Development of an optical biosensor assay for detection of β-lactam antibiotics in milk using the penicillin-binding protein 2x*

An assay was developed for the detection of residues of penicillins and cephalosporins in milk using a surface plasmon resonance (SPR) biosensor. The assay was based on the inhibition of the binding of digoxigenin-labelled ampicillin (DIG-AMPI) to a soluble penicillin-binding protein 2x derivative (PBP 2x*) of Streptococcus pneumoniae. Samples were incubated with PBP 2x* in a first step, whereby β-lactams in positive samples would bind to the PBP 2x*. Non-complexed PBP 2x* was then allowed to form a complex with DIG-AMPI in a second incubation step. The formed DIG-AMPI/PBP 2x*-complexes were detected in a SPR-based biospecific interaction assay (BIA) for digoxigenin with an antibody against digoxigenin immobilised on the sensor chip. Although binding of matrix components to the sensor chip (non-specific binding) occurred, benzylpenicillin, ampicillin, amoxicillin, cloxacillin, cephalexin and cefoperazone could be detected in defatted bulk raw milk samples at concentrations corresponding to the maximum residue limits (MRL) set by the European Union. The influence of matrix components on the performance of the assay was examined in more detail by analysing individual raw milk samples from 19 cows. Compared to bulk raw milk samples, individual samples showed a higher level and variation of matrix interferences. Non-specific binding could be reduced to a lower and more constant level by a heat-treatment step, a centrifugation step and the addition of carboxymethylated dextran to the samples. With this sample preparation, benzylpenicillin could be detected at MRL (4 μg kg⁻¹) in individual raw milk samples. Thus, the assay could be the basis for a screening test for routine use.     

Structural characterization of sulfadiazine metabolites using H/D exchange combined with various MS/MS experiments

Two major metabolites and one minor metabolite of sulfadiazine were found in pig manure, using a special combination of different MS techniques like parent and product ion scans, H/D exchange, accurate mass measurement, and MS/MS experiments with substructures. N4-acetylsulfadiazine and 4-hydroxysulfadiazine were identified as major metabolites. N4-acetylsulfadiazine could be verified by H/D exchange and comparison with product ion spectra of a synthetic reference compound. In the case of 4-hydroxysulfadiazine, the majority of possible isomers could be discounted after H/D exchange. Substructure-specific MS/MS experiments with fragment ions and comparison with product ion spectra of two references revealed the presence of 4-hydroxysulfadiazine. The minor metabolite was characterized to some degree using H/D exchange and tandem mass spectrometry in combination with a high-resolution time of flight mass spectrometer. The aminopyrimidine moiety contained an additional modification with a likely elemental composition of C2H4O and no further acidic hydrogen.     

Bright luminescent lithium and magnesium carbene complexes

We report on the convenient synthesis of a CNC pincer ligand composed of carbazole and two mesoionic carbenes, as well as the corresponding lithium- and magnesium complexes. Mono-deprotonation affords a rare “naked” amide anion. In contrast to the proligand and its mono-deprotonated form, tri-deprotonated s-block complexes show bright luminescence, and their photophysical properties were therefore investigated by absorption- and luminescence spectroscopy. They reveal a quantum yield of 16% in solution at ambient temperature. Detailed quantum-chemical calculations assist in rationalizing the emissive properties based on an Intra-Ligand-Charge-Transfer (ILCT) between the carbazolido- and mesoionic carbene ligands. (Earth-)alkali metals prevent the distortion of the ligand following excitation and, thus, by avoiding non-radiative deactivation support bright luminescence.

Lithium- and magnesium complexes of a CNC pincer ligand composed of carbazole and two mesoionic carbenes are bright luminescent, because the metals prevent molecular distortions upon excitation.     

Crystal Structures of Two Modifications of [3,O-didehydro-mebmt1, val2]-cyclosporin and comparison of three different X-ray data sets

The crystal structure of [3,O-didehydro-MeBmt¹, Val²]cyclosporin (PSC-833; 1 ) was investigated by X-ray analysis. Data were collected from two different crystal modifications. Modification I crystallizes in P3₁21, a = b = 21.419 (2) Å, c = 32.101 (3) Å with one molecule in the asymmetric unit, modification II in P3₂21, a = b = 21.313 (2) Å, c = 62.053(3) Å with two molecules per asymmetric unit. This non-immunosuppressive analogue of cyclosporin A adopts a similar backbone conformation to that found in the crystal structure of cyclosporin A and other analogues. Three different data sets of modification I were collected using an Enraf-Nonius-CAD4 diffractometer with CuK_α radiation at 20°, a Stoe-Siemens four-circle diffractometer with MoK_α radiation at ? 120°, and an EMBL image-plate scanner with synchrotron radiation at 12°. The quality of the data sets was evaluated by internal consistency, independent structure solution, and refinement. The structural parameters reported here for modification I are based on the synchrotron data.     

A Nano-scale Study on Film Stability

Thickness and stability of films formed by different surfactants are studied by means of thin film balance (TFB) technique to observe the formation and stability of the films. It is demonstrated that stable Newton black films (NBFs) can be easily obtained with non-ionic surfactants (C12E5, C12G2) than ionic surfactants (C12TAB, C16TAB). Common black film (CBF) was obtained with C16TAB at 5×10-3 mol/L, while the film of C12TAB is unstable. It has been demonstrated that the horizontal forces should also be taken into consideration for explaining film stability.     

Synthesis,Structure, and Lanthanide Derivatives of an Unusual Hexameric Alcohol: [2,4,6-(CF3)3C6H2CH2OH]6

Hitherto unknown 2,4,6-tris(trifluoromethyl)benzyl alcohol ( 3 ) was synthesized in 41 % yield by treatment of freshly prepared R_FLi ( 2 ) with paraformaldehyde (R_F = 2,4,6-tris(trifluoromethyl)phenyl). According to an X-ray diffraction study the crystal structure of 3 consists of S₆ symmetric cyclic hexamers [2,4,6-(CF₃)₃C₆H₂CH₂OH]₆. Deprotonation of 3 with NaN(SiMe₃)₂ in toluene afforded the unsolvated sodium alkoxide derivative R_FCH₂ONa ( 4 ). Homoleptic lanthanide alkoxides of the type Ln(OCH₂R_F)₃ (Ln = Nd ( 5 ), Sm ( 6 ), Yb ( 7 )) were made by treatment of Ln(C₅H₅)₃ with three equivalents of 3 . Similar reactions in a 1:1 molar ratio afforded the bis(cyclopentadienyl)lanthanide alkoxide derivatives (C₅H₅)₂Ln(OCH₂R_F) (Ln = Nd ( 8 ), Sm ( 9 ), Yb ( 10 )).     

Synthesis and structures of aluminium monohydride and chalcogenides bearing a bidentate [N,O] ligand

The aluminium monohydride (3-tBu-5-Me-2-(O)C(6)H(2)CH(2)-N-2,6-iPr(2)C(6)H(3))AlH(NMe(3))(2) was prepared by treatment of the bidentate salicylaldimine [3-tBu-5-Me-2-(OH)C(6)H(2)CH=N-2,6-iPr(2)C(6)H(3)](1) with a small excess of AlH(3).NMe(3) in high yield. Compound 2 reacted with sulfur and selenium respectively to afford the dimeric aluminium chalcogenide [(3-tBu-5-Me-2-(O)C(6)H(2)CH(2)-NH-2,6-iPr(2)C(6)H(3))Al(micro-E)](2)[E = S (3), E = Se (4)]. During the formation of 2 hydrogen migration from the aluminium centre to the ligand backbone occurred. A possible reaction mechanism for 3 and 4 is discussed and the molecular structures of compounds 2-4 were determined by X-ray structural analyses.     

Evidence of surface charge at the air/water interface from thin-film studies on polyelectrolyte-coated substrates

The stability of thin water films on silicon substrates coated with cationic and anionic polyelectrolytes was investigated by the thin film pressure balance technique. Depending on the surface charge of the substrate, the water films are either stable (on negatively charged wafers) or rupture rapidly (on positively charged wafers). It is supposed that this behavior is due to a negative surface charge of the free water surface. The underlying assumption that the films' stability is due to electrostatic interactions is supported by measurements of the disjoining pressure on silicon wafers with a native oxide layer, which indicates a decrease of the film thickness, and thus decreasing repulsive interaction between the two film interfaces, with increasing ionic strength.     

Effects of Grafting Density and Film Thickness on the Adhesion of Staphylococcus epidermidis to Poly(2‐hydroxy ethyl methacrylate) and Poly(poly(ethylene glycol)methacrylate) Brushes

Thin polymer films that prevent the adhesion of bacteria are of interest as coatings for the development of infection‐resistant biomaterials. This study investigates the influence of grafting density and film thickness on the adhesion of Staphylococcus epidermidis to poly(poly(ethylene glycol)methacrylate) (PPEGMA) and poly(2‐hydroxyethyl methacrylate) (PHEMA) brushes prepared via surface‐initiated atom transfer radical polymerization (SI‐ATRP). These brushes are compared with poly(ethylene glycol) (PEG) brushes, which are obtained by grafting PEG onto an epoxide‐modified substrate. Except for very low grafting densities (ρ = 1%), crystal violet staining experiments show that the PHEMA and PPEGMA brushes are equally effective as the PEG‐modified surfaces in preventing S. epidermis adhesion and do not reveal any significant variations as a function of film thickness or grafting density. These results indicate that brushes generated by SI‐ATRP are an attractive alternative to grafted‐onto PEG films for the preparation of surface coatings that resist bacterial adhesion.