Plasma thiols redox status by laser-induced fluorescence capillary electrophoresis

High concentrations of total plasma thiols such as cysteine and homocysteine are important risk factors for atherosclerosis and cardiovascular diseases. We have recently described a new laser-induced fluorescence capillary electrophoresis (CE-LIF) method to measure total plasma thiols, in which the baseline separation of cysteinylglycine, homocysteine, cysteine, and glutathione was achieved by adding the organic base N-methyl-D-glucamine to the run buffer. However, because the active fractions of homocysteine and cysteine responsible for vascular injuries are still unknown, research calls for a set up of methods able to analyze different forms of plasma thiols. In this paper, we present an improvement of our previous method that allows the measurement of different thiol forms. Total, reduced, and free thiols were measured by varying the order of disulfide reduction with tributylphosphine and proteins precipitation with 5-sulfosalicylic acid. After derivatization with 5-iodoacetamidofluorescein, samples were separated and measured by CE-LIF using a phosphate/borate buffer in the presence of 75 mmol/L N-methyl-D-glucamine. Oxidized thiols and protein bound thiols were calculated by difference, free minus reduced and total minus free form, respectively. Linearity, reproducibility, analytical recovery, and sensitivity were evaluated. The assay was used to measure the thiols redox status in 15 plasma samples from healthy volunteers.     

Multiplexed microRNA detection by capillary electrophoresis with laser-induced fluorescence

In this study, we developed a novel assay that simultaneously detects multiple miRNAs (microRNAs) within a single capillary by combining a tandem adenosine-tailed DNA bridge-assisted splinted ligation with denaturing capillary gel electrophoresis with laser-induced fluorescence. This proposed method not only represents a significant improvement in resolution but also allows for the detection of multiple miRNAs within a single capillary based on the length differences of specified target bridge DNA. The assay's linear range covers three orders of magnitude (1.0 nM to 1.0 pM) with a limit of detection (S/N=3) as low as 190 fM (2.5 zmol). Five miRNAs of Epstein-Barr virus (EBV) were also detected in EBV-infected nasopharyngeal carcinoma cells, while they did not appear in non-virus infected cells. Moreover, the electropherogram indicated that the screening of isomiRs (isomer of miRNA) of BART2 by CE-LIF is feasible by our proposed method. The developed electrophoresis-based method for miRNA detection is fast, amplification-free, multiplexed and cost-effective, making it potentially applicable to large-scale screening of isomiRs.     

Assays for total homocysteine and other thiols by capillary electrophoresis-laser-induced fluorescence detection. I. Preanalytical condition studies

In recent papers, we presented a new analytical method for thiol quantification in serum. It is based on the use of capillary electrophoresis and laser-induced fluorescence to analyze thiol 6-iodoacetamidofluoresceine (IAF) derivatives. Quantitative results of homocysteine, glutathione, cysteine-glycin, and cysteine were shown (Clin. Chem. 45 (1999) 412). A comprehensive comparison of the quantitation of homocysteine in serum, using high-performance liquid chromatography/conventional fluorescence detection and fluorescence polarization immunoassay was also used (E. Caussé et al., Electrophoresis 21 (2000) 2074). Sample preparation prior to derivatization with IAF had never been investigated. In this work we present the results of quantitation of thiols in serum and plasma with three different anticoagulants widely used: ethylenediaminetetraacetic acid (EDTA), heparin, and sodium citrate. We show that serum and EDTA plasma gave the same results. Then serum protein precipitations by acetonitrile, acetone, sulfosalicylic acid, perchloric acid and trichloracetic acid, prior to derivatization by IAF, were also investigated. Their influence on the concentrations of the thiols were determined. Sulfosalicylic acid and acetonitrile precipitations are well adapted, whereas acetone cannot be used.     

Determination of phycobiliproteins by capillary electrophoresis with laser-induced fluorescence detection

Phycobiliproteins are derived from the photosynthetic apparatus of cyanobacteria and eukaryotic algae. They are composed of a protein backbone to which linear tetrapyrrole chromophores are covalently bound. Furthermore, they are water-soluble highly fluorescent, and relatively stable at room temperature and neutral pH. For this reason, capillary electrophoresis-laser induced fluorescence (CE-LIF) seems the idea method for determination of these important proteins. The effects of buffer additives such as sodium dodecyl sulfate (SDS)and putrescine on the separation of the three major phycobiliprotein types, namely allophycocyanin, phycocyanin, and phycoerythrin, with excitation and emission maxima at 652/660, 615/647, and 565(494)/575 nm, respectively, are considered. Detection limits for these proteins by CE-LIF are some 60-500 times better than by absorbance detection. The development of a fast and sensitive CE-LIF assay such as this is of potential significance to our understand ing of chemical and biological oceanographic processes.     

Determination of nitrocellulose by capillary electrophoresis with laser-induced fluorescence detection

The industrial application of nitrocellulose depends on its nitrogen content. When nitrocellulose presents high nitrogen content is used in the manufacture of explosives whereas nitrocellulose with low nitrogen content is used to make a wide range of daily and non-explosive products (e.g. cigarettes, paints, lacquers). This fact makes really important to develop a method for the determination and discrimination of nitrocellulose samples. This work reports, for the first time, the qualitative determination of nitrocellulose previously derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) by capillary electrophoresis (CE-LIF) with laser-induced fluorescence detection (CE-LIF). APTS-labeled nitrocellulose was determined in lowly and highly nitrated nitrocellulose samples present in collodions and smokeless gunpowders, respectively, after their pulverization in liquid nitrogen. The method described enables the visual discrimination of different nitrocelluloses on the basis of the different electrophoretic profiles obtained, and provides a useful tool to determine nitrocellulose. Additionally, the use of field-amplified sample injection (FASI) enabled enhanced sample detection, which made it possible to determine nitrocellulose contained in ∼15 μg of gunpowder.     

N-methyl-D-glucamine improves the laser-induced fluorescence capillary electrophoresis performance in the total plasma thiols measurement

We describe an ultrarapid capillary electrophoresis with laser-induced fluorescence (CE-LIF) method for total plasma thiols measurement. Reduced thiols by 10% tri-n-butylphosphine (TBP) were derivatized in 10 min at room temperature with 5-iodoacetamidofluorescein (5-IAF) as fluorescent reagent. We show that CE-LIF allows a baseline separation of total plasma cysteinylglycine, homocysteine, cysteine, and glutathione in less than 5 min when N-methyl-D-glucamine in run buffer was added. CE was compared with high-performance liquid chromatography (HPLC) with fluorescence detection. The Bland-Altman test and Passing-Bablok regression demonstrates that the results obtained by CE-LIF and by HPLC are highly comparable. The simplified procedure of sample preparation, the short incubation and fast separation times, the high specificity, sensitivity and reproducibility, and the lower cost of analysis suggest that our proposed method can be considered valuable for the automation analysis in a routine laboratory.     

Analysis of baclofen by capillary electrophoresis with laser-induced fluorescence detection

A new analytical method for baclofen (4-amino-3-p-chlorophenylbutyric acid) based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. Naphthalene-2,3-dicarboxaldehyde was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) and a He-Cd laser (excitation at 442 nm, emission at 500 nm). Linearity (r > or = 0.99) over three orders of magnitude was generally obtained and the concentration limit of detection was in the nanomolar level. Coupled with a simple cleanup procedure, the method was successfully applied to the analysis of baclofen in human plasma. Recovery of spiked baclofen in plasma was 98%. The relative standard deviation values on peak size and migration time were 7.9% and 0.4%, respectively. The limit of detection of baclofen in plasma was 10 ng/ml.     

Near-infrared laser-induced fluorescence detection in capillary electrophoresis

As capillary electrophoresis continues to focus on miniaturization, either through reducing column dimensions or situating entire electrophoresis systems on planar chips, advances in detection become necessary to meet the challenges posed by these electrophoresis platforms. The challenges result from the fact that miniaturization requires smaller load volumes, demanding highly sensitive detection. In addition, many times multiple targets must be analyzed simultaneously (multiplexed applications), further complicating detection. Near-infrared (NIR) fluorescence offers an attractive alternative to visible fluorescence for critical applications in capillary electrophoresis due to the impressive limits of detection that can be generated, in part resulting from the low background levels that are observed in the NIR. Advances in instrumentation and fluorogenic labels appropriate for NIR monitoring have led to a growing number of examples of the use of NIR fluorescence in capillary electrophoresis. In this review, we will cover instrumental components used to construct ultrasensitive NIR fluorescence detectors, including light sources and photon transducers. In addition, we will discuss various types of labeling dyes appropriate for NIR fluorescence and finally, we will present several applications that have used NIR fluorescence in capillary electrophoresis, especially for DNA sequencing and fragment analysis.     

Study of DNA-ellipticine interaction by capillary electrophoresis with laser-induced fluorescence detection

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), an alkaloid isolated from Apocynaceae plants, exhibits an antitumor activity, which is exceptionally high against several specific types of tumors. Ellipticine is also interesting as an anticancer drug as it has limited side effects and lacks of hematological toxicity. Various methods to study intercalating activity of this drug have been developed. However, to our best knowledge, capillary electrophoresis (CE) as a technique combining high separation resolution with various detection options has never been used for these purposes. In this study, a novel separation method based on CE with laser-induced fluorescence (CE-LIF) detection has been developed for the determination of ellipticine and for the monitoring of ellipticine-DNA interaction. Sodium acetate (50 mM, pH 4.5) was used as a background electrolyte and LIF detection at λ(ex) = 488 nm. The limit of detection for ellipticine was determined to be 5 × 10?? M. A total of 20% dimethyl sulfoxide was found optimal as sample solvent. Additionally, intercalation of ellipticine into the double-stranded DNA was investigated. Signal corresponding to ellipticine was decreasing and a new peak appeared and was growing. It can be concluded that CE-LIF is a method applicable to in vitro studies of ellipticine-DNA complexes.     

Determination of abscisic acid by capillary electrophoresis with laser-induced fluorescence detection

A novel method based on capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF) was developed for the determination of abscisic acid (ABA), which is an essential phytohormone during plant growth and development. ABA was labeled with 8-aminopyrene-1,3,6-trisulfonate via reductive amination in presence of acetic acid and sodium cyanoborohydride. The derivatization yield was maximized by optimizing several derivatization parameters including derivatization reagent concentration, reaction temperature and time. The conjugate was separated and quantitated by CE-LIF. The linearity of ABA was determined in the range from 0.1 to 10 micromol l(-1) with a correlation of 0.9979. The derivatization limit of detection for ABA was found to be 56 fmol (corresponding to the concentration of 2.8 x 10(-8) mol l(-1)). The detection limit for ABA was 5.5 amol for an injection volume of 5 nl. As a preliminary application, the proposed method was successfully applied to determining trace amount of ABA in the crude extracts of tobacco without extra purification and enrichment procedure and showed a better selectivity and sensitivity than those conventional methods used in determination of ABA.     

Direct immunoassay of estrone by capillary electrophoresis with laser-induced fluorescence detection

An immunoassay for estrone (E(1)) in women's serum, based on the competitive reaction between fluorescein-labeled complete antigen and E(1) with limited amount of anti-estrone monoclonal antibody is described. A thermally reversible hydrogel, poly-N-isopropylacrylamide (pNIPA), was added to the buffer to improve the reproducibility. With a laser-induced fluorescence (LIF) detector, the capillary electrophoretic immunoassay (CEIA) can be applied to determine E(1) at a concentration lower than 19.6 pg/mL. The E(1) levels in ten normal women's serum were measured at the range of 118.6-222.0 pg/mL.     

Single-cell analysis by intracellular immuno-reaction and capillary electrophoresis with laser-induced fluorescence detection

A novel method for single-cell analysis was developed by combining electroporation for intracellular immuno-reaction and capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Human interferon-gamma (IFN-gamma) in natural killer (NK) cells was chosen as the test antigen. Two forms of IFN-gamma in single cells could be well separated and detected with a limit of detection of zeptomole. In this assay, the anti-IFN-gamma monoclonal antibody labeled with fluorescein isothiocyanate (Ab*) was introduced into NK cells by electrophoration for intracellular immuno-reaction. After completion of the intracellular immuno-reaction, the NK cells were chemically pre-perforated with digitonin to lyse easily. Then, one NK cell containing the complexes of IFN-gamma isoantigens with Ab* was electrokinetically injected into the capillary. The cell adsorbed on the tip of capillary was lysed by ultrasonication. Finally, the complexes of the different forms of IFN-gamma in the cell were separated and detected by CE-LIF detection.     

Indirect laser-induced fluorescence detection of diuretics separated by capillary electrophoresis

Indirect LIF detection was applied to the detection of four acidic diuretics separated by CZE. Semiconductor laser was employed to provide the stable excitation of 473 nm. With an optimized electrophoretic buffer system which contained 5 mM of triethylamine, 0.1 microM of fluorescein, and 5% of n-butanol, fast separation of four diuretics (ethacrynic acid, chlorthalidone, bendroflumethiazide, and bumetanide) can be performed within 3 min with the detection limits of 0.2-2 microg/mL. The impacts of buffer components including the concentrations of the electrolytes, fluorescence probe, and the organic additives were demonstrated. The method was applied for the detection of diuretics in urine. As an alternative way for the fast analysis of diuretics, this indirect detection method provided the technical support for future microchip performances, in which diuretics may be detected in the microchip by the common LIF detector without derivatization.     

Solid-state UV laser-induced fluorescence detection in capillary electrophoresis

Two solid-state UV lasers were applied to the laser-induced fluorescence (LIF) detection of various groups of compounds after separation by capillary electrophoresis. These lasers are thermoelectric-cooled, highly compact, and inexpensive. Such lasers provide few mW of quasi-continuous wave (CW) power which are sufficient and stable for LIF detection. Native fluorescence detection of tryptophan-containing proteins and peptides and related indoles was achieved at the nM level with the laser operating at 266 nm. Detection of fluorescamine-labeled amino acids and peptides was also possible at the nM level with the laser operating at 355 nm. Amino acids at a concentration as low as 10 ng/mL could be labeled with fluorescamine. Solid-state UV-LIF detection of the tryptic digest of cytochrome c after fluorescamine derivatization was demonstrated.     

Nonaqueous capillary electrophoresis with laser-induced fluorescence detection: a case study of comparison with aqueous media

A novel method based on separation by nonaqueous capillary electrophoresis (NACE) combined with laser-induced fluorescence (LIF) detection was developed and compared with classic aqueous modes of electrophoresis in terms of resolution of solutes of interest and sensitivity of the fluorescence detection. Catecholamines derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) were chosen as test analytes for their subtle fluorescence properties. In aqueous systems, capillary zone electrophoresis (CZE) was not suitable for the analysis of test analytes due to complete fluorescence quenching of NBD-labeled catecholamines in neat aqueous buffer. The addition of micelles or microemulsion droplets into aqueous running buffer can dramatically improve the fluorescence response, and the enhancement seems to be comparable for micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC). As another alternative, NACE separation was advantageous when performing the analysis under the optimum separation condition of 20 mM sodium tetraborate, 20 mM sodium dodecyl sulfate (SDS), 0.1% (v/v) glacial acetic acid, 20% (v/v) acetonitrile (ACN) in methanol medium after derivatization in ACN/dimethyl sulfoxide (DMSO) (3:2, v/v) mixed aprotic solvents containing 20 mM ammonium acetate. Compared with derivatization and separation in aqueous media, NACE-LIF procedure was proved to be superior, providing high sensitivity and short migration time. Under respective optimum conditions, the NACE procedure offered the best fluorescence response with 5-24 folds enhancement for catecholamines compared to aqueous procedures. In addition, the mechanisms of derivatization and separation in nonaqueous media were elucidated in detail.     

Measuring the activity of farnesyltransferase by capillary electrophoresis with laser-induced fluorescence detection

Enzymatic farnesylation of oncogenic forms of Ras proteins is the initial step in a series of posttranslational modifications essential for Ras activity. The modification is catalyzed by the enzyme, protein farnesyltransferase (PFTase), which transfers a farnesyl moiety from farnesyl diphosphate to the protein. We employed capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection to develop a rapid and sensitive method for the determination of PFTase activity in vitro. The limited substrate specificity of PFTase allowed us to use a fluorescently labeled pentapeptide instead of a Ras protein as a substrate for the enzyme; the product of the enzymatic reaction was the farnesylated pentapeptide. The product was separated from the substrate by CE and quantified with LIF detection. Under optimal conditions, the separation was achieved within 10 min with a resolution of 86. The mass and concentration limits of detection for the farnesylated product were 10(-19) mol and 0.28 nM, respectively. By measuring the rate of accumulation of the farnesylated product, we were able to determine the kinetic parameters of the enzymatic reaction. For yeast PFTase as an enzyme and difluorocarboxyfluorescein-labeled GCVIA peptide as a substrate, the values of k(cat) and K(M) were found to be (3.1 +/- 0.3)x10(-3) s(-1) and (12.0 +/- 1.2) nuM, respectively. Our results suggest that CE-LIF can be efficiently used for the determination of enzymatic activity of PFTase in vitro. After minor modifications, the developed method can be also applied to other reactions of enzymatic prenylation of proteins.     

毛细管电泳-激光诱导荧光检测法测定抗癫痫药加巴喷丁

建立了毛细管电泳-激光诱导荧光(CE-LIF)测定抗癫痫药加巴喷丁的方法。加巴喷丁经4-氯-7-硝基苯并-2-氧杂-1,3-二唑(NBD-Cl)衍生化后,采用10 mmol/L硼砂-10 mmol/L十二烷基硫酸钠(pH 9.75)的缓冲体系,加巴喷丁在6 min内实现高效基线分离。方法的线性范围为0.01～10 mg/L(r=0.9997),检出限为2 μg/L,定量限为10 μg/L。方法的平均回收率为100.2%～103.1%,相对标准偏差为0.15%～1.00%(n=3)。该方法灵敏、快速、准确和可靠,已用于加巴喷丁药物制剂的质量控制。     

The immunoassay of methotrexate by capillary electrophoresis with laser-induced fluorescence detection.

Immunoassay is widely employed as a highly sensitive, specific analytical method for hormones and drugs in biological samples. A technique utilizing capillary electrophoresis with laser-induced fluorescence detection was examined based on the reaction process of these immunoassays in order to develop a protocol characterized by high sensitivity and high speed. The conditions of the antigen-antibody reaction and capillary electrophoresis were variously examined using fluorescein-labeled methotrexate and the antibody of methotrexate. As a result, the immunoassay could be completed within a few minutes. Moreover, detection in the pg range could be accomplished. The sensitivity corresponded to that of radioimmunoassay. A simultaneous multi-component analysis of the immunoassay is also possible due to the high resolving power of capillary electrophoresis. In this study, the possibility of a simultaneous analysis of methotrexate and vancomycin was also investigated.     

Determination of individual microsphere properties by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis with postcolumn laser-induced fluorescence detection was used to individually detect 6.0, 1.0, 0.5, and 0.2 num diameter polystyrene microspheres and individually measure their electrophoretic mobility. The analysis of a nanoliter-size volume from a microsphere suspension results in an electropherogram characterized by several narrow spikes in a well-defined migration time window. Each spike is associated with one microsphere because, when one single microsphere is introduced into the capillary by micromanipulation, the electropherogram has only one spike in the same migration time window. The distributions of individual measurements resulting from an electropherogram were used to evaluate the reproducibility from run to run, observe the effect of sodium dodecyl sulfate (SDS) added to the running buffer, and to investigate the origin of electrophoretic dispersion. As expected from the interactions between microspheres and SDS, the addition of this surfactant to the running buffer narrowed the range and shifted the average electrophoretic mobility to more negative values. After evaluating common sources of broadening in capillary electrophoresis, electrophoretic dispersion was attributed to microsphere heterogeneity. Unlike electropherograms displaying Gaussian-like profiles, the two-dimensional representations of the individual measurements provide a new alternative to evaluate and study electrophoretic-related properties of microspheres.     

Sensitive determination of ephedrine and pseudoephedrine by capillary electrophoresis with laser-induced fluorescence detection

A CE-LIF method was developed for the separation and sensitive detection of ephedrine and pseudoephedrine after derivatization by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol (NBD-C1). The derivatization and separation conditions were investigated in detail and the optimum conditions were obtained. Under the optimum experiment conditions, good linearity relationships (correlation coefficients: 0.9942 for ephedrine and 0.9970 for pseudoephedrine) between the peak heights and concentrations of the analytes were obtained (0.7-140 microM). The detection limits were 0.16 microM for ephedrine and 0.17 microM for pseudoephedrine, which indicated that the sensitivities were at least ten times improved over those reported in the literature obtained by UV detection. The method was applied to the analysis of ephedrine and pseudoephedrine in ephedra herb plants and preparations with good results.