An aqueous two-phase system as a strategy for serum albumin depletion

An aqueous two-phase system, based on the cloud point phenomenon, was proposed for blood plasma albumin depletion prior to gel electrophoresis (SDS-PAGE) analysis. Albumin removal was evaluated using a mixture of Triton^® X-114 and SDS. Variables affecting phase separation and the partition coefficient (K), such as pH (from 5.0 to 7.6) and salt composition, were first optimized to carry out phase separation at room temperature and to promote albumin removal (15 min with ca. 95% efficiency) to the surfactant-rich phase, in a global process that results in reduction of 12% α-helix structures, confirmed through circular dichroism analysis. Gel electrophoresis analyses were carried out to compare crude human plasma composition with human plasma submitted to the cloud point procedure as well as the same plasma submitted to the separation of albumin using a commercially available system based on a chromatographic affinity column. The cloud point extraction procedure allowed identification of 18 protein bands present in the surfactant-poor phase, resulting in a more detailed electrophoretic profile, when compared with crude plasma (10 protein bands).     

Determination of aminoglutethimide enantiomers in pharmaceutical formulations by capillary electrophoresis using methylated-β-cyclodextrin as a chiral selector and computational calculation for their respective inclusion complexes

A capillary electrophoretic method for the separation of the aminoglutethimide (AGT) enantiomers using methylated-β-cyclodextrin (M-β-CD) as chiral selector is described. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, voltage and temperature. Good chiral separation of the racemic mixture was achieved in less than 9 min with resolution factor Rs = 2.1, using a fused-silica capillary and a background electrolyte (BGE) of tris-phosphate buffer solution (50 mmol L⁻¹, pH 3.0) containing 30 mg mL⁻¹ of M-β-CD. The separation was carried out in normal polarity mode at 25 °C, 16 kV and using hydrostatic injection. Acceptable validation criteria for selectivity, linearity, precision, and accuracy/recovery were included. The proposed method was successfully applied to the assay of AGT enantiomers in pharmaceutical formulations. The computational calculations for the inclusion complexes of the R- and S-AGT-M-β-CD rationalized the reasons for the different migration times between the AGT enantiomers.     

Enhanced separation of seven quinolones by capillary electrophoresis with silica nanoparticles as additive

This paper describes the enhanced separation of lomefloxacin, sparfloxacin, fleroxacin, norfloxacin, ofloxacin, gatifloxacin and pazufloxacin by capillary zone electrophoresis (CZE) using silica nanoparticles (SiNPs) as running buffer additive. The impact of SiNPs concentration on the resolution and selectivity of separation was investigated and a given value of SiNPs was finally chosen under the optimum conditions. The addition of the SiNPs to the running buffer enabled electroosmotic flow (EOF) decrease and permitted full interaction between SiNPs and analytes. The influence of separation voltage, pH and buffer concentration on the separation in the presence of SiNPs was examined. Interactions between drugs and nanoparticles during the separation are discussed; the determination of interaction constants is also achieved. A good resolution of seven quinolones was obtained within 15 min in a 50 cm effective length fused-silica capillary at a separation voltage of +10 kV in a 12 mM disodium tetraborate-phosphate buffer (pH 9.08) containing 5.2 μg mL⁻¹ SiNPs.     

Evaluation of the arsenic binding capacity of plant proteins under conditions of protein extraction for gel electrophoretic analysis

As prerequisite for the investigation of arsenic-binding proteins in plants, the general influence of different extraction parameters on the binding behaviour of arsenic to the plant protein pool was investigated. The concentration of the extraction buffer affected the extraction yield both for proteins and for arsenic revealing an optimal buffer concentration of 5 mM Tris/HCl, pH 8. The addition of 1 or 2% (w/v) SDS to the extraction buffer produced a two- to threefold enhancement of the total protein extraction yield but strongly suppressed the simultaneous extraction of arsenic from 80 ± 8% extraction yield obtained without SDS to 48 ± 2% in presence of 2% (w/v) SDS. The arsenic binding capacity of the protein fraction obtained after extraction with Tris buffer and protein precipitation by trichloroacetic acid in acetone was estimated to be 1.4 ± 0.6% independently on the original spiking concentration of arsenic provided in the form of monomethylarsonate to the extracts. Due to the low total protein concentrations of the plant extracts that varied in the range from 75 to 412 μg mL⁻¹ depending on the extraction parameters, high arsenic concentrations of 263-1001 mg (kg protein mass)⁻¹ resulted for spiking concentrations of 10 mg As L⁻¹. The optimized protein isolation procedure was applied to plants grown under arsenic exposure and revealed a similar arsenic binding capacity as for the spiked protein extracts.     

Quantification of testosterone and epitestosterone in biological samples by capillary electrophoresis with immunoaffinity extraction

Testosterone is one of the most common doping drugs abused by athletes. Therefore, it is necessary to develop a sensitive and simple method to monitor testosterone and its epimer epitestosterone. An off-line immunoaffinity extraction followed by capillary electrophoresis for simultaneous determination of testosterone and epitestosterone has been described in this paper. Anti-epitestosterone monoclonal antibody which is specific to both testosterone and epitestosterone had been prepared and immobilized on a Sepharose 4B stationary phase. The immunoaffinity column was used for sample cleanup, extraction and preconcentration. After elution and reconstitution, testosterone and epitestosterone in the sample were separated and quantified by micellar electrokinetic chromatography(MEKC) using the borate buffer (200 mM borate, pH 8.7) containing 40 mM sodium cholate as a chiral selector. The immunoaffinity column was evaluated in different parameters such as the retention mechanism, selectivity, binding capacity, elution protocol, and reusability. The separation of these two compounds by MEKC was also optimized. Limit of detection for testosterone and epitestosterone were 5 and 23 ng mL⁻¹, respectively. It was satisfactory to apply this method to analyze testosterone and epitestosterone in spiked urine sample with the recoveries from 78% to 109%.     

Electrophoretic separation with 2-mm inner diameter fused-silica microcolumn packed with quartz microncrystals and its application

The feasibility of a microcolumn electrophoresis technique was investigated with a 100 mm length, 2 mm I.D. fused-silica microcolumn packed with uniform quartz microncrystals prepared by hydrothermal synthesis. To evaluate the separation technique, tryptophan, phenylalanine and tyrosine were primarily separated by the microcolumn electrophoresis and detected at 216 nm without derivatization by an ordinary spectrophotometer. The separation conditions of the amino acids were optimized. With 1.5 mmol/L disodium phosphate buffer solution (pH 11.5) containing 25% (v/v) methanol and 10% (v/v) acetonitrile, the three amino acids were separated and the separation efficiency of tryptophan was 4.5 × 10⁴ plates/m. The limits of detection were 0.035, 0.22 and 0.20 μmol/L, respectively. The sample capacity of the electrophoretic microcolumn achieved 35 μL. The proposed method was used to determine these amino acids in compound amino acid injection samples without derivatization. For the simplicity and portability of the microcolumn electrophoresis, it is studied as one of the high-performance separation techniques for an in situ and real-time electrokinetic flow analysis system. For its high detection sensitivity and large sample capacity, it can be developed for preparative electrophoresis.     

Fabrication of SU-8 based microchip electrophoresis with integrated electrochemical detection for neurotransmitters

A new SU-8 based microchip capillary electrophoresis (MCE) device has been developed for the first time with integrated electrochemical detection. Embedded electrophoretic microchannels have been fabricated with a multilayer technology based on bonding and releasing steps of stacked SU-8 films. This technology has allowed the monolithic integration in the device of the electrochemical detection system based on platinum electrodes. The fabrication of the chips presented in this work is totally compatible with reel-to-reel techniques, which guarantee a low cost and high reliability production. The influence of relevant experimental variables, such as the separation voltage and detection potential, has been studied on the SU-8 microchip with an attractive analytical performance. Thus, the effective electrical isolation of the end-channel amperometric detector has been also demonstrated. The good performance of the SU-8 device has been proven for separation and detection of the neurotransmitters, dopamine (DA) and epinephrine (EP). High efficiency (30,000-80,000 N/m), excellent precision, good detection limit (450 nM) and resolution (0.90-1.30) has been achieved on the SU-8 microchip. These SU-8 devices have shown a better performance than commercial Topas (thermoplastic olefin polymer of amorphous structure) microchips. The low cost and versatile SU-8 microchip with integrated platinum film electrochemical detector holds great promise for high-volume production of disposable microfluidic analytical devices.     

新型芯片电泳柱端安培检测系统的构建与评价

自行设计开发了一套便于与电泳芯片集成的一体式柱端安培检测池系统．该系统由整块透明有机玻璃精密加工而成,包括电泳芯片支架和安培检测池两部分,芯片可通过芯片插槽和不锈钢夹具固定在芯片支架上,各种检测用电极可直接通过螺母固定在安培检测池中．以100μmol／L的DA为模式分析物,分别采用直径为100、300和500μm的铂金圆盘电极与表观直径为240μm的碳纤维电极作为工作电极均在该装置上实现了良好组装和高灵敏检测．采用碳纤维工作电极对该系统的检测参数进行了优化．测试结果表明该系统在电化学清洗程序下连续六次测定100μmol／L多巴胺的峰电流相对标准偏差为3．2％,保留时间相对标准偏差为0．5％,DA的检测限为0．4μmol／L（按照S／N=3计）．该系统体积小巧,测试稳定,检测灵敏度较高,工作电极更换方便,适合作为芯片电泳柱端安培检测通用平台．     

Polymorphisms of COTL1 gene identified by proteomic approach and their association with autoimmune disorders

To select candidate genes, we attempted to comparative analysis of protein levels between rheumatoid arthritis (RA) patients and healthy controls by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). We identified 17 proteins that showed up- or down-regulated spots in RA patients. We found that coactosin-like1 (COTL1) were highly expressed in RA patients compared with healthy controls. We performed a case-control study to determine whether the COTL1 gene polymorphisms were associated with RA and systemic lupus erythematosus (SLE). The genotype frequency of c.-1124G>T and the allelic frequency of c.484G>A in RA patients, and the genotype frequency of c.484G>A in SLE patients were significantly different from healthy controls (P = 0.009, 0.027, and 0.025, respectively). We also investigated the correlation with the levels of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibody in RA patients, and anti-nuclear antibodies (ANA) in SLE patients. The c.484G>A polymorphism in RA patients has significant association with the levels of anti-CCP antibody (P = 0.03). Our findings demonstrated that c.-1124G>T and c.484G>A polymorphisms of the COTL1 gene might be associated with the genetic susceptibility of autoimmune disorders.     

Electrophoretic methods for separation of nanoparticles

This article reviews progress in the application of electrophoretic techniques for the separation of nanoparticles. Numerous types of nanoparticles have recently been synthesised and integrated into different products and procedures. Consequently, analytical methods for the efficient characterisation of nanoparticles are now required. Several studies have revealed that gel electrophoresis can readily be used for separating nanoparticles according to their size or shape. However, many other studies focused on separation of nanoparticles by CE. In some cases nanoparticles could be separated by CZE, simply using pure buffer as the BGE. In other studies, buffer additives (most often SDS) were used, enabling fast separations of metallic nanoparticles by size. Other CE methods also allowed for separation of nanoparticle conjugates with biomolecules. Dielectrophoresis is yet another electrophoretic technique useful in separation and characterisation of nanoparticles; particularly nanotubes. Detection methods often used after electrophoretic separation include UV/Vis absorption and fluorescence spectroscopy. Examples of recent and relevant older reports are presented here. The authors conclude that electrophoretic methods for nanoanalysis can provide inexpensive and efficient tools for quality assurance and safety control; and as a consequence, they can augment transfer of nanotechnologies from research to industry.