Preparation of an IMI dye (imidazole functional group) containing a 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole fluorophore for labeling of phosphomonoesters

We are studying dye-imidazole conjugates ("IMI dyes") as reagents for labeling phosphomonoesters such as nucleotides. Previously we have employed a BODIPY dye in our IMI reagents, and analyzed the labeled products by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) involving an argon ion laser. (The BODIPY fluorophore is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene). Here we broaden the technology by preparing a DBD-IMI dye [DBD = 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole], and using a helium-cadmium laser. While DBD-IMI (IMI3) is about 50x more stable photolytically than a BODIPY-IMI dye (IMI2, a conjugate of a BODIPY dye with histamine, was tested), the detection limit for IMI2 (5.10(-11) M; S/N = 5, CE-LIF with an argon ion laser) is tenfold better than that for IMI3 (5.10(-10) M, S/N = 5, helium-cadmium laser). IMI3 conjugates of the four major DNA nucleotides were prepared and detected by CE-LIF.     

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.     

Dimethylditetradecylammonium bromide (2C14DAB) as a self-assembled surfactant coating for detection of protein–dye complexes by CE-LIF

A self-assembled column coating for capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF) has been evaluated for the separation and quantitation of protein–dye complexes. This semi-permanent coating, composed of dimethylditetradecyl-ammonium bromide (2C₁₄DAB), is inexpensive and easily assembled onto the column and it allows for better peak resolution and greater control over electroosmotic flow. The versatility of long-chained surfactant coatings was determined particularly with respect to their use with fluorescent probes, different pH buffers, and different proteins. Studies were performed to determine the stability of the coating under various pH and buffer conditions. Red-1c, a red luminescent squarylium dye, was used for on-column protein labeling concurrently with the surfactant coating and LIF detection. Protein–Red-1c complexes were excited with a 650-nm diode laser and their emission detected by a photomultiplier tube with a 664-nm filter. A comparison of pre-column labeling and on-column labeling of a two-model protein system (human serum albumin and β-lactoglobulin A) revealed higher efficiencies and greater sensitivities for both proteins using on-column labeling and coated columns. A linear relationship between peak height and protein concentration was obtained by CE-LIF for this on-column labeling method with 2C₁₄DAB-coated columns and the Red-1c probe.     

Detection and separation of nucleoside-5'-monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser-induced fluorescence detection

We investigated the separation and detection of the 5'-monophosphates of 2'-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5'-monophosphates (2'-deoxyguanosine-5'-monophosphate, 2'-deoxyadenosine-5'-monophosphate, 2'-deoxycytidine-5'-monophosphate, and thymidine-5'-monophosphate) were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE-LIF after digestion of DNA or an oligonucleotide to 5'-monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf-thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'-monophosphates or with NP 1 to 5'-monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive. Moreover, using the same assay, 2'-deoxyuridine and 2'-deoxy-5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'-deoxyribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF has the potential to determine DNA damage and genomic DNA methylation.     

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.     

Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection

Two new red luminescent asymmetric squarylium dyes (designated "Red-1c and Red-3") have been shown to exhibit absorbance shifts to longer wavelengths upon the addition of protein, along with a concomitant increase in fluorescence emission. Specifically, the absorbance maxima for Red-1c and Red-3 dyes are 607 and 622 nm, respectively, in the absence of HSA, and 642 and 640 nm in the presence of HSA, making the excitation of their protein complexes feasible with inexpensive and robust diode lasers. Fluorescence emission maxima, in the presence of HSA, are 656 and 644 nm for Red-1c and Red-3, respectively. Because of the inherently low fluorescence of the dyes in their free state, Red-1c and Red-3 were used as on-column labels (that is, with the dye incorporated into the separation buffer), thus eliminating the need for sample derivatization prior to injection and separation. A comparison of precolumn and on-column labeling of proteins with these squarylium dyes revealed higher efficiencies and greater sensitivities for on-column labeling, which, when conducted with a basic, high-salt content buffer, permitted baseline resolution of a mixture of five model proteins. LOD for model proteins, such as transferrin, alpha-lactalbumin, BSA, and beta-lactoglobulin A and B, labeled with these dyes and analyzed by CE with LIF detection (CE-LIF) were found to be dependent upon dye concentration and solution pH, and are as low as 5 nM for BSA. Satisfactory linear relationships between peak height (or peak area) and protein concentration were obtained by CE-LIF for this on-column labeling method with Red-3 and Red-1c.     

RNA analysis by MEKC with LIF detection

We have developed and validated a procedure of high sensitivity for the analysis of RNA. The procedure is based on the separation and detection of the 5'-monophosphates of ribonucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using CE with LIF. BODIPY conjugates of the four common ribonucleoside-5'-monophosphates were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions. After digestion of RNA or oligoribonucleotides to 5'-monophosphates by nuclease P1 and fluorescence labeling BODIPY conjugates were detected and resolved by CE-LIF without further purification steps. Comparative CE-LIF analyses with DNA digested to deoxyribonucleoside-5'-monophosphates showed that the assay is equally efficient and sensitive for RNA analysis. Conditions to determine the modified ribonucleosides inosine, xanthosine, pseudouridine and 2'-O-methyladenosine were also established. The limits of detection were in the range of 80-200 pM. After calibrating the assay with oligoribonucleotides, pseudouridine was quantified in total RNA of Drosophila, human liver, human kidney and t-RNA of Saccharomyces cerevisiae. These studies demonstrate good potential of fluorescence labeling of ribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF to determine RNA composition with high accuracy and sensitivity.     

Sensitive determination of erythrosine and other red food colorants using capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was applied to separation and sensitive determination of red food colorants. Diode pumped frequency-doubled Nd:YAG laser (532 nm) was used as an excitation source in a laboratory-built CE-LIF system. For highly fluorescent erythrosine B (E127), an extrapolated limit of detection (LOD) of 0.4 ng mL(-1) (S/N=3) was achieved. Extrapolated LODs of other tested red additives, such as carmoisine, E122 (0.5 microg mL(-1)); amaranth, E123 (0.2 microg mL(-1)); ponceau 4R, E124 (0.3 microg mL(-1)) and red 2G, E128 (0.3 microg mL(-1)) were about one-order lower compared to results obtained with CE with absorbance detection in UV/vis (CE-UV/vis). The main advantages of using CE-LIF for analysis of food samples are high selectivity and minimization of matrix effect. To our knowledge, this is the first use of CE-LIF for determination of red food colorants.     

In-capillary non-covalent labeling and determination of tomato systemin with quantum dots in capillary electrophoresis with laser-induced fluorescence detection

Quantum dots (QDs), with their superior size-dependent fluorescence properties, have been employed as non-covalent fluorescent labels for the determination of tomato systemin (TomSys) by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. The optimum conditions of in-capillary labeling and CE separation were investigated in detail, and complete separation of QDs-labeled TomSys from free QDs labels was achieved. Satisfactory results were obtained in terms of linearity (R(2)=0.998), sensitivity (limit of detection, 66 fmol) and repeatability (run-to-run RSDs of migration time and peak area, 0.9 and 4.6%, respectively; day-to-day RSDs of migration time and peak area, 3.1 and 11.9%, respectively). The established CE-LIF method was later applied in the detection of TomSys spiked in the sample of tomato leaves, which showed the applicability of the proposed method in the analysis of the target plant peptide hormone in the complex matrix.     

Laser-induced fluorescence detection schemes for the analysis of proteins and peptides using capillary electrophoresis

Over the past few years, a large number of studies have been prepared that describe the analysis of peptides and proteins using capillary electrophoresis (CE) and laser-induced fluorescence (LIF). These studies have focused on two general goals: (i) development of automatic, selective and quick separation and detection of mixtures of peptides or proteins; (ii) generation of new methods of quantitation for very low concentrations (nm and subnanomolar) of peptides. These two goals are attained with the use of covalent labelling reactions using a variety of dyes that can be readily excited by the radiation from a commonly available laser or via the use of noncovalent labelling (immunoassay using a labelled antibody or antigen or noncovalent dye interactions). In this review article, we summarize the works which were performed for protein and peptide analysis via CE-LIF.     

Fluorimetric studies and noncovalent labeling of protein with the near-infrared dye HITCI for analysis by CE-LIF

1,1',3,3,3',3'-Hexamethylindotricarbocyanine iodide (HITCI) is a commercially available, positively charged, indocarbocyanine dye used typically as a laser dye in the near infrared (NIR). The absorbance and fluorescence properties of HITCI in a variety of solvent systems were determined. Results indicate that the fluorescence of HITCI is not significantly affected by the pH. Titration of HITCI with human serum albumin (HSA) and trypsinogen was carried out to investigate the interactions between this dye and proteins. These studies revealed that the absorbance and fluorescence properties of the dye change upon binding to protein in a wide range of solution pH's. The potential use of HITCI as a noncovalent protein labeling probe, therefore, was explored. Determination and separation of HITCI and HITCI-protein complexes was performed by capillary electrophoresis with diode-laser induced fluorescence detection (CE-LIF). Both pre-column and on-column noncovalent labeling methods are demonstrated.     

Determination of gabapentin in human plasma by capillary electrophoresis-laser induced fluorescence detection with and without solid-phase extraction

We have developed two methods for the quantitation of gabapentin in human plasma. They are based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) with and without solid-phase extraction (SPE) and the derivatizing reagent 5-(4,6-dichlorotriazinyl)amino fluoresencin. The conditions for derivatization, separation and extraction were investigated in detail, and the optimal labeling conditions include a temperature of 40?°C, a reaction time of 30?min, and the use of a borate buffer of pH 9.0 as the reaction medium. A borate buffer of pH 9.2 served as a background electrolyte for CE separations. The CE-LIF and SPE-CE-LIF methods have linear ranges of 5–200?nmol?L^?1 and 0.2–10?nmol?L^?1, respectively, and the limits of detection are 0.5 and 0.02?nmol?L^?1, respectively. The SPE-CE-LIF method was successfully applied to the determination of gabapentin in blood plasma samples.

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.     

Analysis of subcellular sized particles. Capillary electrophoresis with post-column laser-induced fluorescence detection versus flow cytometry

Flow cytometry (FCM) and more recently capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) have both been used for subcellular particle analysis but their analytical performance has not been compared. In this work, we compare a commercial FCM with an in-house built CE-LIF instrument using fluorescently labeled microspheres and isolated mitochondria. As evidenced by the relative standard deviation (RSD) of the individual fluorescence intensities, FCM is two-fold better than CE-LIF for microspheres with > or =1.5 x 10(6) molecules of equivalent soluble fluorescein (MESF). However, FCM has a comparatively low signal-to-noise ratio (S/N) and high RSD for microspheres with <1.5 x 10(6) MESF. CE-LIF, on the other hand, produces S/N ratios that are >25 times higher than FCM for all the microspheres tested and a lower RSD for microspheres with <1.5 x 10(6) MESF. When 10-N-nonyl acridine orange (NAO)-labeled mitochondria are analyzed, the S/N ratios of both techniques are similar. This appears to result from photobleaching of NAO-labeled mitochondria as they are detected by the LIF detector of the CE-LIF instrument. Both techniques have a niche in subcellular analysis; FCM has the advantage of collecting data for thousands of particles quickly, whereas CE-LIF consumes less than a nanoliter of sample and provides the electrophoretic mobility for individual particles.     

Handling and detection of 25 amol of near-infrared dye deoxynucleotide conjugates by capillary electrophoresis with laser-induced fluorescence detection

Near-infrared dyes are attractive as labeling reagents to enhance sensitivity in trace analysis largely because background fluorescence is low in this spectral region. Here we demonstrate, towards a goal of detecting DNA adducts in small biological samples, that some near-infrared (IR) dye-labeled deoxynucleotides can be separated and detected with high sensitivity by capillary electrophoresis (CE)-laser-induced fluorescence detection (LIF) in a realistic way (handling detection limit of 25 amol) for near-IR dye-labeled deoxynucleotides. This detection limit is achieved by polarity-switching injection of 2.0 microl from a volume of 5.0 microl, in which the compounds are 5 x 10(-12) mol/l in 50% aqueous methanol. Although the adenine and cytosine-containing conjugates co-migrated, the other three (guanine, N2-ethylguanine and thymine) were resolved.     

Determination of 2-methyl derivatives of tamoxifen in cell culture medium using high-performance liquid chromatography and electrochemical detection

The 2-methyl derivatives of tamoxifen (2-methyltamoxifen and 2-methyl-4-hydroxytamoxifen) were extracted from a cell culture medium at pH 5.4 (Earle's Minimum Essential Medium) with an internal standard (tamoxifen) on a phenyl sorbent cartridge. The compounds were then separated by high-performance liquid chromatography on a nitrile column eluted with acetonitrile-methanol-0.05 M sodium dihydrogenphosphate (19:4:11.6:69,v/v) containing 0.11 mmol/l disodium EDTA and determined by electrochemical detection at +1.1 V vs. Ag/AgCl/3 M NaCl. The absolute detection limits were 50 pg for 2-methyl-4-hydroxytamoxifen and 100 pg for tamoxifen and 2-methyltamoxifen at a sensitivity of 1 nA/V.     

In-capillary non-covalent labeling of insulin and one gastrointestinal peptide for their analyses by capillary electrophoresis with laser-induced fluorescence detection

The potential of the commercially available dye sypro orange for in-capillary derivatization was evaluated for the detection of insulin and one gastrointestinal peptide (Arg-Arg-gastrin) by capillary electrophoresis with laser induced fluorescence (CE-LIF). The fluorescent emission intensity (lambda(ex) = 488 nm, lambda(em) = 610 nm) of this probe is very low in aqueous medium, and increases strongly in less polar solvent, e.g. methanol. The hydrophobic character of the two analyzed peptides is too low to induce sufficient interaction with the fluorescent probe for good sensitivity when the latter is alone in the background electrolyte. Thus, the potential of several neutral, zwitterionic, cationic and anionic surfactants to favor probe/peptide interactions has been evaluated. It was demonstrated that a borate buffer (pH 8.5) containing tetradecyltrimethylammonium bromide (TTAB) in sub-micellar conditions can be considered as the most suitable buffer for insulin CE-LIF analysis. In addition, the method showed a good linearity between insulin concentration and the peak area of the labeled insulin, allowing quantitative measurements. The sensitivity achieved so far is comparable with that achieved with UV absorption detection, but even at this level it is interesting for microchip analysis, in which fluorescence detection is much more commonly available than UV absorption detection.     

Capillary electrophoresis with laser-induced fluorescence detection for detailed studies on N-linked oligosaccharide profile of therapeutic recombinant monoclonal antibodies

Total N-linked oligosaccharide profiling method for recombinant monoclonal antibody (rmAb) using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and an approach for detailed structural analysis of N-linked oligosaccharide were developed. A CE-LIF method using 2-aminobenzoic acid (2-AA) as a fluorogenic reagent allowed sensitive detection of several minor peaks besides typical asialo-biantennary complex type oligosaccharides in the analysis of N-linked oligosaccharide from a commercial rmAb pharmaceutical, rituximab. These minor peaks were successfully assigned as sialo-biantennary complex type and high-mannose type oligosaccharides by comparison with the migration times of 2-AA derivatized oligosaccharides which were separately fractionated and determined by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In development of biopharmaceuticals, it is important to evaluate these minor oligosaccharides, because some of these minor glycans are likely to influence immunogenicity and clearance rate in vivo. The repetitive analysis using CE-LIF showed excellent precision in relative corrected peak areas. These results demonstrate that the present CE-LIF method is applicable for both structural characterization and quantitative profiling of N-linked oligosaccharides derived from rmAb pharmaceuticals. The present method will be a powerful tool for rapid, quantitative and exhaustive evaluation of N-linked oligosaccharides in various stages of rmAb pharmaceutical development such as clone selection, bioprocess control, and routine lot release testing to ensure product efficacy and consistency.     

Determination of amino acid neurotransmitters in human cerebrospinal fluid and saliva by capillary electrophoresis with laser-induced fluorescence detection

A CE-LIF detection method has been developed to identify and quantitate six amino acid neurotransmitters including glutamic acid, aspartic acid, gamma-aminobutyric acid, glycine, taurine, and glutamine. N-hydroxysuccinimidyl fluorescein-O-acetate, a fluorescein-based dye, was employed for the derivatization of these neurotransmitters prior to CE-LIF analysis. Different parameters which influenced separation and derivatization were optimized in detail. Under optimum conditions, linearity was achieved within concentration ranges of up to three orders of magnitudes for those analytes with correlation coefficients from 0.9989 to 0.9998. The LODs ranged from 0.06 nM to 0.1 nM, and are thus superior or equivalent to those previously reported in the literature using CE-LIF detection. The proposed method has been successfully applied to the determination of amino acid neurotransmitters in biological samples such as human cerebrospinal fluid and saliva with satisfactory recoveries.