Multiplex sorting of foodborne pathogens by on-chip free-flow magnetophoresis

This study reports multiplex sorting of Salmonella typhimurium and Escherichia coli 0157, from broth cultures and from pathogen-spiked skinned chicken breast enrichment broths by employing microfluidic free-flow magnetophoresis. Magnetic beads of different sizes and magnetite content, namely Dynabeads anti-salmonella and Hyglos-Streptavidin beads together with the corresponding pathogen-specific biotinylated recombinant phages, were utilised as affinity solid phases for the capture and concentration of viable S. typhimurium and E. coli 0157. Following optimisation, the protocol was used to demonstrate continuous magnetophoretic sorting of the two pathogen-bound magnetic bead populations from mixed cultures and from pathogen-spiked chicken pre-enrichment broths under the influence of a Halbach magnet array. For example, in the latter case, a pure population of S. typhimurium-bound Dynabeads (72% recovery) was sorted from a 100 μL mixture containing E. coli 0157-bound Hyglos beads (67% recovery) within 1.2 min in the presence of 0.1% Tween 20. This proof-of-principle study demonstrates how more than one pathogen type can be simultaneously isolated/enriched from a single food pre-enrichment broth (e.g. Universal food enrichment broth).     

Ca2+‐complex stability of GAPAGPLIVPY peptide in gas and aqueous phase,investigated by affinity capillary electrophoresis and molecular dynamics simulations and compared to mass spectrometric results

Strong, sequence‐specific gas‐phase bindings between proline‐rich peptides and alkaline earth metal ions in nanoESI‐MS experiments were reported by Lehmann et al. (Rapid Commun. Mass Spectrom. 2006, 20, 2404–2410), however its relevance for physiological‐like aqueous phase is uncertain. Therefore, the complexes should also be studied in aqueous solution and the relevance of the MS method for binding studies be evaluated. A mobility shift ACE method was used for determining the binding between the small peptide GAPAGPLIVPY and various metal ions in aqueous solution. The findings were compared to the MS results and further explained using computational methods. While the MS data showed a strong alkaline earth ion binding, the ACE results showed nonsignificant binding. The proposed vacuum state complex also decomposed during a molecular dynamic simulation in aqueous solution. This study shows that the formed stable peptide–metal ion adducts in the gas phase by ESI‐MS does not imply the existence of analogous adducts in the aqueous phase. Comparing peptide–metal ion interaction under the gaseous MS and aqueous ACE conditions showed huge difference in binding behavior.     

Enantiomeric quality control of antihistamines in pharmaceuticals by affinity electrokinetic chromatography with human serum albumin as chiral selector

The present paper deals with the enantiomeric separation of six antihistaminic enantiomers by affinity electrokinetic chromatography (AEKC)-partial filling technique using human serum albumin (HSA) as chiral selector. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and HSA plug length (SPL) was carried out since there are interactions between variables that could not be considered in an univariate optimization. The estimated and experimental resolution values obtained for antihistaminic enantiomers varied from 1.13 (for orphenadrine) to 2.15 (for brompheniramine). The optimum experimental conditions for enantioresolution of each compound were: brompheniramine, pH 8.5, [HSA] 180 μM, SPL 180 s; chlorcyclizine, pH 6.5, [HSA] 180 μM, SPL 150 s; chlorpheniramine, pH 8.25, [HSA] 160 μM, SPL 150 s; hydroxyzine, pH 7.0, [HSA] 180 μM, SPL 150 s; and orphenadrine, pH 7.8, [HSA] 160 μM, SPL 150 s. pH and the quadratic term of pH seem to be the most critical factors that determine enantioresolution of antihistamines. The validity of the developed methodologies to enantiomeric quality control of antihistamines in pharmaceutical formulations is demonstrated analyzing the content of brompheniramine, chlorpheniramine and hyroxyzine enantiomers in commercially available pharmaceutical formulations containing racemic mixtures of compounds. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed methodologies make them suitable for quality control of the enantiomeric composition of antihistamines in pharmaceutical preparations.     

Miniaturized monolithic disks for immunoadsorption of cardiac biomarkers from serum

Immunoadsorbers based on 2.0 × 6.0 mm i.d., epoxy-bearing, methacrylate-based monolithic disks were developed in order to target myoglobin and N-terminal pro-natriuretic peptide (NT-proBNP), two biomarkers involved in cardiovascular disease. In both cases, antibodies were successfully coupled to the polymeric disk material. The developed immunoadsorbers permitted the selective isolation of myoglobin and NT-proBNP from human serum. Myoglobin was successfully isolated and detected from serum samples at concentrations down to 250 fmol μL⁻¹. However, the affinity of the antibodies was not sufficient for the analysis of low-concentration clinical samples. Frontal analysis of anti-NT-proBNP disks revealed the ability of the immunoadsorber to bind up to 250 pmol NT-proBNP, which is more than sufficient for the analysis of clinical samples. Anti-NT-proBNP disks showed good stability over more than 18 months and excellent batch-to-batch reproducibility. Moreover, anti-NT-proBNP disks permitted the isolation of NT-proBNP at concentrations down to 750 amol μL⁻¹ in serum, corresponding to concentrations of strongly diseased patients. Using reversed-phase trapping columns, the detection of NT-proBNP eluted from immunoadsorbers by mass spectrometry was achieved for concentrations down to 7.8 fmol μL⁻¹.     

Estimation of binding constants of receptors and ligands by affinity capillary electrophoresis

An estimation method for determination of binding constants of receptors to ligands by affinity capillary electrophoresis was evaluated. On the basis of the theories of pseudostationary phase or so-called dynamic stationary phase, the retention factor (k) was used to represent the interaction between the receptor and ligand. k could be easily deduced from the migration times of the ligand and the receptor. Then, with the linear relationship of k versus the concentration of ligand in the running buffer, the binding constant K _b was calculated from the slope and intercept. In order to test its feasibility, the calculation method was demonstrated using three model systems: the interactions between vancomycin and N-acetyl-d-Ala-d-Ala, ristocetin and N-acetyl-d-Ala-d-Ala, and carbonic anhydrase B and an arylsulfonamide. Estimated binding constants were compared with those determined by other techniques. The results showed that this estimation method was reliable. This calculation method offers a simple and easy approach to estimating binding constants of ligands to receptors.     

磁性珠状纤维素亲和吸附剂的制备与应用

采用反相悬浮包埋技术制备了粒径小于300um、粒径分布窄和湿态孔度高(85%～90%)的高顺磁性珠状纤维素,经高碘酸钠活化后,与具有生物活性的绒毛膜促性腺激素偶联,得磁性亲和吸附剂(每克磁性珠状纤维素上固载300～400IU绒毛膜促性腺激素).     

亲和毛细管电泳激光诱导荧光法测定牛血清白蛋白与其单克隆抗体的结合常数

生物分子之间的特异性相互作用是生物界普遍存在的现象．研究这些现象,对揭示生物化学作用机理、药物研究等具有重要意义．结合常数Ｋｂ是描述生物分子之间特异性相互作用最主要的参数,测定结合常数的传统方法包括平衡透析、凝胶过滤色谱和分光光度法等［１］．亲和毛细管电泳（Ａｆｆｉｎｉｔｙｃａｐｉｌｌａｒｙｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ,简称ＡＣＥ）是近几年发展起来的毛细管电泳的一个分支,在研究生物分子之间特异性相互作用等方面有很好的应用前景［２～５］．与上述传统方法相比,ＡＣＥ具有测定速度快,样品用量少,有多…     

Membrane affinity chromatography for analysis and purification of biopolymers

Summary Affinity columns suitable for HPLC were prepared by immobilization of various ligands of protein A, human IgG, human IgM and pectinase on GMA modified cellulose membrane. The adsorption capacity, affinity efficiency and activity recovery of various IgGs on these affinity columns were measured. It was observed that the length of the coupling arm plays a very important role in affinity efficiency, and the effect of eluent flow-rate on adsorption capacity was very small. The protein A column was exploited for the process monitoring of dog IgG in clinical experiments on immuno-adsorption therapy. A pectinase column was used for the determination of polygalacturonase inhibiting proteins first purified on a hydroxyapatite column. It took only about 2.5 min for analysis at a flow-rate of 1.0 mL min⁻¹. The high speed analysis of biopolymers could be performed at a flow rate of 6.0 mL min⁻¹ within 15 s. Membrane affinity chromatography gives good reproducibility, high efficiency, low column-pressure and is rapid. It can also be used for micro-scale purification of biopolymers.     

Untersuchung der Phasenbeziehungen in quaternären Systemen Bi2O3/Bi2Ch/Bi2Ch (Ch = S,Se, Te)

Investigations of the Phase Relations in the Quaternary Systems Bi₂O₃/Bi₂Ch /Bi₂Ch (Ch = S, Se, Te) The stability ranges in the pseudobinary systems Bi₂O₂S/Bi₂O₂Se, Bi₂O₂S/Bi₂O₂Te and Bi₂O₂Se/Bi₂O₂Te have been studied by solid state and chemical transport reactions. A complete mixed crystal Bi₂O₂(Te_xSe_1–x), 0 ≤ x ≤ 1 exists between the ternary compounds Bi₂O₂Te and Bi₂O₂Se. The thermal behaviour of the mixed crystal and the coexistence ranges have been determined by x‐ray and thermal analysis.