Probing single secretory vesicles with capillary electrophoresis.

Secretory vesicles obtained from the atrial gland of the gastropod mollusk Aplysia californica were chemically analyzed individually with a combination of optical trapping, capillary electrophoresis separation, and a laser-induced fluorescence detection. With the use of optical trapping, a single vesicle that had attoliters (10(-18) liters) of volume was introduced into the tapered inlet of a separation capillary. Once the vesicle was injected, it was lysed, and its components were fluorescently labeled with naphthalene-2, 3-dicarboxaldehyde before separation. The resultant electropherograms indicated distinct variations in the contents of single vesicles.

Temporal and spatial monitoring of exocytosis with native fluorescence imaging microscopy.

Exocytosis of rat peritoneal mast cells (RPMCs) was monitored temporally and spatially using native fluorescence imaging microscopy with 305-nm laser excitation. Real time chemical images of the relative amounts of serotonin and protein released from each cell are obtained. Individual cells released different amounts of material and the time delay of the release event after stimulation by polymyxin varied from cell to cell. Release consisted of a main burst of activity followed by slow sustained secretion over many seconds. The images show that different regions of a given cell behave asynchronously in releasing material into the surrounding medium. On rare occasions, highly localized fluorescence bursts can be seen in the vicinity of the cell. Presumably, these are due to delayed release of fluorescent mediators from single granules, following detachment of the latter from the cell. These quantitative fluorescence measurements allow one to follow the time-course of the physiologically important parameter---the amount of material that is secreted into the body fluid on stimulation.

UV- and visible-excited fluorescence of nucleic acids separated by capillary electrophoresis.

UV- and visible-excited fluorescence detection strategies were compared for nucleic acids separated by capillary electrophoresis (CE). A dual-polymer sieving matrix consisting of hydroxypropylmethylcellulose and poly(vinylpyrrolidone) was used to separate DNA fragments from a 100-base pair ladder and RNA from individual cells. Two nucleic acid dyes, SYBR Gold and SYBR Green I, were evaluated for their performance at both UV (275 nm) and visible (488 nm) excitation wavelengths. While SYBR Gold-bound RNA from single cells yielded a substantially reduced UV-excited signal compared to that with visible excitation (as expected), the sensitivity of SYBR Gold-bound double-stranded DNA was comparable for UV and Vis excitation wavelengths. This study reveals the first demonstration of using SYBR Gold dyes for DNA detection following separation with CE and also the first example of SYBR-based detection of RNA sampled and separated from individual cells.

Separation of hemoglobin variants in single human erythrocytes by capillary electrophoresis with laser-induced native fluorescence detection.

Single human red blood cells, in which the hemoglobin (Hb) molecules exist in their native, tetrameric states, were analyzed. Upon injection and lysis of a cell, the tetramers were dissociated on-column into their respective polypeptide chains, separated, and detected by laser-induced native fluorescence detection with 275-nm excitation. This technique was applied to the determination of hemoglobin variants as found in adult (normal and elevated Hb A1) and fetal erythrocytes. Normal adult cells contained 9.6% and 4.8% glycated beta- and alpha-chains, respectively. Cells with elevated Hb A1 gave 30% and 12%, respectively. The amounts of glycated Hb and total Hb in a given cell were found to be uncorrelated.

Continuous cell introduction for the analysis of individual cells by capillary electrophoresis.

Instrumentation for high-throughput analysis of single cells by capillary electrophoresis is described. A flow-based interface that uses electroosmotic flow (EOF) provides continuous injection of intact cells through an introduction capillary into a cell lysis junction and migration of the resulting cell lysate through a separation capillary for analysis. Specifically, two capillaries were coupled together with 5-mm-long Teflon tubing to create a approximately 5-microm gap, and the junction was immersed in a buffer reservoir. High voltage was applied across both capillaries so that cells were continuously pumped into the first capillary by EOF. Individual cells were lysed on-column at the junction without detergents, presumably owing to mechanical disruption caused by a dramatic change in flow properties at the gap. After each cell was lysed at the junction, the major proteins hemoglobin and carbonic anhydrase were separated by capillary electrophoresis and the resultant analyte zones were detected by laser-induced native fluorescence using 275-nm excitation. The detection limits of hemoglobin and carbonic anhydrase were 37 and 1.6 amol, respectively, which correlate well with the literature. The instrumentation was evaluated with intact red blood cells. The averaged time for complete analysis (i.e., continuous injection, lysis, separation, and detection) of one human erythrocyte was less than 4 min with this capillary-based setup. Moreover, this instrumentation simplifies the introduction of individual, intact cells without the use of a microscope.

In-situ sampling and separation of RNA from individual mammalian cells.

In this investigation RNA was directly sampled and separated at the single-cell level (without extraction) by capillary electrophoresis (CE). Laser-induced fluorescence (LIF) was employed to detect ethidium bromide-labeled RNA molecules under native conditions. Hydroxypropylmethylcellulose was used as a matrix for molecular sieving. Additives to the polymer solution included poly(vinylpyrrolidone) to eliminate the electroosmotic flow and mannitol to enhance the separation. Peak identities were confirmed as RNA by enzymatic treatment with RNase I. The individual Chinese Hamster Ovary (CHO-K1) cells were injected into a capillary and the cells were lysed online with sodium dodecyl sulfate (SDS) solutions before running electrophoresis. Low molecular mass (LMM) RNAs as well as larger fragments (tentatively identified as 18S and 28S ribosomal RNA by comparison with the literature) were detected with this system, which corresponds to a detected amount of approximately equals 10-20 pg of RNA/cell. A Proteinase K study showed that proteins incorporated with RNA molecules were eliminated by SDS treatment and thus did not influence the migration of RNA. Experiments were also performed with this technique to detect nucleic acid damage. Changes in the peak pattern were detected in the cells treated with hydrogen peroxide, which meant that strand breaks occurred in DNA and RNA. It was found that 60 mM caused the most severe damage to the nucleic acids.

Measurement of single-cell gene expression using capillary electrophoresis.

Capillary electrophoresis with laser-induced fluorescence detection was used to monitor gene expression in individual mammalian cells using the reverse transcriptasepolymerase chain reaction. Specifically, beta-actin expression in single LNCaP (prostate cancer) cells was measured. A sieving matrix containing hydroxypropyl methyl cellulose was used to effect size-based separation. Ethidium bromide fluorescence of the product DNA was used as the detection scheme and yielded excellent sensitivity. The beta-actin product, resulting from an individual cell lysed by a freeze-thaw method, gave an average signal-to-noise ratio (S/N) of 77+/-27 (n = 2). Chemical lysis of a single cell, using a dilute solution of SDS, gave a S/N of 26+/-2 (n = 2), roughly 3-fold lower than for freeze-thaw lysis. An initial detection limit (not considering fully optimized conditions) was calculated from an amplified cDNA standard to correspond to a concentration of approximately 133 starting molecules/nL (of beta-actin mRNA).

Analysis of single erythrocytes by injection-based capillary isoelectric focusing with laser-induced native fluorescence detection.

A modified version of capillary isoelectric focusing (cIEF) was developed to separate hemoglobin variants contained within single human erythrocytes. Laser-induced native fluorescence with 275 nm excitation was used to detect the separated hemoglobins. In this method, baseline fluctuations were minimized and detection sensitivity was improved by using dilute solutions of anolyte, catholyte, and carrier ampholytes (with methylcellulose). Since electroosmotic flow was used for mobilization of the focused bands, separation and detection were integrated into a single step. The capillary was first filled with only ampholyte solution, and the cell (or standard) was injected as in capillary zone electrophoresis. The approximately 90 fl injection volume for individual cells is 7 x 10(4) times lower than those previously reported. Adult (normal and elevated A1), sickle (heterozygous), and fetal erythrocytes were analyzed with the amounts of hemoglobins AO, A1c, S and F determined. The pH gradient for cIEF was linear (r = 0.9984), which allowed tentative identification of Hb Fac. Variants differing by as little as 0.025 pl units were resolved.

Screening and characterization of biopharmaceuticals by high-performance capillary electrophoresis with laser-induced native fluorescence detection.

High-performance capillary electrophoresis (HPCE) with laser-induced native fluorescence (LIF) detection is used to address significant problems in the quality control of biopharmaceuticals. All of the biopharmaceuticals studied can be detected at subnanomolar levels with linear dynamic ranges of at least 3 orders of magnitude. HPCE/LIF can determine impurities in "purified" biopharmaceuticals present in amounts less than 0.01% (i.e., at 4 x 10(-11) M) that of the major component. With HPCE/LIF, detection sensitivity is thus no longer a concern in the assaying of active ingredients in biopharmaceutical dosage formulations. The peptide mapping of biopharmaceuticals present at 1 x 10(-7) M (or an injected limit of detection of 60 amol) is presented. Also, kinetic information on the reaction of a recombinant enzyme-drug with its substrate present at the micromolar level has been extracted from electropherograms acquired in real-time.

Monitoring exocytosis and release from individual mast cells by capillary electrophoresis with laser-induced native fluorescence detection.

The complex temporal evolution of on-column exocytotic release of serotonin and proteins from individual rat peritoneal mast cells (RPMCs) was monitored by using capillary electrophoresis. Laser-induced native fluorescence detection with 275-nm excitation was used, and a detection limit of 1.7 amol (S/N = 3; rms) was obtained for serotonin. A physiological running buffer was used to ensure that the cell remained viable throughout. The secretagogue was polymyxin B sulfate (Pmx). Following the injection of a single mast cell into the capillary, electromigration of Pmx toward and past the cell induced degranulation and release of serotonin. The time course of release was registered in the electropherograms with subsecond resolution. Subsequent introduction of SDS caused the cell to lyse completely and allowed the residual serotonin to be quantified. The average amount of serotonin observed per RPMC was 1.6 +/- 0.6 fmol; the average percentage of serotonin released was 28 +/- 14%. Events that are consistent with released serotonin from single submicrometer granules (250 aL each) were evident, each of which contained an average amount of 5.9 +/- 3 amol.

Separation and characterization of amines from individual atrial gland vesicles of Aplysia californica.

Several amine-containing components of individual vesicles from the atrial gland of Aplysia californica were identified with capillary electrophoresis (CE). On-line derivatization with naphthalene-2,3-dicarboxaldehyde was performed, and the derivatized amine-containing components were detected with laser-induced fluorescence (LIF). Amino acids, including taurine, that had not been determined previously in atrial gland vesicles were observed by using CE-LIF, and their identities were confirmed with CE, HPLC, NMR, and electrospray ionization mass spectrometry. The finding that taurine is packaged and stored into secretory vesicles supports the hypothesis that taurine may exhibit neuromodulatory activity. The bioactive peptides, well-known to be in atrial gland vesicles, were detected in lysed vesicle samples fractionated with HPLC and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These peptides were also observed in single-vesicle runs with CE-LIF. The atrial gland vesicles (ranging from 0.5 to 2 microns diameter and 65 aL to 4 fL volume, respectively) studied in this work represent the smallest biological entities to be analyzed chemically on an individual basis.