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.     

Studying protein-drug interaction by microfluidic chip affinity capillary electrophoresis with indirect laser-induced fluorescence detection

We developed a microfluidic chip-affinity CE method based on indirect LIF detection to study protein-drug interactions. The interaction between heparin and BSA was quantitatively studied, as a model system. In our method, sodium fluorescein was chosen as background, and redistilled water as marker to monitor EOF. The electrophoretic mobility changes of BSA were measured, with various concentrations of heparin added to the running buffer. Each run was completed within 80 s. The binding constant was determined to be (1.24 +/- 0.05) x 10(3) M(-1), which was in good agreement with that reported in the literature.     

Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser-induced fluorescence detection

Carbonyl-modified proteins are markers of oxidative damage. Here, we report a new method for detecting and quantifying carbonylated proteins by capillary sieving electrophoresis (CSE) with LIF detection (CSE-LIF). Alexa 488 hydrazide is used for the specific labeling of carbonyls while 3-(2-furoyl) quinoline-2-carboxaldehyde (FQ) is used for protein labeling. BSA subjected to metal-catalyzed oxidation is used to optimize the labeling reactions, confirm the separation power of CSE, and characterize the response of the LIF detector. The method is capable of detecting femtomole (fmol) amounts of carbonyls in proteins with molecular masses ranging from 26 to 30 kDa. Using this method, we determined that mitochondrial proteins isolated from skeletal muscle contains 2.1 +/- 0.1 (average +/- SD; n = 3) nmol carbonyl/mg protein. The methodology described here should be compatible with the analysis of single cells and needle biopsies taken from oxidative stress animal models.     

Determination of the cardiolipin content of individual mitochondria by capillary electrophoresis with laser-induced fluorescence detection

We report the application of capillary electrophoresis (CE) with postcolumn laser-induced fluorescence (LIF) detection to measure the cardiolipin content of individual mitochondria from cultured NS1 cells. Mitochondria were isolated by differential centrifugation and stained with the fluorescent dye 10-N-nonyl acridine orange which stoichiometrically binds to cardiolipin in a 1:1 or 2:1 ratio depending on the dye concentration. The green fluorescence resulting from the 1:1 complex was chosen for analysis because it is substantially more intense than the red fluorescence resulting from the 2:1 complex. Two dye concentrations that resulted in maximal and submaximal formation of the 1:1 10-N-nonyl acridine orange-cardiolipin complex were identified by spectrofluorometry. Individual mitochondria stained with both dye concentrations were separated and detected by CE with LIF detection. The data from mitochondria dosed with the lower dye concentration, where it is assumed that all the dye added to the mitochondrial sample was bound to cardiolipin, were used to derive a sensitivity factor relating fluorescence intensity of a mitochondrial event to its cardiolipin content. Using this factor, the cardiolipin contents of individual mitochondria stained with the higher dye concentration were determined, and ranged from 1.2 to 920 amol, with a median value of 4 amol. These results suggest a new strategy for estimating the organellar content of compounds that can be fluorescently tagged.     

Electrophoretic behavior of individual nuclear species as determined by capillary electrophoresis with laser-induced fluorescence detection

We determined the feasibility of using capillary electrophoresis with postcolumn laser-induced fluorescence (CE-LIF) detection to characterize electrophoretic properties of isolated cell nuclei and impurities present in nuclear fractions. These fractions were isolated from NS-1 mouse hybridoma cells, stained with hexidium iodide, a DNA intercalating dye, and analyzed by CE-LIF detection. The corresponding electropherograms display two features: (i) broad peaks (6-90 s wide) caused by the cell culturing media and by free-DNA intercalated with hexidium iodide, and (ii) a large number of narrow peaks (180 ms wide), resulting from DNA associated with individual intact or disrupted nuclei. We confirmed that the narrow peaks were not caused by contaminating mitochondria. The overall electrophoretic mobility range of disrupted nuclei is 0 to -5 x 10(-4)cm(2)/Vs, while intact nuclei seem to have mobilities in the -1.5 to -3.5 x 10(-4)cm(2)/Vs range. Furthermore, the highly sensitive CE-LIF method reveals a high abundance of disrupted nuclei that cannot be directly observed by confocal microscopy.     

Analysis of individual platelet-derived microparticles,comparing flow cytometry and capillary electrophoresis with laser-induced fluorescence detection

Platelet-derived microparticles (PMPs) formed by vesiculation during platelet activation seem to play a role in blood coagulation and in pathological disease states. Flow cytometry is currently the gold standard to characterize platelets and PMPs. Using this technique we distinguished between platelets and PMPs based on size and the presence of phosphatidyl serine (PS); PMPs were arbitrarily defined to be smaller than one micrometer and capable of forming a stable complex with fluorescently-labeled Annexin V, a protein that forms a calcium-dependent complex with PS. Further confirmation of PMP and platelet identity was done by use of fluorescently-labeled antibodies against CD41a, a glycoprotein found on the surface of both platelets and PMPs. In this report we also introduce the use of capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) for the analysis of fluorescently labeled platelets and PMPs. While both flow cytometry and CE-LIF can measure individual fluorescent events, only CE-LIF allowed us to calculate individual electrophoretic mobilities of activated platelets and PMPs that were then represented as distributions. A comparison between distributions suggests that PMPs have less negative mobilities. The fact that activated platelet preparations include PMPs partially obscure the interpretation of the data. While PMP and platelet number ml(-1) determined by flow cytometry is lower than the same parameter determined by CE-LIF, signal-to-noise ratio was 20 fold better for flow cytometry than for CE-LIF. This is the first time that a direct comparison between these two techniques is reported.     

Capillary electrophoresis of aminoglycosides with argon-ion laser-induced fluorescence detection

A new analytical method for aminoglycosides (kanamycin, bekanamycin, paromomycin and tobramycin) based on capillary electrophoretic separation and argon-ion laser-induced fluorescence detection has been developed. 6-Carboxyfluorescein succinimidyl ester (CFSE) was used for precolumn derivatization of the non-fluorescent aminoglycosides. Optimal separation and detection were obtained with an electrophoretic buffer of 30 mM sodium borate (pH 9.0) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). The concentration limits of detection in aqueous solution were 3.9-8.2 nM. Combined with a simple cleanup procedure, this method can be applied to the determination of aminoglycosides in human plasma. A calibration curve ranging from 0.15 to 30 microM was shown to be linear. The limits of detection of aminoglycosides in human plasma were between 14.4 and 24.0 nM. Recoveries of spiked aminoglycosides in human plasma were between 92 and 105%.     

Simultaneous laser-induced fluorescence and contactless-conductivity detection for microfluidic chip

A combined detection system involving simultaneous LIF and contacfless-conductometric measurements at the same place of the microfluidic chip was described. The LIF measurement was designed according to the confocal principle and a moveable contactless-conduetivity detector was used in C^4D. Both measurements were mutually independent and advantageous in analyses of mixtures. Various experimental parameters affecting the response were examined and optimized. The performances were demonstrated by simultaneous detection of Rhodamine B. And the results showed that the combined detection system could be used sensitively and reliably.     

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.     

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。     

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.     

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.     

Bio-assay based on single molecule fluorescence detection in microfluidic channels

A rapid bioassay is described based on the detection of colocalized fluorescent DNA probes bound to DNA targets in a pressure-driven solution flowing through a planar microfluidic channel. By employing total internal reflection excitation of the fluorescent probes and illumination of almost the entire flow channel, single fluorescent molecules can be efficiently detected leading to the rapid analysis of nearly the entire solution flowed through the device. Cross-correlation between images obtained from two spectrally distinct probes is used to determine the target concentration and efficiently reduces the number of false positives. The rapid analysis of DNA targets in the low pM range in less than a minute is demonstrated.     

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.     

Detection of recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of recombinant hirudin (r-hirudin), a specific and reproducible analysis method is required. Capillary electrophoresis (CE) is rapidly becoming an important procedure for the analysis of biological molecules. Recently, CE combined with immunoassay has emerged as a new analytical technique. CE-based immunoassay (CEIA) is a sensitive and specific method combining laser-induced fluorescence (LIF) and immunoassay. Therefore, in this study, we specifically investigated fluorescence labeling and determination of r-hirudin by CEIA with a LIF detector using labeled r-hirudin and polyclonal antibody. r-Hirudin was labeled with fluorescein isothiocyanate (FITC). FITC-labeled r-hirudin was purified using high-performance liquid chromatography (HPLC). The method is based on preincubation of r-hirudin and antibody for 20 min, followed by CE analysis using an uncoated capillary. Free and bound r-hirudin were separated within 5 min using CE with high reproducibility. This study demonstrated that the CEIA method could be applied to quantitative analysis of r-hirudin in biological fluids.     

Capillary electrophoresis of FITC labeled amino acids with laser-induced fluorescence detection

FITC labeled amino acids have been separated using a home-huilt capillary electrophoresis with a laserinduced fluorescence detection (CE-LIF) system. Seventeen peaks can now be generated from the twenty common amino acids. The key conditions lie in the optimization of pH, buffer electrolytes and buffer additives.     

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.     

Analysis of amino acids in individual human erythrocytes by capillary electrophoresis with electroporation for intracellular derivatization and laser-induced fluorescence detection

A method for monitoring amino acids in single erythrocytes is described. For intracellular derivatization, reagent fluorescein isothiocyanate (FITC) was introduced into living cells by electroporation. For an 8 microm erythrocyte, the analytes were diluted by a factor of only 1.6. After completion of the derivatization reaction, a single cell was injected into the separation capillary tip and lysed there. The derivatized amino acids were separated by capillary electrophoresis, followed by laser-induced fluorescence detection. Nine amino acids were quantitatively determined, with amounts of amino acids ranging from 3.8-32 amol/single cell.     

Determination of the DNA methylation level in tumor cells by capillary electrophoresis and laser-induced fluorescence detection

An analytical method to determine the genome-wide DNA methylation in only 100 ng DNA is presented. The analysis is based on DNA isolation and hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with a fluorescence dye (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride, Bodipy FL EDA). The separation of the derivatives was carried out by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. To calculate the methylation level, the derivatization factor and the quantum yields of the Bodipy conjugates of 2'-desoxycytidine-3'-monophosphate (dCMP) and 2'-desoxy-5-methylcytidine-3'-monophosphate (5m-dCMP) were determined by measurement of methylated Lambda DNA. The assignment was made by cochromatography with the synthesized and characterized standard compound 5m-dCMP. After optimization of the method it was possible to determine the methylation level in 100-ng DNA samples with a standard deviation of less than 5%.