A multianalyte ELISA for immunochemical screening of sulfonamide,fluoroquinolone and ß-lactam antibiotics in milk samples using class-selective bioreceptors

A multianalyte ELISA has been developed for the simultaneous determination of the most frequently used antibiotic families in the veterinary field following the typical planar microarray configuration, where the identity of the target analyte is encoded by its location in the detection platform (Master et al. in Drug Discovery Today 11:1007-1011, 2006). To accomplish this aim, two individual enzyme-linked immunosorbent assays for sulfonamide and fluoroquinolone antibiotics and an enzyme-linked receptor assay for ss-lactam antibiotics have been combined. The strategy uses microplates coated with the corresponding haptenized proteins in specific sections of the microplate. The samples are mixed with a cocktail containing the bioreagents, and distributed in the wells of the microplate. Identification of the antibiotic present in a particular sample is consequently accomplished by detecting a positive response on the corresponding microplate section. Since the bioreceptors used show a wide recognition of the congeners of each antibiotic family, the multianalyte method is able to detect more than 25 different antibiotics from the three most important antibiotic families. The detectability reached in full-fat milk samples is below the European maximum residue limits. The accuracy and reliability of this multiplexed bioanalytical method have been demonstrated by analyzing blind spiked samples.     

Oxygen dependence of two-photon activation of zinc and copper phthalocyanine tetrasulfonate in Jurkat cells

Abstract Photodynamic therapy (PDT), the use of light-activated drugs, is a promising treatment of cancer as well as several nonmalignant conditions. However, the efficacy of one-photon (1-gamma) PDT is limited by hypoxia, which can prevent the production of the cytotoxic singlet oxygen ((1)O(2)) species, leading to tumor resistance to PDT. To solve this problem, we propose an irradiation protocol based on a simultaneous, two-photon (2-gamma) excitation of the photosensitizer (Ps). Excitation of the Ps triplet state leads to an upper excited triplet state T(n) with distinct photochemical properties, which could inflict biologic damage independent of the presence of molecular oxygen. To determine the potential of a 2-gamma excitation process, Jurkat cells were incubated with zinc or copper phthalocyanine tetrasulfonate (ZnPcS(4) or CuPcS(4)). ZnPcS(4) is a potent (1)O(2) generator in 1-gamma PDT, while CuPcS(4) is inactive under these conditions. Jurkat cells incubated with either ZnPcS(4) or CuPcS(4) were exposed to a 670 nm continuous laser (1-gamma PDT), 532 nm pulsed-laser light (2-gamma PDT), or a combination of 532 and 670 nm (2-gamma PDT). The efficacy of ZnPcS(4) to photoinactivate the Jurkat cells decreased as the concentration of oxygen decreased for both the 1-gamma and 2-gamma protocols. In the case of CuPcS(4), cell phototoxicity was measured only following 2-gamma irradiation, and its efficacy also decreased at a lower oxygen concentration. Our results suggest that for CuPcS(4) the T(n) excited state can be populated after 2-gamma irradiation at 532 nm or the combination of 532 and 670 nm light. Dependency of phototoxicity upon aerobic conditions for both 1-gamma and 2-gamma PDT suggests that reactive oxygen species play an important role in 1-gamma and 2-gamma PDT.     

Online large volume sample staking preconcentration and separation of enantiomeric GHRH analogs by capillary electrophoresis

A capillary electrophoresis method is proposed to analyze the four most well-known growth hormone–releasing hormone (GHRH) analogs that are misused by athletes. Dimethyl-β-cyclodextrin used as a chiral selector allowed, for the first time, the separation of those basic peptide analogs, including enantiopeptides (sermorelin and CJC-1293) that differ by the chirality of only one amino acid. To increase the method sensitivity, electrokinetic preconcentration methods have been investigated. The large volume sample stacking with polarity switching (PS-LVSS) method with an injected sample volume corresponding to 80% of the capillary one was found superior to the sweeping in terms of signal enhancement factor (SEF). Acid and organic solvent addition to the sample (0.1 mM phosphoric acid with 30% methanol) led to a twofold signal improvement, when compared to water as a matrix. We increased capillary dimensions to provide a signal enhancement through the injection of a larger sample volume. Finally, using a combination of the optimized PS-LVSS preconcentration with the chiral capillary zone electrophoresis (CZE), the GHRH analogs were separated and limits of detection between 75 and 200 ng/mL were reached. This method was successfully applied to urine after a desalting step. An optimized C18 SPE was used for that purpose in order to provide low sample conductivity (<130 µS/cm) and preserve the efficiency of LVSS preconcentration. SEF of 640 was obtained with desalted urine spiked with sermorelin by comparison to the CZE (without preconcentration) method.     

Revisiting the determination of langmuir parameters--application to tetrahydrothiophene adsorption onto activated carbon

The selection of a proper sorbent for a given application is a complex problem. The design and efficiency of adsorption processes require an equilibrium adsorption model. Linear transformation is one of the methods available to estimate the adjustable parameters of isotherm models but possesses limitations compared to nonlinear regressions. A different approach to calculate predicted equilibrium isotherm values leading to an alternative nonlinear regression is presented in this paper and compared with usual regression methods. Adsorption isotherm data of gaseous THT onto three activated carbon materials constitute an experimental basis for the discussion. Assessment of the goodness-of-fit of the Langmuir model is supported by different selected test functions. The new nonlinear approach did not obtain the best results for each test function, but raises questions about the inherent combined error in regression procedures.     

Adsorption of simple fluid on silica surface and nanopore: effect of surface chemistry and pore shape

This paper reports a molecular simulation study on the adsorption of simple fluids (argon at 77 K) on hydroxylated silica surfaces and nanopores. The effect of surface chemistry is addressed by considering substrates with either partially or fully hydroxylated surfaces. We also investigate the effect of pore shape on adsorption and capillary condensation by comparing the results for cylindrical and hexagonal nanopores having equivalent sections (i.e., equal section areas). Due to the increase in the polarity of the surface with the density of OH groups, the adsorbed amounts for fully hydroxylated surfaces are found to be larger than those for partially hydroxylated surfaces. Both the adsorption isotherms for the cylindrical and hexagonal pores conform to the typical behavior observed in the experiments for adsorption/condensation in cylindrical nanopores MCM-41. Capillary condensation occurs through an irreversible discontinuous transition between the partially filled and the completely filled configurations, while evaporation occurs through the displacement at equilibrium of a hemispherical meniscus along the pore axis. Our data are also used to discuss the effect of surface chemistry and pore shape on the BET method. The BET surface for fully hydroxylated surfaces is much larger (by 10-20%) than the true geometrical surface. In contrast, the BET surface significantly underestimates the true surface when partially hydroxylated surfaces are considered. These results suggest that the surface chemistry and the choice of the system adsorbate/adsorbent is crucial in determining the surface area of solids using the BET method.     

Embedded silver ions-containing liposomes in polyelectrolyte multilayers: cargos films for antibacterial agents

A new antibacterial coating made of poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films and liposome aggregates loaded with silver ions was designed. Liposomes filled with an AgNO 3 solution were first aggregated by the addition of PLL in solution. The obtained micrometer-sized aggregates were then deposited on a PLL/HA multilayer film, playing the role of a spacer with the support. Finally, HA/PLL/HA capping layers were deposited on top of the architecture to form a composite AgNO 3 coating. Release of encapsulated AgNO 3 from this composite coating was followed and triggered upon temperature increase over the transition temperature of vesicles, found to be equal to 34 degrees C. After determination of the minimal inhibitory concentration (MIC) of AgNO 3 in solution, the antibacterial activity of the AgNO 3 coating was investigated against Escherichia coli. A 4-log reduction in the number of viable E. coli cells was observed after contact for 120 min with a 120 ng/cm (2) AgNO 3 coating. In comparison, no bactericidal activity was found for PLL/HA films previously dipped in an AgNO 3 solution and for PLL/HA films with liposome aggregates containing no AgNO 3 solution. The strong bactericidal effect could be linked to the diffusion of silver ions out of the AgNO 3 coating, leading to an important bactericidal concentration close to the membrane of the bacteria. A simple method to prepare antibacterial coatings loaded with a high and controlled amount of AgNO 3 is therefore proposed. This procedure is far superior to that soaking AgNO 3 or Ag nanoparticles into a coating. In principle, other small bactericidal chemicals like antibiotics could be encapsulated by this method. This study opens a new route to modify surfaces with small solutes that are not permeating phospholipid membranes below the phase transition temperature.     

Selective hetero-trisfunctionalization of the large rim of a biomimetic calix[6]arene using host-guest chemistry as a synthetic tool

A calix[6]arene has been selectively functionalized by three different groups at the large rim. The strategy relies on the hostguest recognition chemistry of a biomimetic metal complex at the small rim (so-called "funnel complex") and the Huisgen cycloaddition. The intramolecular thermal reaction proceeds with a high efficiency, chemio- and regioselectivity, allowing the monofunctionalization of one aromatic unit among three. Thanks to the high yield and selectivity of the reaction, it can be applied successively twice on the same compound, which opens the route for inherently chiral calix[6]arenes. This methodology not only is of wide potential for obtaining and exploiting calix[6]arenes that are dissymmetrized at the large rim, but also stands as an exemplary strategy for the selective appending of a functional group on a host platform.     

A ditopic calix[6]arene ligand with N-methylimidazole and 1,2,3-triazole substituents: synthesis and coordination with Zn(II) cations

The first member of a new class of ditopic calix[6]arene has been synthesized, which is decorated with three N-methylimidazoles at the small rim and three 1,4-disubstituted-1,2,3-triazoles at the large rim. The coordination of a first Zn(II) cation selectively takes place at the small rim. Addition of a second equivalent results in the complexation of the three triazoles, providing a rare example of 1,2,3-triazole ligands embedded within a supramolecular system.