Detection of green fluorescent protein in a single bacterium by capillary electrophoresis with laser-induced fluorescence

Green fluorescence protein (GFP) is a common reporter used to monitor protein expression in single cells. However, autofluorescence from endogenous components can mask the signal from GFP, particularly at low expression levels in prokaryotes. We employ capillary electrophoresis with laser-induced fluorescence for the analysis of the expression of green fluorescent protein in a single bacterium. Capillary electrophoresis separates GFP from native cellular autofluorescent components, reducing the background signal and improving detection limits. Our system provides 100 ymol (60 copies) limits of detection for GFP. To demonstrate the performance of this instrument, we employ a model system of Deinococcus radiodurans that has been engineered to express GFP under the control of the recA promoter. We report resolution and detection of GFP and autofluorescent components in a single D. radiodurans bacterium. This paper presents the first example of expression of GFP in D. radiodurans and the first detection of GFP in a single bacterium by capillary electrophoresis.     

Two-photon nitric oxide laser-induced fluorescence measurements in a diesel engine

A two-photon nitric oxide (NO) laser-induced fluorescence (LIF) technique was developed and applied to study in-cylinder diesel combustion. The technique prevents many problems associated with in-cylinder, single-photon NO planar-laser-induced fluorescence measurements, including fluorescence interference from the Schumann-Runge bands of hot O2, absorption of a UV excitation beam by in-cylinder gases, and difficulty in rejecting scattered laser light while simultaneously attempting to maximize fluorescence signal collection. Verification that the signal resulted from NO was provided by tuning of the laser to a vibrational off-resonance wavelength that showed near-zero signal levels, which resulted from either fluorescence or interference at in-cylinder pressures of as much as 20 bar. The two-photon NO LIF signal showed good qualitative agreement with NO exhaust-gas measurements obtained over a wide range of engine loads.     

Measuring the doxorubicin content of single nuclei by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

Individual nuclei isolated from the human leukemia CCRF-CEM and CEM-C2 cells treated with doxorubicin (DOX) were in-column lysed with a sodium dodecyl sulfate (SDS) containing buffer, their contents were then separated by micellar electrokinetic capillary chromatography using the same lysing buffer, and the DOX content was detected by laser-induced fluorescence. Use of a microscope for the selection of one nucleus from the nuclear preparation decreases the possibility of introduction of other subcellular components that are commonly found as impurities in subcellular fractions. The presence of SDS in the running buffer made negligible the DNA's quenching effect on DOX fluorescence, which often compromises quantification of DOX by direct imaging, making it possible to carry out the first direct measurement of the doxorubicin content of isolated nuclei. On average, nuclei from CCRF-CEM and CEM/C2 cell lines contained 85 +/-64 (n = 6) and 91 +/- 51 (n =7) amol of DOX, respectively. These values correspond to 74 and 65% of the average total cellular content as determined by single-cell analysis of the corresponding cell types. It is envisioned that this approach could become an important bioanalytical tool to investigate the effect of treatments with fluorescent drugs targeting the nucleus.     

Pteridine analysis in urine by capillary electrophoresis using laser-induced fluorescence detection

Pteridines are a class of compounds excreted in urine, the levels of which are found to elevate significantly in tumor-related diseases. For the first time, we have developed a method, based on high-performance capillary electrophoresis (HPCE) and laser-induced fluorescence (LIF) detection, to monitor the pteridine levels in urine. HPCE provides better separation than high-performance liquid chromatography and the LIF detector enables us to detect minute amounts of pteridines in body fluid. Eight different pteridine derivatives were well separated in 0.1 M Tris-0.1 M borate-2 mM EDTA buffer (pH 8.75) using a 60-cm fused-silica capillary (50-micron i.d., 35-cm effective length), six of which were detected and characterized in urine samples from normal persons and different cancer patients. The detection limits of these pteridines are under 1 x 10(-10) M. The levels of neopterin, pterine, xanthopterin, and pterin-6-carboxylic acid were found to be significantly elevated in urine excreted by cancer patents, while the level of isoxanthopterin dropped in these patients. No significant change of biopterin level was found between healthy individuals and cancer patients. This method can be used in clinical laboratories either for cancer monitoring or for precancer screening.     

Instrumentation for medium-throughput two-dimensional capillary electrophoresis with laser-induced fluorescence detection

In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 +/- 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates prepared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 +/- 0.7 transfers in the CSE dimension and 0.46 +/- 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 +/- 1.2 transfers in the CSE dimension and 0.88 +/- 0.55 s in the MECC dimension. Spot capacity was 300.     

Studies of protein--DNA interactions by capillary electrophoresis/laser-induced fluorescence polarization

Protein-DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 x 10(6) M(-1)) and the 37-mer (23 x 10(6) M(-1)). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multiple protein-DNA complexes that differ in stoichiometry was observed. The results demonstrate the applicability of the method to study complex interactions between protein and DNA.     

Ascorbic acid assays of individual neurons and neuronal tissues using capillary electrophoresis with laser-induced fluorescence detection

Ascorbic acid is an important cellular metabolite involved in many biochemical pathways. A method to quantitate ascorbic acid and dehydroascorbic acid in individual neurons and neuronal tissues is described with detection limits of 320 pM (430 zmol). The method uses microvial sampling, derivatization with 4,5-dimethyl-1,2-phenylenediamine, capillary electrophoresis separation, and laser-induced fluorescence detection and quantifies the ascorbic acid and dehydroascorbic acid levels with less than a 15-min total analysis time including sample preparation and derivatization. Ascorbic acid and dehydroascorbic acid levels are measured using functionally characterized and identified neurons of Aplysia californica, Pleurobranchaea californica, and Lymnaea stagnalis -three well-recognized models in cellular and system neuroscience. Multiple assays of a particular identified neuron (e.g., metacerebral cells from Aplysia) show a high level of reproducibility, while endogenous intracellular concentrations of ascorbate are neuron-specific. Ascorbic acid concentrations in the neurons studied range from 0.19 to 6.2 mM for Aplysia and 0.12 to 0.22 mM for Lymnaea. In contrast, concentrations of ascorbic acid observed in heterogeneous tissues such as ganglia (with connective tissues, glia, blood vessels, neuropile, and areas with intercellular spaces), 4-190 microM, are significantly lower than the single-cell values.     

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

Individual liposome measurements by capillary electrophoresis with postcolumn laser-induced fluorescence detection facilitated the determination of liposome property distributions, two-dimensional plots, and an improved characterization of a liposomal preparation. This advancement in liposome analysis was feasible by using a high-sensitivity postcolumn laser-induced fluorescence detector wired for millisecond response. For each individual liposome containing fluorescein, peak height and migration time were determined. From these measurements the individual entrapped volumes and electrophoretic mobilities were determined. Distribution analysis of these properties facilitated comparison of various liposome dilutions and indicated that the method is reproducible and unaffected by the density of liposomes (10(7)-10(9) liposomes/mL) in the suspension. Furthermore, liposomes showed entrapped volumes that vary from 0.3 to 13 fL with apparent radius varying from 370 nm to 1.8 microns. Two-dimensional plots of reduced mobility versus kappa R (Debye parameter x liposome radius) revealed that the liposomes resuspended from a dried film of phospholipids are heterogeneous in regard to the surface charge density of individual liposomes. The described method has the potential of becoming a new tool for characterization of commercial liposomal preparations and theoretical studies.     

Binding stoichiometry of DNA adducts with antibody studied by capillary electrophoresis and laser-induced fluorescence

Four oligonucleotides (fluorescently labeled and unlabeled 16- and 90-mer), each containing a single adduct of benzo[a]pyrene diol epoxide (BPDE), were synthesized and used to study the binding stoichiometry between the DNA adduct and its antibody. The free oligonucleotide and its complexes with mouse monoclonal antibody were separated using capillary electrophoresis and detected with laser-induced fluorescence (LIF). Two complexes, representing the 1:1 and 1:2 stoichiometry between the antibody and the DNA adduct, were clearly demonstrated. The stoichiometry depended upon the relative concentrations of the antibody and the DNA adducts. A new approach examining the binding of the antibody with a mixture of a tetramethylrhodamine (TMR)-labeled and unlabeled BPDE-16-mer revealed insights on ligand redistribution and exchange between the labeled and unlabeled BPDE-16-mer oligonucleotides in the complexes. The observation of this unique behavior has not been possible previously with other binding studies. A mixture of the antibody with the TMR-labeled BPDE- 16-mer and an unlabeled BPDE-90-mer further revealed the formation of three fluorescent complexes: antibody with one TMR-BPDE-16-mer molecule, antibody with two TMR-BPDE- 16-mer molecules, and antibody with one TMR-BPDE-16-mer and one BPDE-90-mer. The three complexes clearly demonstrated binding stoichiometry and ligand redistribution/exchange.     

Sheath-flow cuvette for high-sensitivity laser-induced fluorescence detection in capillary electrophoresis

The sheath-flow cuvette is a key component in a high-sensitivity post-column laser-induced fluorescence detector for capillary electrophoresis. Most designs are based on commercial cuvettes originally manufactured for use in a flow cytometer. In these devices, a quartz flow chamber is held in a stainless-steel fixture that is difficult to machine and subjects the cuvette to a torque when sealed, which frequently leads to damage of the flow chamber. In this report we present a design for a cuvette that may easily be constructed. This design uses compression to hold and seal the quartz flow chamber without applying torque. The system produces detection limits (3sigma) of 115 yoctomoles (70 copies) for FQ-labeled carbonic anhydrase.     

Distribution of zeptomole-abundant doxorubicin metabolites in subcellular fractions by capillary electrophoresis with laser-induced fluorescence detection

Doxorubicin (DOX) treatment of NS-1 mouse hybridoma cells results in the formation of zeptomole amounts of metabolites per cell that are difficult to determine by confocal microscopy or HPLC. The native fluorescence of DOX and its metabolites together with laser-induced fluorescence detection (HF) has previously been used to detect a maximum of four components. In this study, we use capillary electrophoresis with postcolumn LIF (CE-LIF) to separate and detect 12 components attributed to DOX metabolism, resulting from treatment of NS-1 cells with 25 microM DOX for 8 h. The so-called metabolites 8 and 10 have been identified as doxorubicinone (DOXone) and 7-deoxydoxorubicinone (7-deoxyDOXone), respectively, by comigration with the corresponding synthetic standard. Due to comigration of DOX with doxorubicinol (DOXone), the presence of DOXone had to be determined separately by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The rest of the metabolites remain unidentified and are referred to by their number assignment. In comparison with the whole cell lysate, fractionation by differential centrifugation results in a better separation resolution of metabolites due to reduced amounts of metabolites in each fraction. This approach was chosen to compare the distribution of 13 metabolites in three subcellular fractions that form a pellet at < 1,400 g, 1,400-14,000 g, and > 14, 000 g and that generically are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosolic components, respectively. The most abundant metabolite, DOXone, was estimated to be 90 +/- 15, 18 +/- 2, and 60 +/- 12 amol/cell (n = 5) in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively. In contrast, the total amount of other metabolites in a given fraction varied from 0 to 1,300 zmol. 7-DeoxyDOXone is the only metabolite that was present at similar levels in the three fractions. Other salient observations are metabolites 3, 7, and 11 are not detectable in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively; metabolite 9 and DOXone are more abundant in the nuclear-enriched fraction than in the other two fractions. The observations presented here suggest that subcellular fractionation followed by CE-LIF could be a powerful diagnostic for monitoring drug distribution, which is highly relevant to DOX cytoxicity studies.     

Determination of cell viability in single or mixed samples using capillary electrophoresis laser-induced fluorescence microfluidic systems

The advent of high-efficiency microbial separations will have a profound effect on both chemistry and microbiology. For the first time, it appears that it may be possible to obtain qualitative and quantitative information on microbial systems with the accuracy, precision, speed, and throughput that currently is found for chemical systems. Recently it was suggested that an analytical separations-based approach for determining the viability of cells would be advantageous. The feasibility of such an approach is demonstrated using CE-LIF of two bacteria and yeast. The analytical procedures and figures of merit are outlined. High-throughput analyses and evaluation of microorganisms now appear to be possible.     

Carbohydrate analysis of a chimeric recombinant monoclonal antibody by capillary electrophoresis with laser-induced fluorescence detection

A general method for the analysis of asparaginyl-linked (N-linked) carbohydrate moieties of an IgG1 monoclonal antibody is described here. The antibody, rituximab, is a mouse/human chimeric antibody to human CD20 antigen. The glycans present on rituximab are neutral complex biantennary oligosaccharides with zero, one, and two terminal galactose residues (G0, G1, and G2, respectively). To monitor the variation of the glycosylation during manufacture, the glycans were first enzymatically released from the antibody via digestion with peptide-N-glycosidase F, then derivatized with a charged fluorophore, 8-aminopyrene-1,3,6-trisulfonic acid and further separated by capillary electrophoresis with laser-induced fluorescence detection. All observed glycans were fully resolved, including the positional isomers of G1. The exact nature of the isomers in terms of the location of the terminal galactose was further characterized via multiple enzymatic digestion steps including mannosidase with activity toward specific Man(alpha 1,3) linkage. The optimization and several key parameters, i.e., enzymatic digestion and derivatization, in the assay development will be discussed. Moreover, to ensure that the assay can be used in routine lot release testing, the assay was validated and found to be accurate and precise. The analytical approach described is suitable for characterization as well as routine testing of the N-linked glycan content in any IgG1 monoclonal antibody and glycoproteins in general.     

Comparisons of laser-saturated, laser-induced, and planar laser-induced fluorescence measurements of nitric oxide in a lean direct-injection spray flame

We report quantitative, spatially resolved laser-saturated fluorescence (LSF), linear laser-induced fluorescence (LIF), and planar laser-induced fluorescence (PLIF) measurements of nitric oxide (NO) concentration in a preheated, lean direct-injection spray flame at atmospheric pressure. The spray is produced by a hollow-cone, pressure-atomized nozzle supplied with liquid heptane, and the overall equivalence ratio is unity. NO is excited by means of the Q(2)(26.5) transition of the gamma(0, 0) band. LSF and LIF detection are performed in a 2-nm region centered on the gamma(0, 1) band. PLIF detection is performed in a broad ~70-nm region with a peak transmission at 270 nm. Quantitative radial NO profiles obtained by LSF are presented and analyzed so as to correct similar LIF and PLIF profiles. Excellent agreement is achieved among the three fluorescence methodologies.     

Simultaneous laser-induced fluorescence and scattering detection of individual particles separated by capillary electrophoresis

This technical note describes a detector capable of simultaneously monitoring scattering and fluorescence signals of individual particles separated by capillary electrophoresis. Due to its nonselective nature, scattering alone is not sufficient to identify analyte particles. However, when the analyte particles are fluorescent, the detector described here is able to identify simultaneously occurring scattering and fluorescent signals, even when contaminating particles (i.e., nonfluorescent) are present. Both fluorescent polystyrene particles and 10-nonyl acridine orange (NAO)-labeled mitochondria were used as models. Fluorescence versus scattering (FVS) plots made it possible to identify two types of particles and a contaminant in a mixture of polystyrene particles. We also analyzed NAO-labeled mitochondria before and after cryogenic storage; the mitochondria FVS plots changed with storage, which suggests that the detector reported here is suitable for monitoring subtle changes in mitochondrial morphology that would not be revealed by monitoring only fluorescence or scattering signals.     

Temperature measurements of single droplets by use of laser-induced phosphorescence

A novel technique for measuring droplet temperatures has been demonstrated. Laser-induced phosphorescence from thermographic phosphors, seeded to distilled water and iso-octane, was used to measure temperatures of single falling droplets. The phosphors were excited by the fourth and third harmonics of a Nd:YAG laser. The subsequent emission was evaluated by spectral and temporal investigations of the thermographic phosphors Mg4FGeO6:Mn and La2O2S:Eu, respectively. The spectral and the temporal methods permitted temperature measurements of free-falling droplets up to 433 K. Results from both methods, which show an estimated accuracy of better than 1%, are presented.     

Magnetic beads based immunoaffinity capillary electrophoresis of total serum IgE with laser-induced fluorescence detection

A magnetic beads based immunoaffinity capillary electrophoresis method for total Immunoglobulin E quantification in serum has been developed. The method combines speed, automation ability, and minimal sample consumption. Only 1 microL of serum is required while the whole immunoaffinity capillary electrophoresis method is performed in less than 50 min. The concomitant use of online immunocapture, transient isotachophoresis, and laser-induced fluorescence detection provides a sensitivity in the low picomolar range and a highly linear fluorescence response over 4 orders of magnitude (IgE concentration ranging from 2.4 to 2400 ng/mL). After validation with a reference material, the method has been successfully applied to the quantification of total IgEs in patient sera. The results compared well with classical ImmunoCap data.     

Nuclear localization of aminoacyl-tRNA synthetases using single-cell capillary electrophoresis laser-induced fluorescence analysis

Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes whose function in specific aminoacylation of tRNAs is central to the process of protein translation, which occurs in the cytoplasm of all living cells. In addition to their well-established cytoplasmic localization, fluorescence microscopy studies and analysis of the aminoacylation state of nuclear tRNAs have revealed that synthetases are localized in the nuclei of cells from several species including Xenopus laevis and Saccharomyces cerevisiae. Whether nuclear localization of aaRSs is a general phenomenon that occurs in all eukaryotic cells is an open question. In the work described here, human methionyl-tRNA synthetase (MRS) and human lysyl-tRNA synthetase (KRS) were expressed in human-derived DeltaH2-1 osteosarcoma cells as enhanced green fluorescent protein (EGFP) fusion proteins. The subcellular localization of these EGFP-aaRSs was first probed by fluorescence microscopy using cells that coexpressed EGFP-aaRS and a nuclear marker fusion protein, nuDsRed. As expected, both aaRSs were present in the cytosol, while only EGFP-MRS was also clearly localized in the nucleus. To confirm these findings, and to investigate a potentially more sensitive, general method for nuclear localization studies, capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze single DeltaH2-1 cells expressing both EGFP-aaRS and nuDsRed. While cytosolic EGFP signals were detected for both EGFP-MRS and EGFP-KRS, only EGFP-MRS was found in the nucleus, along with nuDsRed. The detection of EGFP-MRS in nuclei of DeltaH2-1 cells demonstrates the feasibility of using CE-LIF analysis in nuclear localization studies of proteins in mammalian cells.     

Quantitative determination of native proteins in individual human erythrocytes by capillary zone electrophoresis with laser-induced fluorescence detection.

Intracellular fluid within single human erythrocytes is analyzed by capillary electrophoresis with laser-excited native protein fluorescence. Good signal-to-noise is achieved, allowing even minor components to be quantified. Non-Gaussian distributions were found for total protein, fraction carbonic anhydrase, fraction hemoglobin A0, and an unidentified component. Variations among a group of 29 cells for each quantity are as much as 1 order of magnitude, even though erythrocytes are known to be fairly homogeneous in size distribution. Variations in fraction hemoglobin A0 reflect differences in in vitro oxidation rates to methemoglobin. A positive correlation was observed between carbonic anhydrase and hemoglobin A0 for individual cells. This is consistent with the presence of erythrocytes of different ages within the population, with the older cells being less capable of maintaining enzyme activity and preventing oxidative damage.     

Multiplex single strand conformation polymorphism analysis by capillary electrophoresis with on-the-fly fluorescence lifetime detection

This paper describes the use of on-the-fly fluorescence lifetime detection (OFLD) for multiplex single strand conformation polymorphism (SSCP) analysis by capillary electrophoresis (CE). The dye labels studied for multiplex SSCP-OFLD-CE analyses included RG, NBD, and BODIPY-FL. The dyes were first investigated for a model system of "Wild Type" and "Mutant" 43-base fragments designed to vary by a single A/T substitution. Two dye pairs, BODIPY-FL/ RG and BODIPY-FL/NBD, were then used to detect the G20210A mutation in the human prothrombin gene. Mobility correction was required for the BODIPY-FL/RG system. Three "blind" analyses were performed of three mixtures that combined a control fragment (wild type-BODIPY-FL) with two "unknown" fragments selected among four possibilities (wild type or mutant labeled with NBD or RG). In each multiplex analysis, the "origin" of the unknown fragments was correctly identified on the basis of fluorescence lifetime of the dye label and the presence or absence of the mutation was correctly determined on the basis of conformation-induced differences in migration time.