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Biomedical subjects

R N Zare

Publications and source records attributed to R N Zare.

At least 55 records · Page 3Linked to original sources
PubMed1995

Use of 2,3-naphthalenedicarboxaldehyde derivatization for single-cell analysis of glutathione by capillary electrophoresis and histochemical localization by fluorescence microscopy.

We report that 2,3-naphthalenedicarboxaldehyde reacts rapidly with glutathione and its precursor, gamma-glutamylcysteine, to form highly fluorescent derivatives under physiological conditions. In contrast to previous accounts of 2,3-naphthalenedicarboxaldehyde labeling of primary amines, no additional CN- ion or any other additional nucleophile is required. The fluorescence spectral properties of the chromophores (lambda exc max = 472 nm, lambda em max = 528 nm) make these derivatives amenable to excitation and detection by optical instrumentation that is optimized for fluorescein wavelengths. This selective labeling chemistry enabled quantitative determination and histochemical localization of glutathione in neurobiological samples. Intracellular glutathione was labeled by incubating cultured cells or cell suspensions in a 2,3-naphthalenedicarboxaldehyde-supplemented, DMSO-containing physiological buffer (pH = 7.4) for 2-10 min. Applications include imaging of cultured NG 108-15 cells (mouse neuroblastoma x rat glioma) and primary glial and neuronal cell cocultures (rat hippocampus) using epiluminescent and confocal fluorescence microscopy. Quantitative determination of glutathione in single NG 108-15 cells was accomplished using laser-induced fluorescence detection and capillary electrophoresis.

Animals↗
PubMed1995

Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system.

A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3-acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

Animals↗
PubMed1995

Single cells as biosensors for chemical separations.

A biosensor system based on the response of living cells was demonstrated that can detect specific components of a complex mixture fractionated by a microcolumn separation technique. This system uses ligand-receptor binding and signal-transduction pathways to biochemically amplify the presence of an analyte after electrophoretic separation. The transduced signal was measured by means of two approaches: (i) fluorescence determination of intracellular calcium concentrations in one or more rat PC-12 cells and (ii) measurement of transmembrane current in a Xenopus laevis oocyte microinjected with messenger RNA that encodes a specific receptor. This analysis system has the potential to identify biologically active ligands present in a complex mixture with exceptional sensitivity and selectivity.

Acetylcholine↗
PubMed1995

Analysis of underivatized amino acids by capillary electrophoresis using constant potential amperometric detection.

A mixture of native (underivatized) amino acids is separated by capillary electrophoresis under alkaline conditions (pH approximately 12) and amperometrically detected with a copper-disk microelectrode. A simple design facilitates capillary-electrode alignment without the need for micropositioning equipment. The limits of detection for the amino acids are in the low microM concentration range, and the signal response is linear over 2-3 orders of magnitude. This procedure is applied to analyze the amino acid hydrolysis products from cytochrome c.

Amino Acids↗
PubMed1994

Probing individual molecules with confocal fluorescence microscopy.

Confocal fluorescence microscopy coupled with a diffraction-limited laser beam and a high-efficiency detection system has been used to study the diffusive movement and emission process of individual fluorescent molecules in the liquid phase at room temperature. The high detection sensitivity achieved at fast data acquisition speeds (greater than 1 kilohertz) allows real-time observation of single-molecule fluorescence without statistical analysis. The results show fluorescence-cycle saturation at the single-molecule level and multiple recrossings of a single molecule into and out of the probe volume as well as the triplet state.

Fluorescein↗
PubMed1994

Microcolumn sample injection by spontaneous fluid displacement.

The withdrawal of a capillary structure from a sample solution causes a droplet to be formed at the end of the capillary. Because of the interfacial pressure difference across the curved surface of the droplet, the droplet is driven into the entrance of the capillary, thereby causing injection of the sample. Assuming negligible sample penetration by diffusive or convective mixing, this injection is intrinsically the smallest possible for a capillary. Moreover, the injection volume can be varied by changing the shape of the capillary structure, specifically the outer diameter of the capillary. This injection method eliminates the need for external pressure differences, applied fields across the capillary, or precise timing, thus offering several advantages over conventional procedures. Studies using capillary electrophoresis as the separation procedure show that approximately 3.5 nl (66 microns I.D. capillary) sample volumes can be injected by hand with a reproducibility of 5.8 +/- 0.7% R.S.D. Parameters that affect the variability of the injection are discussed.

Electrophoresis↗
PubMed1994

Spontaneous injection in microcolumn separations.

The phenomenon of spontaneous (ubiquitous) injection in microcolumn separations has been characterized to improve quantitative precision for ultramicrosampling and to enhance separation efficiency. By combining fluorescence imaging, video microscopy, and measurements from capillary electrophoresis, we demonstrate that spontaneous injection is caused primarily by an interfacial pressure difference formed at the inlet of the capillary. This complex injection mechanism has been modeled with some simple assumptions based on fluid dynamics. In particular, studies showed that extraneous injection is reduced up to 12-fold by etching the capillary inlet or by using a thin-walled capillary. Variations by a factor of 2 in the injection length can result from delays between sample introduction and reinsertion into the inlet vial if the timing is not controlled precisely. Evidence is presented that evaporation of buffer from the inlet can reduce the injection length by more than 1 order of magnitude.

Image Processing, Computer-Assisted↗
PubMed1994

Field programming to achieve uniform sensitivity for on-line detection in electrophoresis.

Under constant applied electric field, an analyte band traverses an on-line detection zone in an electrophoretic separation at a velocity inversely proportional to the detection time. Because analyte bands migrate with different velocities, the sensitivity of the detection system to different species can vary significantly. One approach for overcoming this problem is to change the applied electric field, E, as a function of the separation time, tau, so that the velocity of an analyte band when it passes through the detection zone is independent of the analyte's electrophoretic mobility. A possible solution is to make E(tau) grow exponentially, but this procedure causes band compression. The preferred solution is to make both E(tau) and the distance between the starting position of the sample and the detection zone increase linearly with separation time. This approach can be implemented with either slab-gel or capillary electrophoresis.

Electromagnetic Fields↗
PubMed1994

Selective determination of adenine-containing compounds by capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis coupled with laser-induced fluorescence detection provides a selective analysis of mixtures of adenine-containing compounds (adenine, adenosine, cAMP, AMP, ADP, and ATP) that are derivatized using chloroacetaldehyde as a fluorogenic reagent. The components can be detected with linear response over the concentration range of 10(-4) to 10(-9) M, and the detection limit corresponds to 5 to 10 x 10(-19) mol for each nucleotide. Analysis is achieved in less than 10 min with high separation efficiencies (up to 400,000 theoretical plates) and shows good reproducibility for migration times (0.5 to 1.0%) and peak heights (2.8 to 7.7%). To demonstrate the rapid analysis of small-volume samples, the adenine-containing compounds present in an oocyte from Xenopus laevis are determined.

Adenine↗
PubMed1993

Optimizing fluorescence detection in chemical separations for analyte bands traveling at different velocities.

In many separation techniques, such as field flow fractionation, liquid chromatography, and electrophoresis, chemical species form bands that migrate at distinct velocities. If these bands are to be quantified on-line using a shot-noise-limited detection system, then attention must be given to the data-digitization rate and to the removal rate of molecules from the analyte pool as a result of the detection process. A theory is developed for calculating the signal-to-noise ratio under such conditions, and it is specialized to the case of fluorescence detection in capillary electrophoresis. Using standard detection procedures in which the data-digitization rate and excitation intensity remain constant for the duration of a separation, detection sensitivity can vary by more than a factor of five for bands that arrive at the detection zone between migration times tau fast and 10 tau fast, where tau fast is the time after the start of the separation that the fastest migrating band arrives at the detection zone. To compensate for different band velocities, both the data-digitization rate and the excitation intensity must be decreased as separation time (tau) increases by the factor tau fast/tau. Only when these corrections are made can uniform sensitivity with the highest possible signal-to-noise ratio be achieved for each peak. These predictions are experimentally tested and compare favorably to observations.

Electrophoresis↗
PubMed1992

Analysis of free intracellular nucleotides using high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) with UV absorbance detection (254 nm) has been applied for analyzing intracellular free ribonucleotides. The nucleotide profiles obtained from peripheral blood lymphocytes differ from those obtained from Molt4 human leukemic cells. With a 140 mM borate buffer, pH 9.4, a nearly complete profile can be obtained in 25 min. HPCE has comparable resolution to that of high-performance liquid chromatography (HPLC) but is faster in terms of time per sample run (25 min vs 45 min) and requires much less sample (nanoliter range for HPCE vs microliter range for HPLC).

Chromatography, High Pressure Liquid↗
PubMed1992

Organic compounds in the Forest Vale, H4 ordinary chondrite.

We have analyzed the H4 ordinary chondrite Forest Vale for polycyclic aromatic hydrocarbons (PAHs) using two-step laser mass spectrometry (L2MS) and for amino acids using a standard chromatographic method. Indigenous PAHs were identified in the matrices of freshly cleaved interior faces but could not be detected in pulverized silicates and chondrules. No depth dependence of the PAHs was found in a chipped interior piece. Amino acids, taken from the entire sample, consisted of protein amino acids that were nonracemic, indicating that they are terrestrial contaminants. The presence of indigenous PAHs and absence of indigenous amino acids provides support for the contention that different processes and environments contributed to the synthesis of the organic matter in the solar system.

Amino Acids↗
PubMed1991

Fluorescence detection in capillary zone electrophoresis using a charge-coupled device with time-delayed integration.

A fluorescence detection system for capillary zone electrophoresis is described in which a charged-coupled device (CCD) views a 2-cm section of an axially illuminated capillary column. The CCD is operated in two readout modes: a snapshot mode that acquires a series of images in wavelength and capillary position, and a time-delayed integration mode that allows long exposure times of the moving analyte zones. By use of the latter mode, the ability to differentiate a species based on both its fluorescence emission and migration rate is demonstrated for fluorescein and sulforhodamine 101. The detection limit for fluorescein isothiocyanate (FITC) is 1.2 X 10(-20) mol; detection limits for FITC-amino acids are in the (2-8) X 10(-20) mol range.

Amino Acids↗
PubMed1991

Protocol for resolving protein mixtures in capillary zone electrophoresis.

The separation of protein mixtures by capillary zone electrophoresis can be plagued by wall adsorption of the protein components, causing peak broadening and distortion. A method is presented for overcoming this problem by adding ethylene glycol to the protein sample and by choosing the running buffer and protein sample to be at different pH values and molarities. This protocol appears to work for a wide class of proteins having different molecular weights and pI values. The method has been applied to the analysis of proteins in human serum. Compared to the traditional method of agarose gel electrophoresis, the present method is more rapid and offers better resolution, suggesting its potential as a clinical diagnostic of certain disease states.

Blood Proteins↗