Review on screening and analysis techniques for hemoglobin variants and thalassemia

Thalassemia involves gene mutation that causes the production of an insufficient amount of normal structure globin chains while Hb variant involves gene mutation that causes the change in type or number of amino acid of the globin chain. It has been reported that some 200 million people worldwide had hemoglobinopathies of some sort. Attempts to develop effective and economical techniques for screening and analysis of thalassemia and Hb variants have become very important. In this review, we report the different techniques available, ranging from initial screening to extensive analysis, comparing advantages and disadvantages. Some indirect studies related to thalassemia indication and treatment follow-up are also included. We hope that information on these various techniques would be useful for some scientists who are working on development of a new technique or improving the existing ones.     

Attomole protein analysis by CIEF with LIF detection

We have coupled CIEF with an LIF detector that is based on a post‐column sheath flow cuvette. We employed Chromeo P503 as a fluorogenic reagent to label proteins before analysis. This reagent reacts with the ε‐amine of lysine residues, preserving the cationic nature of the residue; labeled proteins generate extremely sharp peaks in CIEF. A set of four standard proteins generated a linear relationship between migration time and pI. A protein homogenate prepared from a Barrett's esophagus cell line resolved over 100 components in a 40 min separation. Detection limits for Chromeo P503‐labeled β‐lactoglobulin were 5 amol injected into the capillary. Fluorescent impurities present in the ampholytes generated a large background signal that degraded the detection limit by four orders of magnitude compared with other forms of capillary electrophoresis with this detector.     

CE-based noncompetitive immunoassay for immunoglobulin G in bovine colostrum products

A CE-based noncompetitive immunoassay for IgG in bovine colostrum products was established. FITC-labeled protein G (FITC-PrG) was tagged through noncovalent bindings to the Fc region of the mouse monoclonal antibovine IgG (Ab). The FITC-PrG, Ab, and IgG formed a sandwiched immunocomplex FITC-PrG-Ab-IgG under optimal incubation conditions. The immunocomplex was separated and analyzed by CZE with LIF detection in less than 2 min in an uncoated fused-silica capillary. Addition of PEG 20,000 (PEG 20M) in the running buffer significantly suppressed analyte adsorption and thus improved the reproducibility and the resolution. The precision of the method was 5.1% (n = 7). A linear relationship was established for the IgG concentration in the range of 1-5 mg/L with a linear correlation coefficient (r = 0.9917). The LOD was 0.1 mg/L (S/N = 3). The method was successfully applied for the determination of IgG in bovine colostrum products and satisfactory results were achieved.     

A sensitive method for simultaneous determination of four macrolides by CE with electrochemiluminescence detection and its applications in human urine and tablets

A sensitive method for simultaneous determination of azithromycin (AZI), acetylspiramycin (ACE), erythromycin (ERY), and josamycin (JOS) was developed by CE coupled with electrochemiluminescence detection with Ru(bpy)₃²⁺. The parameters related to separation and detection were investigated in detail. The four macrolides were well separated and detected within 6 min under the optimized conditions. The LOD (S/N=3) of AZI, ACE, ERY, and JOS were 1.2×10^?9, 7.1×10^?9, 3.9×10^?8 and 9.5×10^?8 mol/L, respectively. The LOQ (S/N=10) of AZI, ACE, ERY, and JOS in human urine were 8.2×10^?8, 2.5×10^?7, 8.9×10^?7 and 1.2×10^?6 mol/L, respectively. The recoveries of the four macrolides in human urine and pharmaceutical tablet samples were 85.0–104.0% at different concentration levels.     

Intact and middle-down CIEF of commercial therapeutic monoclonal antibody products under non-denaturing conditions

A two-step CIEF with chemical mobilization was developed for charge profiling of the therapeutic mAb rituximab under non-denaturing separation conditions. CIEF of the intact mAb was combined with a middle-down approach analyzing Fc/2 and F(ab´)₂ fragments after digest with a commercial cysteine protease (IdeS). CIEF methods were optimized separately for the intact mAb and its fragments due to their divergent pIs. Best resolution was achieved by combining Pharmalyte (PL) 8–10.5 with PL 3–10 for variants of intact rituximab and of F(ab´)₂ fragments, respectively, whereas PL 6.7–7.7 in combination with PL 3–10 was used for Fc/2 variants. Charge heterogeneity in Fc/2 dominates over F(ab´)₂. In addition, a copy product of rituximab, and adalimumab were analyzed. Both mAbs contain additional alkaline C-terminal lysine variants as confirmed by digest with carboxypeptidase B. The optimized CIEF methods for intact mAb and Fc/2 were tested for their potential as platform approaches for these mAbs. The CIEF method for Fc/2 was slightly adapted in this process. The pI values for major intact mAb variants were determined by adjacent pI markers resulting in 9.29 (rituximab) and 8.42 (adalimumab). In total, seven to eight charge variants could be distinguished for intact adalimumab and rituximab, respectively.     

Thirty years of capillary electrophoresis in food analysis laboratories: potential applications

CE has generated considerable interest in the research community since instruments were introduced by different trading companies in the 1990s. Nowadays, CE is popular due to its simplicity, speed, highly efficient separations and minimal solvent and reagent consumption; it can also be included as a useful technique in the nanotechnology field and it covers a wide range of specific applications in different fields (chemical, pharmaceutical, genetic, clinical, food and environmental). CE has been very well evaluated in research laboratories for several years, and different new approaches to improve sensitivity (one of the main drawbacks of CE) and robustness have been proposed. However, this technique is still not well accepted in routine laboratories for food analysis. Researching in data bases, it is easy to find several electrophoretic methods to determine different groups of analytes and sometimes they are compared in terms of sensitivity, selectivity, precision and applicability with other separation techniques. Although these papers frequently prove the potential of this methodology in spiked samples, it is not common to find a discussion of the well-known complexity of the matrices to extract analytes from the sample and/or to study the interferences in the target analytes. Summarizing, the majority of CE scientific papers focus primarily on the effects upon the separation of the analytes while ignoring their behavior if these analytes are presented in real samples.     

Advances and analytical applications in chemiluminescence coupled to capillary electrophoresis

The present review presents the state of the art of the developments, key strategies and analytical applications of chemiluminescence detection coupled to CE (CE‐CL). Different parts considering the most common CL systems have been included, such as the tris(2,2′‐bipyridine)ruthenium(II) system, the luminol and derivatives reaction, the peroxyoxalate CL or direct oxidations. New advances in homemade configurations and applications in different fields such as clinical, pharmaceutical, environmental and food analysis have been included. The focus is on studies which appeared from 2000 to the end of 2009.     

Optimization of urinary pteridine analysis conditions by CE‐LIF for clinical use in early cancer detection

The second leading cause of death in the US is cancer and early discovery of the disease has translated into reduced fatality rates. We have identified and performed a systematic investigation of a method for urinary pteridine analysis by using CE‐LIF, which is believed to possess the potential to diagnose the presence of cancer even earlier than existing methodologies. Through system enhancements, we have been able to improve the resolution of the two least resolved sets of peaks (6,7‐dimethylpterin versus 6‐biopterin and D ‐(+)‐neopterin versus 6‐hydroxymethylpterin) from 0.85 to 2.48 and 0.90 to 3.58, respectively. Additionally, we have discovered that the preparation of the urine samples in previous works was inadequate, and we have corrected the method to fully oxidize the pteridines in the urine, resulting in significantly less variability in quantification and greater ease of defining p‐values for healthy versus cancer patients. Finally, we have performed validation steps of spike and recovery and short‐term aging studies to demonstrate the method's robustness. As a result, we present an optimized and validated method ready for transfer from discovery phase to clinical trial that can potentially act as a non‐invasively pre‐screening test for cancer.     

毛细管电泳分析中的富集技术及其应用

毛细管电泳（CE）具有分析速度快、分离效率高、样品消耗少、成本低廉等优点,已被应用于无机离子、有机小分子、蛋白质、核酸及细胞等的分析中。CE中最常用的检测方式是紫外检测（UV）,但由于常规进样样品体积小、检测光程短,CE-UV的灵敏度往往不能满足复杂样品中痕量物质直接分析的要求。CE中的在柱富集技术包括堆积、动态pH界面、吹扫和瞬间等速电泳等,可在很大程度上提高CE-UV的检测灵敏度;另外,固相和液相微萃取技术及其与在柱富集技术相结合应用在CE中也能净化样品基质,进一步提高富集倍数,改善分析灵敏度,从而拓宽了CE-UV在复杂样品分析中的应用范围。     

CIEF method optimization: Development of robust and reproducible protein reagent characterization in the clinical immunodiagnostic industry

Several method parameters have been refined for application of CIEF methods to provide optimal capillary robustness and performance longevity while maintaining desired analytical output for the ever increasing characterization scrutiny of protein reagents used in clinical assay formulations. Demonstrated here are significant modifications to the existing protocols in order to attain a robust, reproducible method that achieves as much as a 20‐fold increase in the number of consecutive runs before capillary degradation. Not only is it a concern for the rudimentary analysis of acidic and basic components of the isoform profile for monoclonal antibodies, but a comprehensive identification of each individual isoform to obtain a characteristic fingerprint is necessary for minor distinguishable properties between multiple proteins in unambiguous identification. In order to maintain the integrity of these modifications, extensive studies were conducted on an implemented system suitability standard protein with specifically defined parameters indicating either sufficient or poor separation performance.     

Highly sensitive voltammetric biosensor for nitric oxide based on its high affinity with hemoglobin

Although heme protein-based, amperometric nitric oxide (NO) biosensors have been well documented in previous studies, most have been conducted in anaerobic conditions. Herein we report a novel hemoglobin-based NO biosensor that is not only very sensitive but also usable in air. The heme protein was entrapped in a sodium montmorillonite film, which was immobilized at a pyrolytic graphite electrode surface. Film-entrapped hemoglobin can directly exchange electrons with the electrode, and this process has proven to favor the catalytic reduction of oxygen. In addition, NO induced a cathodic potential shift of the catalytic reduction peak of oxygen. This potential shift was proportional to the logarithm of NO concentration ranging from 4.0 × 10⁻¹¹ to 5.0 × 10⁻⁶ mol/L. The detection limit has been estimated to be 20 pM, approximately four orders lower than previously reported amperometric detectors.     

Interactions of hemoglobin with lecithin liposomes

In this paper, the interaction of hemoglobin (Hb) with lecithin liposomes is studied by UV-vis spectroscopy, fluorescent spectroscopy, and transmission electron microscopy. The adsorption of Hb on liposomes is likely to be related to the hydrophobic interaction between Hb and liposomes, which brings about the increase of the microenvironmental polarity (I ₁/I ₃) and the decrease of the fluorescence polarization (P) of lecithin liposomes. These results are considered to be that the adsorption of Hb on liposomes makes the spaces between the lecithin molecules increase, and a temporary gap is consequently formed in the liposomal bilayer membranes. The leakage of aqueous-space marker from the liposomes is increased with the addition of Hb.     

血红蛋白催化荧光法测定其氢供体底物酪氨酸

利用血红蛋白(Hb)的过氧化物酶特性,催化H2O2氧化氢供体底物酪氨酸(Tyr)的荧光反应,实验发现NH3配体对该反应产物的荧光强度有增强作用。通过反应机理的探讨,获得了反应速率方程。在NH3 NH4Cl的缓冲体系中优化了反应条件,建立了新的测定酪氨酸的催化荧光分析法。酪氨酸浓度在2.0×10-7～2.0×10-5mol/L范围内呈现良好的线性关系(r=0.9995),方法的检出限为4.63×10-9mol/L。已用于实际样品的测定。     

催化荧光光度法测定血红蛋白

基于血红蛋白(Hb)对过氧化氢氧化靛蓝胭脂红体系的催化作用,建立了模拟酶催化荧光光度法测定血红蛋白的新方法并对催化氧化反应的机理进行了初步探讨。实验发现体系的荧光强度与血红蛋白浓度在6.20×10-11~7.75×10-8mol/L范围内线性关系良好,方法的检出限为1.67×10-11mol/L。方法用于人血中血红蛋白含量的测定,回收率为98.8%~103.2%。     

亲水作用色谱及其在环境分析中的应用进展

亲水作用色谱(HILIC)是一种分离极性和亲水性化合物的液相色谱模式,其作为反相液相色谱(RPLC)的重要补充,近年来越来越受到各个领域的关注和重视。这不只是因为强极性化合物的分离问题在各个领域引起了重视,而且因为与RPLC比较,HILIC具有流动相组成黏度低、色谱柱渗透性好、与质谱联用的灵敏度高及反压较低等优势。本文简要概述了HILIC的发展历程、特点及保留机理,重点介绍了HILIC用于环境分析的最新进展,评述了HILIC及RPLC用于污染物分析的优缺点,并指出了HILIC用于环境分析的未来发展趋势。     

Electrode modified with toluidine blue-doped silica nanoparticles, and its use for enhanced amperometric sensing of hemoglobin

Three-dimensionally structured, silica based, organic–inorganic hybrid nanoparticles (NPs) were prepared by a simple and feasible water-in-oil (W/O) microemulsion method and a promising platform for bioelectrochemical analysis was obtained. The commonly used phenathiazine organic compound, toluidine blue (TB) was readily captured in the three-dimensional cage of the inorganic SiO₂ network, which was considered to serve as a protective “shell” toward the embedded TB. A TEM image indicated the size of the thus prepared TB-doped SiO₂ (TB@SiO₂) NPs was 21 ± 3 nm. UV–visible and IR spectroscopy confirmed successful formation of the organic–inorganic composite and possible interaction between TB and SiO₂, which favored enhanced stability of the hybrid. A sensitive amperometric sensor for hemoglobin (Hb) biomolecules based on TB@SiO₂ NPs conjugated with a biopolymer chitosan (CHIT) membrane was then developed. The surface of the silica NPs was highly biocompatible and the TB captured inside maintained its high electron-transfer efficiency. Dye leakage of TB from the TB@SiO₂ hybrid was proved to be minimal, owing to the inorganic SiO₂ network and the force of interaction between TB and SiO₂. The amperometric sensor had a detection limit of 2.5 × 10⁻⁹ mol L⁻¹ (S/N = 3) with a linear range from 5.0 × 10⁻⁹ to 3.0 × 10⁻⁶ mol L⁻¹ for Hb. When it was applied to determine the concentration of a clinical blood sample, satisfactory results were obtained which were in good agreement with those obtained by the standard method.     

Flavonoids biosynthesis in plants and its further analysis by capillary electrophoresis

Flavonoids represent an important bioactive component in plants. Accumulation of flavonoids often occurs in plants subjected to abiotic stresses, including the adaptation of plants to the environment and in overcoming their stress conditions. This fact makes their analysis and determination an attractive field in food science since they can give interesting information on the quality and safety of foods. In this study, we discuss reports on plants flavonoids biosynthesis against abiotic stresses and advances in analytical capillary electrophoresis used for their identification and quantification in plants.     

血红蛋白在海藻酸钠膜中的电化学和电催化行为

用海藻酸钠(SodiumAlginate,SA)将血红蛋白(Hb)固定在热裂解石墨电极表面,制备了Hb SA膜修饰电极。包埋在海藻酸钠膜中的血红蛋白与电极直接传递电子。在pH7.0的磷酸盐缓冲溶液中可得到一对可逆的血红蛋白辅基血红素Fe(Ⅲ) Fe(Ⅱ)电对氧化还原峰,式电势为-0.364V(vs.SCE)。其式电势随溶液pH值增加而负移且成线性关系,直线斜率为-36.0mV pH,说明血红蛋白的电子传递过程伴随有质子的转移。并研究了Hb SA膜修饰电极对O2、H2O2和NO的电催化性质。     

Sixteen capillary electrophoresis applications for viral vaccine analysis

A broad range of CE applications from our organization is reviewed to give a flavor of the use of CE within the field of vaccine analyses. Applicability of CE for viral vaccine characterization, and release and stability testing of seasonal influenza virosomal vaccines, universal subunit influenza vaccines, Sabin inactivated polio vaccines (sIPV), and adenovirus vector vaccines were demonstrated. Diverse CZE, CE-SDS, CGE, and cIEF methods were developed, validated, and applied for virus, protein, posttranslational modifications, DNA, and excipient concentration determinations, as well as for the integrity and composition verifications, and identity testing (e.g., CZE for intact virus particles, CE-SDS application for hemagglutinin quantification and influenza strain identification, chloride or bromide determination in process samples). Results were supported by other methods such as RP-HPLC, dynamic light scattering (DLS), and zeta potential measurements. Overall, 16 CE methods are presented that were developed and applied, comprising six adenovirus methods, five viral protein methods, and methods for antibodies determination of glycans, host cell-DNA, excipient chloride, and process impurity bromide. These methods were applied to support in-process control, release, stability, process- and product characterization and development, and critical reagent testing. Thirteen methods were validated. Intact virus particles were analyzed at concentrations as low as 0.8 pmol/L. Overall, CE took viral vaccine testing beyond what was previously possible, improved process and product understanding, and, in total, safety, efficacy, and quality.