared using high speed mixers. Two surfactant systems were studied: a comb-type silicone-polyether surfactant, and a surfactant system employing a mixture of the surface active protein human serum albumin (HSA, in the internal phase) and an alkoxysilane-modified silicone TES-PDMS in the silicone oil (continuous phase). Little difference in particle sizes was noted between the two surfactant types for a given mixing protocol, but dual-blade turbulent mixing led to relatively monodisperse particles of approximately 2–5 m in diameter while high speed Dremel mixers led to bimodal particle distributions. Prior to spontaneous demulsification of the latter emulsions stabilized by HSA/TES-PDMS (the 3225C emulsions remain stable), they proved very difficult to break. The addition of dibutyltin dilaurate to the HSA/TES-PDMS-stabilized emulsions led to catastrophic collapse of the emulsion and formation of a silicone elastomer at the bulk water/oil interface. This makes unlikely the possibility that silicone elastomers, formed by protein-catalyzed crosslinking of the alkoxysilane in albumin/TES-PDMS-stabilized emulsions, are involved in stabilizing the emulsion. The nature of the stabilization of the interface is discussed.     

Highly-functionalised difluorinated cyclohexane polyols via the Diels-Alder reaction: regiochemical control via the phenylsulfonyl group

A difluorinated dienophile underwent cycloaddition reactions with a range of furans to afford cycloadducts which could be processed regio- and stereoselectively via episulfonium ions, generated by the reaction between their alkenyl groups and phenylsulfenyl chloride. The oxabicyclic products were oxidised to the phenylsulfonyl level and ring opened via E1(C)B or reductive desulfonative pathways to afford, ultimately, difluorinated cyclohexene or cyclohexane polyols.     

Interaction of formic and acetic acid with ice surfaces between 187 and 227 K. Investigation of single species- and competitive adsorption

The physical adsorption of formic (HC(O)OH) and acetic (CH(3)C(O)OH) acid on ice was measured as a function of concentration and temperature. At low concentrations, the gas-ice interaction could be analysed by applying Langmuir adsorption isotherms to determine temperature dependent partition constants, K(Lang). Using temperature independent saturation coverages (N(max)) of (2.2 +/- 0.5) x 10(14) molecule cm(-2) and (2.4 +/- 0.6) x 10(14) molecule cm(-2) for HC(O)OH and CH(3)C(O)OH, respectively, we derive K(Lang)(HC(O)OH) = 1.54 x 10(-24) exp (6150/T) and K(Lang)(CH(3)C(O)OH) = 6.55 x 10(-25) exp (6610/T) cm(3) molecule(-1). Via a van't Hoff analysis, adsorption enthalpies were obtained for HC(O)OH and CH(3)C(O)OH. Experiments in which both acids or HC(O)OH and methanol interacted with the ice surface simultaneously were adequately described by competitive adsorption kinetics. The results are compared to previous measurements and used to calculate the equilibrium partitioning of these trace gases to ice surfaces under conditions relevant to the atmosphere.     

Simplicial collapsibility,discrete Morse theory,and the geometry of nonpositively curved simplicial complexes

Understanding the conditions under which a simplicial complex collapses is a central issue in many problems in topology and combinatorics. Let K be a finite simplicial complex of dimension three or less endowed with the piecewise Euclidean geometry given by declaring edges to have unit length, and satisfying the property that every 2-simplex is a face of at most two 3-simplices in K. Our main result is that if |K| is nonpositively curved [in the sense of CAT(0)] then K simplicially collapses to a point. The main tool used in the proof is Forman’s discrete Morse theory, a combinatorial analog of the classical smooth theory developed in the 1920s. A key ingredient in our proof is a combinatorial analog of the fact that a minimal surface in has nonpositive Gauss curvature.      

Photolysis of CH3C(O)CH3 (248 nm, 266 nm), CH3C(O)C2H5 (248 nm) and CH3C(O)Br (248 nm): pressure dependent quantum yields of CH3 formation

The formation of CH(3) in the 248 or 266 nm photolysis of acetone (CH(3)C(O)CH(3)), 2-butanone (methylethylketone, MEK, CH(3)C(O)C(2)H(5)) and acetyl bromide (CH(3)C(O)Br) was examined using the pulsed photolytic generation of the radical and its detection by transient absorption spectroscopy at 216.4 nm. Experiments were carried out at room temperature (298 +/- 3 K) and at pressures between approximately 5 and 1500 Torr N(2). Quantum yields for CH(3) formation were derived relative to CH(3)I photolysis at the same wavelength in back-to-back experiments. For acetone at 248 nm, the yield of CH(3) was greater than unity at low pressures (1.42 +/- 0.15 extrapolated to zero pressure) confirming that a substantial fraction of the CH(3)CO co-product can dissociate to CH(3) + CO under these conditions. At pressures close to atmospheric the quantum yield approached unity, indicative of almost complete collisional relaxation of the CH(3)CO radical. Measurements of increasing CH(3)CO yield with pressure confirmed this. Contrasting results were obtained at 266 nm, where the yields of CH(3) (and CH(3)CO) were close to unity (0.93 +/- 0.1) and independent of pressure, strongly suggesting that nascent CH(3)CO is insufficiently activated to decompose on the time scales of these experiments at 298 K. In the 248 nm photolysis of CH(3)C(O)Br, CH(3) was observed with a pressure independent quantum yield of 0.92 +/- 0.1 and CH(3)CO remained below the detection limit, suggesting that CH(3)CO generated from CH(3)COBr photolysis at 248 nm is too highly activated to be quenched by collision. Similar to CH(3)C(O)CH(3), the photolysis of CH(3)C(O)C(2)H(5) at 248 nm revealed pressure dependent yields of CH(3), decreasing from 0.45 at zero pressure to 0.19 at pressures greater than 1000 Torr with a concomitant increase in the CH(3)CO yield. As part of this study, the absorption cross section of CH(3) at 216.4 nm (instrumental resolution of 0.5 nm) was measured to be (4.27 +/- 0.2) x 10(-17) cm(2) molecule(-1) and that of C(2)H(5) at 222 nm was (2.5 +/- 0.6) x 10(-18) cm(2) molecule(-1). An absorption spectrum of gas-phase CH(3)C(O)Br (210-305 nm) is also reported for the first time.     

Fabrication of an ammonia gas sensor using inkjet-printed polyaniline nanoparticles

This work details the fabrication and performance of a sensor for ammonia gas analysis which has been constructed via the inkjet-printed deposition of polyaniline nanoparticle films. The conducting films were assembled on interdigitated electrode arrays and characterised with respect to their layer thickness and thermal properties. The sensor was further combined with heater foils for operation at a range of temperatures. When operated in a conductimetric mode, the sensor was shown to exhibit temperature-dependent analytical performance to ammonia detection. At room temperature, the sensor responded rapidly to ammonia (t₅₀ = 15 s). Sensor recovery time, response linearity and sensitivity were all significantly improved by operating the sensor at temperatures up to 80 °C. The sensor was found to have a stable logarithmic response to ammonia in the range of interest (1-100 ppm). The sensor was also insensitive to moisture in the range from 35 to 98% relative humidity. The response of the sensor to a range of common potential interferents was also studied.