Interface of an array of five capillaries with an array of one-nanoliter wells for high-resolution electrophoretic analysis as an approach to high-throughput chemical cytometry

We report a system that allows the simultaneous aspiration of one or more cells into each of five capillaries for electrophoresis analysis. A glass wafer was etched to create an array of 1-nL wells. The glass was treated with poly(2-hydroxyethyl methacrylate) to control cell adherence. A suspension of formalin-fixed cells was placed on the surface, and cells were allowed to settle. The concentration of cells and the settling time were chosen so that there was, on average, one cell per well. Next, an array of five capillaries was placed so that the tip of each capillary was in contact with a single well. A pulse of vacuum was applied to the distal end of the capillaries to aspirate the content of each well into a capillary. Next, the tips of the capillaries were placed in running buffer and potential was applied. The cells lysed upon contact with the running buffer, and fluorescent components were detected at the distal end of the capillaries by laser-induced fluorescence. The electrophoretic separation efficiency was outstanding, generating over 750,000 theoretical plates (1,800,000 plates/m). In this example, AtT-20 cells were used that had been treated with TMR-G(M1). The cells were allowed to metabolize this substrate into a series of products before the cells were fixed. The number of cells found in each well was estimated visually under the microscope and was described by a Poisson distribution with mean of 0.98 cell/well. This system provides an approach to high-throughput chemical cytometry.     

One-dimensional protein analysis of an HT29 human colon adenocarcinoma cell

A single HT29 human colon adenocarcinoma cell was introduced into a fused-silica capillary and lysed, and the protein content was fluorescently labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. The labeled proteins were separated by capillary electrophoresis in a submicellar buffer and detected by laser-induced fluorescence in a postcolumn sheath-flow cuvette. Several dozen components were resolved. A number of experiments were done to verify that these components were proteins. Most components of the single-cell electropherogram had the same mobility as components present in the 30-100 kDa fraction of a protein extract prepared from the cell culture. One component was identified as a approximately 100 kDa protein by co-injecting the sample with purified protein obtained from an SDS-PAGE gel. Protein expression varied significantly between cells, but the average expression was consistent with that observed from a protein extract prepared from 10(6) cells.     

Biarsenical-tetracysteine motif as a fluorescent tag for detection in capillary electrophoresis

Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, relatively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine whether the biarsenical labeling scheme could be extended to fluorescent detection of analytes in capillary electrophoresis. Recombinant protein or synthesized peptides containing the optimized tetracysteine motif "-C-C-P-G-C-C-" were labeled with biarsenical dyes and then analyzed by micellar electrokinetic capillary chromatography (MEKC). The biarsenical-tetracysteine complex was stable and remained fluorescent under standard MEKC conditions for peptide and protein separations. The detection limit following electrophoresis in a capillary was less than 3 x 10(-20) mol with a simple laser-induced fluorescence system. A mixture of multiple biarsenical-labeled peptides and a protein were easily resolved. Demonstrating that the label did not interfere with bioactivity, a peptide-based enzyme substrate conjugated to the tetracysteine motif and labeled with a biarsenical dye retained its ability to be phosphorylated by the parent kinase. The feasibility of using this label for chemical cytometry experiments was shown by intracellular labeling and subsequent analysis of a recombinant protein possessing the tetracysteine motif expressed in living cells. The extension of the biarsenical-tetracysteine tag to fluorescent labeling of peptides and proteins in chemical separations is a valuable addition to biochemical and cell-based investigations.     

Capillary isoelectric focusing and fluorometric detection of proteins and microorganisms dynamically modified by poly(ethylene glycol) pyrenebutanoate

The nonionogenic pyrene-based tenside, poly(ethylene glycol) pyrenebutanoate, was prepared and applied in capillary isoelectric focusing with fluorometric detection. This dye was used here as a buffer additive in capillary isoelectric focusing for a dynamic modification of the sample of proteins and microorganisms. The values of the isoelectric points of the labeled bioanalytes were calculated with use of the fluorescent pI markers and were found comparable with pI of the native compounds. The mixed cultures of proteins and microorganisms, Escherichia coli CCM 3954, Staphylococcus epidermidis CCM 4418, Proteus vulgaris, Enterococcus faecalis CCM 4224, and Stenotrophomonas maltophilia, the strains of the yeast cells, Candida albicans CCM 8180, Candida krusei, Candida parapsilosis, Candida glabrata, Candida tropicalis, and Saccharomyces cerevisiae were reproducibly focused and separated by the suggested technique. Using UV excitation for the on-column fluorometric detection, the minimum detectable amount was down to 10 cells injected on the separation capillary.     

Probing single molecules in single living cells

Single-molecule detection in single living cells has been achieved by using confocal fluorescence microscopy and externally tagged probe molecules. The intracellular background fluorescence is substantially higher than that in aqueous buffer, but this background is continuous and stable and does not significantly interfere with the measurement of single-molecule photon bursts. As a result, single-molecule data have been obtained on three types of fluorescent probes at spatially resolved locations (e.g., cytoplasm and nucleus) inside human HeLa cells. First, the iron transport protein transferrin labeled with tetramethylrhodamine undergoes rapid receptor-mediated endocytosis, and single transferrin molecules are detected inside living cells. Second, the cationic dye rhodamine 6G (R6G) enters cultured cells by a potential-driven process, and single R6G molecules are observed as intense photon bursts when they move in and out of the intracellular laser beam. Third, we report results on synthetic oligonucleotides that are tagged with a fluorescent dye and are taken up by living cells via a passive, nonendocytic pathway.     

Fluorescent liposomes as contrast agents for in vivo optical imaging of edemas in mice

This study assesses if specially designed fluorescent liposomes can be used as contrast agent for near-infrared fluorescence (NIRF) optical imaging of cultured macrophages in vitro and for NIRF imaging of inflammatory processes, like edema, in an in vivo mouse model. Fluorescent liposomes are prepared by the film hydration and extrusion method using cholesterol, L-phosphatidylcholine, and the NIR fluorescent dye DY-676-C(18) ester. Photon correlation spectroscopy and flow cytometry reveal that fluorescent liposomes are structurally stable for up to 133 days. Distinct uptake/labeling of cultured murine J774 macrophages is demonstrated by confocal laser scanning microscopy (CLSM), flow cytometry, and macroscopic NIRF imaging system at wavelengths >670 nm. Moreover, CLSM analysis reveals fluorescence signals within intracellular compartments. Ear edema is induced in mice (n = 16) by subcutaneous injection of zymosan A. Whole-body NIRF imaging is performed after intravenous injection (0-24 h) of fluorescent liposomes (55 nmol dye per kg body weight). Distinctly higher fluorescence intensities (1613.6 +/- 61.7 a.u.) are detected at inflamed areas of diseased mice as compared to controls (892.8 +/- 19.4 a.u.). Furthermore, cell isolated from ear lavage reveals the presence of labeled F4/80 positive tissue macrophages. Taken together, the results indicate both that mouse macrophages labeled with fluorescent liposomes can be detected in vitro with fluoro-optical methods and that in vivo optical imaging of inflammatory processes with fluorescent liposomes as contrast agent is feasible. Possibly, early stages of other inflammatory diseases could also be detected by the proposed diagnostic tool in the long term.     

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.     

Generation of focused electric field patterns at dielectric surfaces

We here report on a concept for creating well-defined electric field gradients between the boundaries of capillary electrode (a capillary of a nonconducting material equipped with an interior metal electrode) outlets, and dielectric surfaces. By keeping a capillary electrode opening close to a boundary between a conducting solution and a nonconducting medium, a high electric field can be created close to the interface by field focusing effects. By varying the inner and outer diameters of the capillary, the span of electric field strengths and the field gradient obtained can be controlled, and by varying the slit height between the capillary rim and the surface, or the applied current, the average field strength and gradient can be varied. Field focusing effects and generation of electric field patterns were analyzed using finite element method simulations. We experimentally verified the method by electroporation of a fluorescent dye (fluorescein diphosphate) into adherent, monolayered cells (PC-12 and WSS-1) and obtained a pattern of fluorescent cells corresponding to the focused electric field.     

Imaging of Zn2+ release from pancreatic beta-cells at the level of single exocytotic events

Regulated secretion of Zn2+ from isolated pancreatic beta-cells was imaged using laser-scanning confocal microscopy. In the method, beta-cells were incubated in a solution containing the novel fluorescent Zn2+ indicator FluoZin-3. Zn2+ released from the cells reacted with the dye to form a fluorescent product, which was detected by the confocal microscope. The new dye is much brighter than Zinquin, previously used for this application, allowing detection limits of 10-40 nM and temporal resolution of 16 ms/image. The high temporal resolution allowed imaging of isolated fluorescent transients that occurred at the edge of the cells following stimulation with 20 mM glucose or 40 mM K+. Fluorescent transients took 16-50 ms to reach a peak from the initial rise and returned to baseline after 170 +/- 50 ms (n = 78 transients from 15 cells). It was concluded that the transients correspond to detection of exocytotic release of Zn2+. Analysis of the temporal and spatial dispersion of the transients indicates that the release of Zn2+ is not diffusion limited but is instead kinetically controlled in agreement with previous observations of insulin release detected by amperometry.     

Monitoring the uptake of glycosphingolipids in Plasmodium falciparum-infected erythrocytes using both fluorescence microscopy and capillary electrophoresis with laser-induced fluorescence detection

The metabolism of glycosphingolipids by the malaria-causing parasite Plasmodium falciparum plays an important role in the progression of the disease. We report a new and highly sensitive method to monitor the uptake of glycosphingolipids in infected red blood cells (iRBCs). A tetramethylrhodamine-labeled glycosphingolipid (GM1-TMR) was used as a substrate. Uptake was demonstrated by fluorescence microscopy. The iRBCs were lysed with a 15% solution of saponin and washed with phosphate buffered saline to release intact parasites. The parasites were further lysed and the resulting homogenates were analyzed by capillary electrophoresis with laser-induced fluorescence detection. The lysate from erythrocytes infected at 1% parasitemia generated a signal 20 standard deviations larger than uninfected erythrocytes, which suggests that relatively low infection levels can be studied with this technique.     

Nonaqueous capillary electrophoresis of fatty acids derivatized with a near-infrared fluorophore

Saturated linear fatty acids, derivatized with a near-infrared absorbing fluorescent dye, were separated in 100% methanol with 12.5 mM tetraethylammonium chloride added as a charge carrier. Separation at 380 V/cm was acceptable for acids that differed in length by a single carbon. The labeled linear fatty acids behaved as random coils in the nonaqueous separation medium, as shown in a fit to a simple theoretical expression. However, even in 100% methanol with a trimethylsilylated capillary, significant adsorption to the capillary wall occurred, which reduced resolution and slowed the separation. Addition of water to the methanol medium caused significant differences in separation behavior of high molecular weight acids (>C16). Addition of a cetyltrimethylammonium bromide surfactant to the separation medium dynamically coated the capillary and greatly improved the separation. The surfactant also interacted with the acyl tail, apparently causing it to collapse. Resolution in an optimal separation medium (20 mM surfactant) ranged from 1.6 to 1.1, depending on chain length, and theoretical plate heights were under 4 microm (N > 10(5)). Resolution was more than adequate to separate stearic (C18:0) from oleic (C18:1) acid, as well as other unsaturated C18 homologues.     

Fluorescence-labeled peptide pI markers for capillary isoelectric focusing

Nineteen fluorescent pH standards or pI markers ranging pH 3.64-10.12 were developed for use in capillary isoelectric focusing using laser-induced fluorescence detection. Tetra- to tridecapeptides containing one cysteine residue were designed to focus sharply at their respective isoelectric points by including amino acids that contain charged side chains, the pKa values of which are close to the corresponding pI values. An iodoacetylated derivative of tetramethylrhodamine was coupled to the thiol group of cysteine to yield fluorescent pI markers. The pI values of the labeled peptides were precisely determined after isoelectric focusing on polyacrylamide gel slabs by direct measurement of the pH of the focused bands. The markers were subjected to capillary isoelectric focusing for 10-15 min in coated capillaries under conditions of low electroosmosis and were detected by means of a scanning laser-induced fluorescence detector down to a level of subpicomolar range. The markers permitted the calibration of a wide-range pH gradient formed in a capillary by fluorescence detection for the first time and should facilitate the development of highly sensitive analytical methods based on a combination of capillary isoelectric focusing and laser-induced fluorescence detection.     

Cell cycle-dependent protein fingerprint from a single cancer cell: image cytometry coupled with single-cell capillary sieving electrophoresis

Study of cell cycle-dependent protein expression is important in oncology, stem cell research, and developmental biology. In this paper, we report the first protein fingerprint from a single cell with known phase in the cell cycle. To determine that phase, we treated HT-29 colon cancer cells with Hoescht 33342, a vital nuclear stain. A microscope was used to measure the fluorescence intensity from one treated cell; in this form of image cytometry, the fluorescence intensity is proportional to the cell's DNA content, which varies in a predictable fashion during the cell cycle. To generate the protein fingerprint, the cell was aspirated into the separation capillary and lysed. Proteins were fluorescently labeled with 3-(2-furoylquinoline-2-carboxaldehyde, separated by capillary sieving electrophoresis, and detected by laser-induced fluorescence. This form of electrophoresis is the capillary version of SDS-PAGE. The single-cell electropherogram partially resolved approximately 25 components in a 30-min separation, and the dynamic range of the detector exceeded 5000. There was a large cell-to-cell variation in protein expression, averaging 40% relative standard deviation across the electropherogram. The dominant source of variation was the phase of the cell in the cell cycle; on average, approximately 60% of the cell-to-cell variance in protein expression was associated with the cell cycle. Cells in the G1 and G2/M phases of the cell cycle had 27 and 21% relative standard deviations in protein expression, respectively. Cells in the G2/M phase generated signals that were twice the amplitude of the signals generated by G1 phase cells, as expected for cells that are soon to divide into two daughter cells. When electropherograms were normalized to total protein content, the expression of only one component was dependent on cell cycle at the 99% confidence limit. That protein is tentatively identified as cytokeratin 18 in a companion paper.     

Staining method for protein analysis by capillary gel electrophoresis

A novel staining method and the associated fluorescent dye were developed for protein analysis by capillary SDS-PAGE. The method strategy is to synthesize a pseudo-SDS dye and use it to replace some of the SDS in SDS-protein complexes so that the protein can be fluorescently detected. The pseudo-SDS dye consists of a long, straight alkyl chain connected to a negative charged fluorescent head and binds to proteins just as SDS. The number of dye molecules incorporated with a protein depends on the dye concentration relative to SDS in the sample solution, since SDS and dye bind to proteins competitively. In this work, we synthesized a series of pseudo-SDS dyes, and tested their performances for capillary SDS-PAGE. FT-16 (a fluorescein molecule linked with a hexadodecyl group) seemed to be the best among all the dyes tested. Although the numbers of dye molecules bound to proteins (and the fluorescence signals from these protein complexes) were maximized in the absence of SDS, high-quality separations were obtained when co-complexes of SDS-protein-dye were formed. The migration time correlates well with protein size even after some of the SDS in the SDS-protein complexes was replaced by the pseudo-SDS dye. Under optimized experimental conditions and using a laser-induced fluorescence detector, limits of detection of as low as 0.13 ng/mL (bovine serum albumin) and dynamic ranges over 5 orders of magnitude in which fluorescence response is proportional to the square root of analyte concentration were obtained. The method and dye were also tested for separations of real-world samples from E. coli.     

Microfabricated system for parallel single-cell capillary electrophoresis

Performing single-cell electrophoresis separations using multiple parallel microchannels offers the possibility of both increasing throughput and eliminating cross-contamination between different separations. The instrumentation for such a system requires spatial and temporal control of both single-cell selection and lysis. To address these problems, a compact platform is presented for single-cell capillary electrophoresis in parallel microchannels that combines optical tweezers for cell selection and electromechanical lysis. Calcein-labeled acute myloid leukemia (AML) cells were selected from an on-chip reservoir and transported by optical tweezers to one of four parallel microfluidic channels. Each channel entrance was manufactured by F2-laser ablation to form a 20- to 10-microm tapered lysis reservoir, creating an injector geometry effective in confining the cellular contents during mechanical shearing of the cell at the 10-microm capillary entrance. The contents of individual cells were simultaneously injected into parallel channels resulting in electrophoretic separation as recorded by laser-induced fluorescence of the labeled cellular contents.     

Capillary electrophoretic separation of nuclei released from single cells

We report here the first capillary electrophoresis analysis of intact nuclei released on-column from single cells. Expression of the nuclear-targeted protein nuDsRed2 and the plasma membrane-bound farnesylated enhanced green fluorescent protein in cultured human DeltaH2-1 cells allowed fluorescent monitoring of the fate of these subcellular compartments upon injection of a single cell into the separation capillary. On-column treatment with digitonin allowed for the separation of the plasma membrane from the nucleus as indicated by their selective laser-induced fluorescence detection in two separate spectral regions. The data suggest that less than 0.1% of the plasma membrane remains bound to individually detected nuclei. In digitonin-treated cells, the electropherograms consisted of a prominent fluorescent peak attributed to nuDsRed2 localized to the nucleus and a collection of weakly fluorescent events (barely distinguishable from scattering) that seem to indicate additional localization of this protein to other subcellular regions. Taken together, this report points to the feasibility of studying intact organelles released from a single mammalian cell by capillary electrophoresis, which is a prerequisite to understanding the relevance of subcellular heterogeneity in biological systems.     

Determination of intracellular species at the level of a single erythrocyte via capillary electrophoresis with direct and indirect fluorescence detection.

Intracellular contents reflect the specific history of a cell including innate physiological heterogeneity as well as differing levels of exposure to environmental influences. A method capable of analyzing a variety of species from within a single human erythrocyte is demonstrated. Guided by a microscope, individual cells can be drawn into open capillaries of 10-microns i.d. On contact with a low ionic strength buffer solution, the cell lyses and releases its intracellular fluid. The ionic components are then separated by capillary electrophoresis. For glutathione, microderivatization with a fluorescent reagent can be accomplished in vitro with monobromobimane. The effects of extracellular oxidizing and reducing agents on the glutathione levels can thus be followed. For sodium and potassium, or any other ionic species, charge displacement of a fluorescent cation results in indirect fluorescence detection. The two detection modes are suitable for intracellular components present at low-attomole and sub-femtomole levels, respectively.     

Color-tuned FRET polystyrene microspheres by single wavelength excitation

Fluorescent monodisperse polystyrene microspheres were prepared by two-stage dispersion polymerization, which successfully covalently labeled microspheres with two dyes without disturbing the final particle size and size distribution. By varying the dye concentrations, microspheres show tuned colors with different fluorescent intensity under a single wavelength excitation. Fluorescence resonance energy transfer (FRET) between two labeled dyes was proved to contribute to the emission of the longer-wavelength dye at a shorter-wavelength excitation. There is no dye leakage for microspheres because of the covalent incorporation of dye molecules. The microsphere matrix provides good protection of dye molecules and blocks the influence of media outside on the fluorescence of microspheres. Single microsphere shows intense fluorescence due to a large number of encapsulated dye molecules. These uniform barcoding fluorescent microspheres have potential application in multiplexed bioanalysis.     

Monitoring electroosmotic flow by periodic photobleaching of a dilute, neutral fluorophore

Electroosmotic flow has been monitored in a capillary using a method based on periodic photobleaching of a neutral, fluorescent buffer additive. Rhodamine B was determined to be neutral between pH 6.0 and 10.8 and was added to the running buffer at a concentration of 400 nM. Rhodamine B was photobleached by opening a shutter under computer control for 250 ms every 5.00 s, to expose the dye to a laser beam and create a photobleached zone. The time was measured for the photobleached zone to migrate 6.13 mm to a downstream laser-induced fluorescence detector, to determine the rate of electroosmotic flow in the entire capillary. The flow rate was sampled every 5.00 s, and the precision of the flow measurements was 0.7% or better. Three fluorescent compounds were separated and detected by capillary electrophoresis with laser-induced fluorescence detection, while simultaneously monitoring the electroosmotic flow rate.     

Light-emitting-diode-induced fluorescence detection of fluorescent dyes for capillary electrophoresis microchip with cross-polarization method

A cross-polarization scheme is presented to filter out the excitation light from the emission spectrum of fluorescent dyes using green light emitting diodes as a light source and a linear charge coupled device as an intensity detector. The excitation light was linearly polarized and was then used to illuminate the fluorescent dyes in the microchannels of a capillary electrophoresis microchip. The detector was shielded by the second polarizer, oriented perpendicular to the excitation light. The fluorescent signals from Rhodamine B dyes were measured in a dilution series with resulting emission signals and four different concentrations of fluorescent dyes were detected simultaneously with the same excitation source and detector. A limit-of-detection of 1 μM was demonstrated for Rhodamine B dye under the optimal conditions.