Inverse-flow derivatization for capillary electrophoresis of DNA fragments with laser-induced fluorescence detection

A novel method is presented to detect DNA fragments separated by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection using inverse-flow derivatization. In electrophoresis, the intercalating dye, thiazol orange was only added to the separation buffer at the positive polarity. The negatively charged DNA fragments migrated from the negative polarity to the positive polarity, while the positively charged dye migrated in the opposite direction. When DNA fragments met with dye ions, the DNA–dye complexes were formed. The complexes continued migrating to the positive end, due to their net negative charges. When the complexes passed through the detection window, the fluorescent signals were generated. Importantly, DNA fragments migrated as their native state before DNA–dye complexes were formed. This procedure was used to detect double stranded DNA (dsDNA) and single stranded DNA (ssDNA) fragments, and polymerase chain reaction (PCR) products. The excellent resolution and good reproducibility of DNA fragments were achieved in non-gel sieving medium. This procedure may be useful in genetic mutation/polymorphism detections.     

Separation and quantitation of phycobiliproteins using phytic acid in capillary electrophoresis with laser-induced fluorescence detection

The similar electrophoretic mobilities and sizes of several of the phycobiliproteins, which are derived from the photosynthetic apparatus of cyanobacteria and eukaryotic algae, render their separation and quantitation a challenging problem. However, we have developed a suitable capillary electrophoresis (CE) method that employs a phytic acid-boric acid buffer and laser-induced fluorescence (LIF) detection with a single 594 nm He-Ne laser. This method takes advantage of the remarkably high quantum yields of these naturally fluorescent proteins, which can be attributed to their linear tetrapyrrole chromophores covalently bound to cysteinyl residues. As such, limits of detection of 1.18 x 10(-14), 5.26 x 10(-15), and 2.38 x 10(-15) mol/l were obtained for R-phycoerythrin, C-phycocyanin, and allophycocyanin proteins, respectively, with a linear dynamic range of eight orders of magnitude in each case. Unlike previously published CE-LIF methods, this work describes the separation of all three major classes of phycobiliproteins in under 5 min. Very good recoveries, ranging from 93.2 to 105.5%, were obtained for a standard mixture of the phycobiliproteins, based on seven-point calibration curves for both peak height and peak area. It is believed that this development will prove useful for the determination of phycobiliprotein content in naturally occurring cyanobacteria populations, thus providing a useful tool for understanding biological and chemical oceanographic processes.     

Use of capillary electrophoresis and laser-induced fluorescence for attomole detection of amino acids

A capillary electrophoresis and laser-induced fluorescence (CE-LIF) method was developed to identify and quantitate at amol (10(-18)) concentration. Amino acids were derivatized with 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde prior to CE-LIF analysis. The assay was developed by varying the sodium borate concentration, buffer pH, operating voltage, and operating temperature. A run buffer system containing 6.25 mM borate, 150 mM sodium dodecyl sulfate, and 10 mM tetrahydrofuran (pH 9.66) at 25 degrees C, and 24 kV provided analysis conditions for a high-resolution, sensitive, and repeatable assay of amino acids. The rate of derivatization, stability of the labeled amino acids, and amino acid quantitation varied for each amino acid. Amino acids were detected with greater efficiency by this method than automated HPLC amino acid analysis. The repeatability of the assay ranged from 0.3 to 0.9% within a day and 0.7 to 1.5% between analysis days. Bacterial amino acid utilization in a chemically defined medium was successfully monitored using this method. This work defines a sensitive and repeatable method for the detection of amino acids during bacterial metabolism.     

Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis.

A low cost, 0.75-mW helium neon laser, operating in the green region at 534.5 nm, is used to excite fluorescence from tetramethylrhodamine isothiocyanate-labelled DNA fragments that have been separated by capillary gel electrophoresis. The detection limit (3 sigma) for the dye is 500 ymol [1 yoctomole (1 ymol) = 10(-24) mol] or 300 analyte molecules in capillary zone electrophoresis; the detection limit for labeled primer separated by capillary gel electrophoresis is 2 zmol [1 zeptomole (1 zmol) = 10(-21) mol]. The Richardson-Tabor peak-height encoded sequencing technique is used to prepare DNA sequencing samples. In 6% T, 5% C acrylamide, 7 M urea gels, sequencing rates of 300 bases/hour are produced at an electric field strength of 200 V/cm; unfortunately, the data are plagued by compressions. These compressions are eliminated with addition of 20% formamide to the sequencing gel; the gel runs slowly and sequencing data are generated at a rate of about 70 bases/hour.     

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.     

Sequencing of antisense DNA analogues by capillary gel electrophoresis with laser-induced fluorescence detection

A method using capillary gel electrophoresis with laser-induced fluorescence detection is described which permits complete sequence determination of antisense DNA analogues of unknown sequence. This method, originally created as a tool to confirm the sequence of antisense oligonucleotides being developed as therapeutic drugs, utilizes data collected under a range of experimental conditions described by the Ogston model as applied to gel electrophoresis. A linear relationship independent of experimental conditions between the relative electrophoretic migration time and the oligonucleotide base number was observed and is shown to be consistent with a simplified version of this model and can be used to facilitate the sequence determination.     

Simultaneous detection of ultraviolet B-induced DNA damage using capillary electrophoresis with laser-induced fluorescence

An immunoassay based on CE–LIF was developed for the simultaneous detection of cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) in genomic DNA irradiated with UVB or natural sunlight. Human cells were first exposed to varying amounts of UVB or natural sunlight to induce DNA damage. Genomic DNA was extracted and incubated with anti-CPD and anti-6-4PP primary antibodies attached to secondary antibodies with a fluorescent quantum dot (QD) reporter that emitted either red or yellow fluorescence. CE was used to separate the unbound antibodies from those bound to the photoproducts, and LIF with appropriate optical filters was used to separate the fluorescence signals from each QD to individual photomultiplier tubes for simultaneous photoproduct detection. Using this strategy, photoproducts were detected from ∼6 ng (200 ng μL⁻¹) of DNA under a low UVB fluence of 65 J m⁻² for CPDs or 195 J m⁻² for 6-4PPs. This assay was also the first to demonstrate the detection of CPDs in human cells after only 15 min of irradiation under natural sunlight.     

Competitive immunoassay for vancomycin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay using capillary electrophoresis with laser-induced fluorescence was developed for vancomycin. Capillary electrophoresis using a Tris-glycine running buffer provided adequate separation of the antibody-bound from the unbound fluorescent probe (tracer) in less than 4 min. Laser-induced fluorescence polarization (LIFP) provided high sensitivity detection and simultaneous monitoring of fluorescence intensity and polarization. A fluorescence polarization value of 0.30 confirmed the formation of the antibody-tracer complex. Calibration curves showed a working linear range of 2-3 orders of magnitude with a minimum detectable concentration of 0.98 ng mL(-1) (or 1.1 fg vancomycin). Clinical samples obtained from patients undergoing vancomycin treatment were analyzed for vancomycin and the results correlated well with a standard immunoassay based on latex particle detection that was routinely used by a hospital laboratory. Only 1/10 of the reagents were needed as compared with the standard immunoassay.     

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.     

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.     

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.     

On-line wall-free cell for laser-induced fluorescence detection in capillary electrophoresis

A wall-free detection method based on liquid junction in a capillary gap was proposed for laser-induced fluorescence (LIF) of capillary electrophoresis (CE). The capillary gap of the wall-free cell was fabricated by etching a 10-mm × 50-μm I.D. fused-silica capillary to obtain a polyimide coating sleeve, decoating about 6 mm at one end of both 50 μm I.D. separation and liquid junction capillary, inserting the treated capillary ends into the coating sleeve oppositely, fixing the capillaries with a gap distance of 140 μm by epoxy glue and removing the coating sleeve by burning. The theoretical model, experimental results and wall-free cell images indicated that the gap distance and applied voltage were main influence factors on the wall-free detection. Since the wall-free cell increased the absorption light path and avoided the stray light from the capillary wall, it improved the ratio of signal to noise and limit of detection (LOD) of CE-LIF. Three flavin compounds of riboflavin (RF), flavin mononucleotide sodium (FMN) and flavin adenine dinucleotide disodium (FAD) were used to evaluate the wall-free detection method. Compared with on-column cell, the LODs of the wall-free cell were improved 15-, 6- and 9-fold for RF, FMN and FAD, respectively. The linear calibration concentrations of the flavins ranged from 0.005 to 5.0 μmol/L. The column efficiency was in the range from 1.0 × 10⁵ to 2.5 × 10⁵ plates. The wall-free detection of CE-LIF was applied to the analysis of the flavins in spinach and lettuce leaves.     

Indirect laser-induced fluorescence detection for capillary electrophoresis using a frequency-doubled diode laser

A blue (452 nm) frequency-doubled diode laser with a quasi-cw optical output power of 10 microW is used for indirect laser-induced fluorescence detection in combination with the capillary electrophoretic separation of inorganic anions. As fluorescing probe ion the anion of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) was selected having an absorption maximum of 454 nm in alkaline medium. Employing a capillary coated with linear acrylamide, baseline separation of eight inorganic anions was possible within 5 min. With a separation buffer containing 50 micromol.L(-1) HPTS and 10 mmol.L(-1) lysine the limits of detection for sulfate, nitrite, nitrate, azide, thiocyanate, and chlorate were between 0.9 and 4.7 micromol.L(-1). Separation of chloride and sulfate was achieved by adding 0.25 mmol.L(-1) calcium hydroxide to the separation buffer. Inorganic anions in several mineral and tap water samples have been determined with the technique developed and results are compared to data obtained by ion chromatography in combination with conductivity detection after conductivity suppression.     

High-sensitivity laser-induced fluorescence detection of native proteins in capillary electrophoresis.

A highly sensitive laser-induced (LIF) detection scheme for native, tryptophan- or tyrosine-containing proteins in capillary electrophoresis (CE) has been demonstrated. The 275.4 nm line from an argon-ion laser is used to excite native protein fluorescence. A limit of detection (LOD) (S/N = 2) of 1 x 10(-10) M for conalbumin represents a 140-fold improvement over earlier reports. With stacking at injection, the LOD is 3 x 10(-12) M. Linear dynamic ranges of at least 5 and 4 orders of magnitude for, respectively, tryptophan and bovine serum albumin are found. The practical performance and blueprint of an easily constructed, rugged, compact and user-friendly LIF detector for CE are shown.     

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.     

Competitive immunoassay for staphylococcal enterotoxin A using capillary electrophoresis with laser-induced fluorescence detection.

Staphylococcal enterotoxins are a family of toxic proteins secreted by S. aureus. Using capillary electrophoresis (CE) linked with laser-induced fluorescence, a highly sensitive and selective assay using antibody-antigen recognition was developed for the determination of Staphylococcal enterotoxin A. Staphylococcal enterotoxin A (SEA) was chemically labeled with fluorescein and the product was used as a fluorescent tracer. A competitive assay was developed to detect SEA at concentrations between 0.3 nM and 6.5 nM with standard deviations of less than 5%. The detection limit was found to be 3 amol with the potential improvement by further optimization of the assay. No cross-reactivity between staphylococcal enterotoxin B and the SEA antibody was found at the concentrations used for the CE immunoassay.     

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.