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Biomedical subjects

R N Zare

Publications and source records attributed to R N Zare.

At least 19 recordsLinked to original sources
PubMed1996

Rapid preparation of giant unilamellar vesicles.

We report here a rapid evaporation method that produces in high yield giant unilamellar vesicles up to 50 microns in diameter. The vesicles are obtained after only 2 min and can be prepared from different phospholipids, including L-alpha-phosphatidylcholine (lecithin), dipalmitoleoyl L-alpha-phosphatidylcholine, and beta-arachidonoyl gamma-palmitoyl L-alpha-phosphatidylcholine. Vesicles can be produced in distilled water and in Hepes, phosphate, and borate buffers in the pH range of 7.0 to 11.5 with ionic strengths up to 50 mM. The short preparation time allows encapsulation of labile molecular targets or enzymes with high catalytic activities. Cell-sized proteoliposomes have been prepared in which gamma-glutamyltransferase (EC 2.3.2.2) was functionally incorporated into the membrane wall.

Drug Stability↗
PubMed1996

Search for past life on Mars: possible relic biogenic activity in martian meteorite ALH84001.

Fresh fracture surfaces of the martian meteorite ALH84001 contain abundant polycyclic aromatic hydrocarbons (PAHs). These fresh fracture surfaces also display carbonate globules. Contamination studies suggest that the PAHs are indigenous to the meteorite. High-resolution scanning and transmission electron microscopy study of surface textures and internal structures of selected carbonate globules show that the globules contain fine-grained, secondary phases of single-domain magnetite and Fe-sulfides. The carbonate globules are similar in texture and size to some terrestrial bacterially induced carbonate precipitates. Although inorganic formation is possible, formation of the globules by biogenic processes could explain many of the observed features, including the PAHs. The PAHs, the carbonate globules, and their associated secondary mineral phases and textures could thus be fossil remains of a past martian biota.

Antarctic Regions↗
PubMed1996

Patch-clamp detection of neurotransmitters in capillary electrophoresis.

Gamma-aminobutyrate acid, L-glutamate, and N-methyl-D-aspartate were separated by capillary electrophoresis and detected by the use of whole-cell and outside-out patch-clamp techniques on freshly dissociated rat olfactory interneurons. These neuroactive compounds could be identified from their electrophoretic migration times, unitary channel conductances, and power spectra that yielded corner frequencies and mean single-channel conductances characteristic for each of the different agonist-receptor interactions. This technique has the sensitivity to observe the opening of a single ion channel for agonists separated by capillary electrophoresis.

Animals↗
PubMed1996

Cell-to-cell scanning in capillary electrophoresis.

A widespread limitation in using cell-based biosensors for repetitive chemical analysis is loss of agonist-induced response caused by receptor desensitization. We overcome this problem by scanning an array of immobilized cells underneath a capillary electrophoresis column outlet. In this way, electrophoretically fractionated components that exit the separation capillary are always directed onto cells previously unexposed to receptor agonists. To demonstrate this concept of response recovery using a scanning format, we have chosen the bradykinin B2 receptor system in the NG108-15 cell line, which is known to undergo desensitization. Whereas four subsequent injections of 250 microM bradykinin separated by 120 s are found to reduce the NG108-15 cell response markedly, scanning to new cells can fully restore the response during the separation. Furthermore, by pretesting individual NG108-15 cells for an agonist response and then later scanning back to the same cell, we achieved a 100% success rate in detecting bradykinin in subsequent electrophoretic separations.

Animals↗
PubMed1996

Automated capillary electrochromatography: reliability and reproducibility studies.

The routine application of capillary electrochromatography (CEC) is demonstrated by incorporating 75 microns I.D. capillaries packed with 3 microns octadecylsilica (ODS) particles into a commercial CZE instrument. A mixture of several neutral compounds is separated into its components with an average efficiency up to 181 000 plates/m in less than 8 min. Hundreds of consecutive runs are performed over a period of weeks from which it is concluded that the reproducibility of the capacity factors is better than 2% and that CEC separations can be achieved in a reliable and routine manner.

Chromatography↗
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↗