Protein A immobilized monolithic capillary column for affinity chromatography

Monolithic capillary columns for affinity chromatography were prepared by an in situ polymerization procedure using glycidyl methacrylate (GMA) as a monomer and trimethylolpropane trimethacrylate (TRIM) and ethylene dimethacrylate (EDMA) as cross-linkers, respectively. Scanning electron microscopy was applied to characterize the morphology of the end of monolithic capillary and mercury intrusion porosimetry to characterize the polymer rod prepared within the confines of a stainless steel column with 50 mm x 4.6 mm i.d. under the same polymerization condition. Obvious differences in the porous properties between the TRIM- and EDMA-based monoliths could be observed. Moreover, the mechanical stability of these two monolithic capillary columns was compared by testing the reproducibility of the column performance. The rod prepared with GMA and TRIM proved to be mechanically more stable than that prepared with GMA and EDMA. Protein A was immobilized on the monolithic rod for affinity chromatography and the experiments were performed on a capillary electrophoresis instrument, using its pressure system as the driving force. Non-specific adsorption was not observed on the TRIM-based affinity column, as proved with bovine serum albumin (BSA) as a test protein. The affinity column prepared with GMA and TRIM was then applied to determine the hIgG concentration in human serum. The correlative coefficient of the calibration curve reached 0.9942. The amount of adsorbed hIgG was unaffected by the flow rate of the loading buffer, which makes this method suitable for fast determination of biomacromolecules in microliter samples. (C) 2002 Elsevier Science B.V All rights reserved.

Bis(2,2 '-bipyridine)(5,6-epoxy-5,6-dihydro-[1,10] phenanthroline)ruthenium: Synthesis and electrochemical and electrochemiluminescence characterization

In this work, an electrochemiluminescence (ECL) reagent bis(2,2'-bipyridine)(5,6-epoxy-5,6-dihydro-[1,10]phenanthroline)ruthenium complex (Ru-1) was synthesized, and its electrochemical and ECL properties were characterized. The synthesis of Ru-1 was confirmed by IR spectra, element analysis, and H-1 NMR spectra. For further study, its UV-vis absorption and fluorescence emission spectra were investigated. Ru-1 also exhibited quasi-reversible Ru-II/Ru-III redox waves in acetonitrile solution. The aqueous ECL behaviors of Ru-1 were also studied in the absence and in the presence of tripropylamine.

Sensitive, Label-Free Protein Assay Using 1-ethyl-3-methylimidazolium Tetrafluoroborate Supported Microchip Electrophoresis with Laser-Induced Fluorescence Detection

Based on the dimer-monomer equilibrium movement of the fluorescent dye Pyronin Y (PY), a rapid, simple, highly sensitive, label-free method for protein detection was developed by microchip electrophoresis with LIF detection. PY formed a nonfluorescent dimer induced by the premicellar aggregation of an anionic surfactant, SDS, however, the fluorescence intensity of the system increased dramatically when proteins such as BSA, bovine hemoglobin, cytochrome c, and trypsin were added to the solution due to the transition of dimer to fluorescent monomer. Furthermore, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF(4)) instead of PBS was applied as running buffers in microchip electrophoresis.

Nanoscale-enhanced Ru(bpy)(3)(2+) electrochemiluminescence labels and related aptamer-based biosensing system

A unique multilabeling at a single-site protocol of the Ru(bpy)(3)(2+) electrochemiluminescence (ECL) system is proposed. Nanoparticles (NPs) were used as assembly substrates to enrich ECL co-reactants of Ru(bpy)(3)(2+) to construct nanoscale-enhanced ECL labels. Two different kinds of NP substrates [including semiconductor NPs (CdTe) and noble metal NPs (gold)] capped with 2-(dimethylamino)ethanethiol (DMAET) [a tertiary amine derivative which is believed to be one of the most efficient of co-reactants of the Ru(bpy)(3)(2+) system] were synthesized through a simple one-pot synthesis method in aqueous media.

Progress in Ru (bpy)(3)(2+) Electrogenerated Chemiluminescence

Among various ECL systems, such as 9,10-diphenylanthracene, lucigenin, tris(2,2'-bipyridyl) ruthenium, peroxyoxalate, luminol, graphene, and nanocrystals, Ru(bpy)(3)(2+) ECL is one of the most widely studied ECL systems in recent years due to its broad applications in immunoassays, DNA probe assays, coreactants analysis, and aptasensors. In this review, the progress in Ru(bpy)(3)(2+) ECL has been summarized on the whole, and the future research trends have been proposed.

Formation of [Ru(bpy)(3)](2+)-containing microstructures induced by electrostatic assembly and their application in solid-state detection of electrochemiluminescence

Tris(2,2'-bipyridine)ruthenium(II) ((Ru(bpy)(3)](2+)) is one of the most extensively studied and used electrochemiluminescent (ECL) compounds owing to its superior properties, which include high sensitivity and stability under moderate conditions in aqueous solution. In this paper we present a simple method for the preparation of [Ru(bpy)(3)](2+)-containing microstructures based on electrostatic assembly The formation of such micro-structures occurs in a single process by direct mixing of aqueous solutions of [Ru(bpy)(3)]Cl-2 and K-3[Fe(CN)(6)] at room temperature. The electrostatic interactions between [Ru(bpy)(3)]Cl-2 cations and [Fe(CN)(6)](3-) anions cause them to assemble into the resulting microstructures. Both the molar ratio and concentration of reactants were found to have strong influences on the formation of these microstructures. Most importantly, the resulting [Ru(bpy)(3)](2+)- containing microstructures exhibit excellent ECL behavior and, therefore, hold great promise for solid-state ECL detection in capillary electrophoresis (CE) or CE microchips.

Luminescent supramolecular microstructures containing Ru(bpy)(3)(2+): Solution-based self-assembly preparation and solid-state electrochemiluminescence detection application

In this correspondence, we report on the first preparation of novel, robust Ru(bpy)(3)(2+)-containing supramolecular microstructures via a solution-based self-assembly strategy, carried out by directly mixing H2PtCl6 and Ru(bpy)(3)Cl-2 aqueous solutions at room temperature. It reveals that both the molar ratio and concentration of reactants have a heavy influence on the morphologies of such microstructures. The electrochemical behavior of the Ru(bpy)(3)(2+) components contained in the solid film of the microstructures formed on the electrode surface is also studied and found to exhibit a diffusion-controlled voltammetric feature. Most importantly, such microstructures exhibit excellent electrochemiluminescence (ECL) behaviors and therefore hold great promise as new luminescent materials for solid-state ECL detection in capillary electrophoresis (CE) or CE microchip.

Analytical applications of the electrochemiluminescence of tris (2.2 '-bipyridyl) ruthenium and its derivatives

This article presents the state of the art of analytical applications of the electrochemiluminescence (ECL) of tris (2,2'-bipyridyl) ruthenium (Ru(bpy)(3)(2+)) and its derivatives. in the last seven years, Ru(bpy)(3)(2+) ECL has attracted much interest from analysts and been successfully exploited as a detector of flow injection analysis (FIA), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and micro total analysis systems (TAS). Immobilization of Ru(bPY)(3)(2+) on a solid surface provides several advantages over the solution-phase ECL procedure, such as the simplicity of experimental design and cost-effectiveness. After a brief discussion of the mechanism of Ru(bpy)(3)(2+) ECL, we discuss its applications in FIA, HPLC, CE and TAS and give special attention to the design of Ru(bpy)(3)(2+) ECL cells and some immobilization techniques of Ru(bpy)(3)(2+); we focus on papers published after 1997.

Investigation of sol-gel transition in Pluronic F127/D2O solutions using a combination of small-angle neutron scattering and Monte Carlo simulation

Physical gelation in the concentrated Pluronic F127/D2O solution has been studied by a combination of small-angle neutron scattering (SANS) and Monte Carlo simulation. A 15% F127/D2O solution exhibits a sol-gel transition at low temperature and a gel-sol transition at the higher temperature, as evidenced by SANS and Monte Carlo simulation studies. Our SANS and simulation results also suggest that the sol-gel transition is dominated by the formation of a percolated polymer network, while the gel-sol transition is determined by the loss of bound solvent. Furthermore, different diffusion behaviors of different bound solvents and free solvent are observed. We expect that this approach can be further extended to study phase behaviors of other systems with similar sol-gel phase diagrams.

Determination of total calcium in plasma by flow injection analysis with tris(2,2 '-bipyridyl)ruthenium(II) electrochemiluminescent detection

We described here a new method for the determination of total calcium in plasma. The method is based on the precipitation of calcium with excess oxalate and the measurement of residual oxalate by flow injection analysis with Ru(bpy)(3)(2+) electrochemiluminescent detection. It has the advantages of extremely stable reagent, user-friendly instrument, high selectivity, good analytical recovery, wide dynamic range, and nice correlation with atomic absorption spectroscopy. The calibration plot for calcium is linear over a concentration range from 0.5 mmol L-1 to 4.8 mmol L-1, which is wider than those obtained by most other methods. The analytical recoveries for plasma calcium are 98.4-101.2% with coefficients of variation (CVs) of 1.96-2.52%. The within-day CVs range from 0.76% to 0.95%, and between-day CVs were from 1.12% to 1.46%. The time for each injection is one minute. Because the proposed method can be readily carried out on increasingly popular instruments for Ru(bpy)(3)(2+) ECL immunoassays and DNA probe assays, Ru(bpy)32+ ECL method is suitable for routine clinical analysis of calcium.

Analysis of five pharmacologically active compounds from the Tibetan medicine Elsholtzia with micellar electrokinetic capillary chromatography

A high performance capillary electrophoresis method with diode array detector detection for the determination of five bioactive ingredients in Tibetan medicine Elsholtzia, namely quercetin, rutin, saussurenoside, kaempferol, and oleanolic acid, has been developed. The effects of several factors, such as the acidity, concentration of running buffer, separation voltage, temperature, and SDS concentration were investigated. The optimal conditions were 44 mmol/L boric acid running buffer (pH 8.5), 45 mmol/L SDS, 16 KV voltage, 20 degrees C, and 10.0% (V/V) of acetonitrile. Under the optimum conditions, five components could be separated with a good baseline resolution within 17 min. The calibration curves showed good linear relationship over the concentration range of 5 x 10(-4)similar to 0.1 mg/mL for quercetin, rutin, saussurenoside, kaempferol, and 1 x 10(-3) similar to 0.1 mg/mL for oleanolic acid. The average recoveries of the method and RSD were ( 99.2%, 3.2%) for quercetin, (102.1%, 2.1%) for rutin, (99.4%, 1.5%) for saussurenoside, (98.9%, 1.8%) for kaempferol, and (99.0%, 2.9%) for oleanolic acid, respectively. The detection limits (S/N = 3) were 1.1 x 10(-4) mg/mL for quercetin, 2.6 x 10(-4) mg/mL for rutin, 1.8 x 10(-4) mg/mL for saussurenoside, 2.9 x 10(-4) mg/mL for kaempferol, and 6.3 x 10(-4) mg/mL for oleanolic acid, respectively. The method was simple, rapid, and reproducible and could be applied for the determination of quercetin, rutin, saussurenoside, kaempferol, and oleanolic acid in Tibetan medicine Elsholtzia, and the assay results were satisfactory.

Comparative analysis of allantoin quercetin, and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in Nitraria tangutorum Bobr. Seed by HPLC-APCI-MS and CE

This paper describes the simultaneous determination of allantoin, quercetin, and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCCA) in Nitraria tangutorum Bobr seed by HPLC-APCI-MS and CE (capillary electrophoresis) methods. The final optimized chromatographic conditions were investigated in a reversed-phase Eclipse XDB-C8 column (150 x 4.6 mm, 5 mu m). A seventeen-minute gradient elution, (A: aqueous acetonitrile 20% (v/v); B: aqueous acetonitrile 60% (v/v); C: pure acetonitrile 100%) at a flow rate of 1.0 mL/min was selected for the separation of three natural products with diode array detection (DAD) at 220 nm. A CE experiment was carried out in a fused silica capillary with 32 mmol/L boric acid (pH 10), 32 mmol/L SDS and acetonitrile (10.0%, v/v). The applied potential and temperature was, respectively, set at 19 kV and 25 degrees C. After development, the validation was performed in parallel for HPLC and CE, with the same standards and sample to avoid differences due to the manipulation. The validation parameters of both techniques were adequate for the intended purpose.

Capillary electrophoretic analysis of pharmacologically active xanthone compounds from Swertia przewalskii pissjauk extract

Pharmacologically active xanthone compounds isolated from Swertia przewalskii pissjauk were well separated by capillary electrophoresis (CE) within 5 min, using a running buffer of 25 mM disodium tetraborate at pH 9.0. Quantitative determination was shown to be possible because regression equations revealed a linear relationship between the peak area of each constituent and its concentration, with correlation coefficients of 0.9972-0.9994. The relative standard deviations were between 0.44%-0.73% for migration times and 2.52%-4.28% for peak areas. The dissociation constant of 1,7-O-beta-D-glucopyranosyl-8-hydroxy-3,7-dimethoxyxanthone, 1,8-dihydroxy-3, 7-dimethoxy-xanthone and 1,7-dihydroxy-3,8-dimethoxyxanthone were also measured by the CE method, giving a value of 9.04, 8.94, and 8.59, respectively.

Separation of peptides by strong cation-exchange capillary electrochromatography

Separation of small peptides on ion-exchange capillary electrochromatography (IE-CEC) with strong cation-exchange packing (SCX) as stationary phase was investigated. It was observed that the number of theoretical plates for small peptides varied from 240 000 to 460 000/m, and the relative standard deviation for t(0) and the migration time of peptides were less than 0.57% and 0.27%, respectively for ten consecutive runs. Unusually high column efficiency has been explained by the capillary electrophoretic stacking and chromatofocusing phenomena during the injection and separation of positively charged peptides. The sample buffer concentration had a marked effect on the column efficiency and peak area of the retained peptides. The influences of the buffer concentration and pH value as well as the applied voltage on the separation were investigated. It has been shown that the electrostatic interaction between the positively charged peptides and the SCX stationary phase played a very important role in IE-CEC, which provided the different separation selectivity from those in the capillary electrophoresis and reversed-phase liquid chromatography. A fast separation of ten peptides in less than 3.5 min on IE-CEC by adoption of the highly applied voltage was demonstrated. (C) 2000 Elsevier Science B.V. All rights reserved.

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.