Michael T. Bowser

In vivomonitoring of amine neurotransmitters using microdialysis with on-line capillary electrophoresis

Microdialysis sampling was coupled via a flow‐gated interface on‐line to capillary electrophoresis with laser‐induced fluorescence (LIF) detection for in vivo monitoring of neuroactive amino acids and amines. In the instrument, analytes are derivatized precolumn with o‐phthaldehyde and β‐mercaptoethanol to form fluorescent isoindole products. The instrument was improved over previous designs by incorporating a sheath‐flow cuvette for reduced background in LIF detection which improved sensitivity by 15‐fold. The methodology was improved by utilizing a voltage ramped injection which allowed generation of 500u2009000 theoretical plates with 20 s separations. Resolution of the isoindole derivatives was further improved by addition of hydroxypropyl‐modified β‐cyclodextrin to the electrophoresis buffer. The new instrumentation and methods allow resolution and detection of glutamate, γ‐aminobutyric acid, glycine, aspartate, serine, taurine, glutamine and dopamine (if levels are elevated) collected from in vivo sampling probes every 20 s. The technique is suited to continuous monitoring for dynamic measurements of these compounds in vivo.

Neuronal release of d-serine: a physiological pathway controlling extracellular d-serine concentration

d‐Serine is thought to be a glia‐derived transmitter that activates N‐methyl d‐aspartate receptors (NMDARs) in the brain. Here, we investigate the pathways for d‐serine release using primary cultures, brain slices, and in vivo microdialysis. In contrast with the notion that d‐serine is exclusively released from astrocytes, we found that d‐serine is released by neuronal depolarization both in vitro and in vivo. Veratridine (50 µM) or depolarization by 40 mM KCl elicits a significant release of endogenous d‐serine from primary neuronal cultures. Controls with astrocyte cultures indicate that glial cells are insensitive to veratridine, but release d‐serine mainly by the opening of volume‐regulated anion channels. In cortical slices perfused with veratridine, endogenous d‐serine release is 10‐fold higher than glutamate receptor‐evoked release. Release of d‐serine from slices does not require internal or external Ca2+, suggesting a nonvesicular release mechanism. To confirm the neuronal origin of d‐serine, we selectively loaded neurons in cortical slices with d‐[3H]serine or applied d‐alanine, which specifically releases d‐serine from neurons. Depolarization with veratridine promotes d‐serine release in vivo monitored by high temporal resolution microdialysis of the striatum. Our data indicate that the neuronal pool of d‐serine plays a major role in d‐serine dynamics, with implications for the regulation of NMDAR transmission. Rosenberg, D., Kartvelishvily, E., Shleper, M., Klinker, C. M. C., Bowser, M. T., Wolosker, H. Neuronal release of d‐serine: a physiological pathway controlling extracellular d‐serine concentration. FASEB J. 24, 2951–2961 (2010). www.fasebj.org

Methods for measuring aptamer-protein equilibria: a review.

Aptamers are single stranded DNA or RNA molecules that have been selected using in vitro techniques to bind target molecules with high affinity and selectivity, rivaling antibodies in many ways. In order to use aptamers in research and clinical applications, a thorough understanding of aptamer-target binding is necessary. In this article, we review methods for assessing aptamer-protein binding using separation based techniques such as dialysis, ultrafiltration, gel and capillary electrophoresis, and HPLC; as well as mixture based techniques such as fluorescence intensity and anisotropy, UV-vis absorption and circular dichroism, surface plasmon resonance, and isothermal titration calorimetry. For each method the principle, range of application and important features, such as sample consumption, experimental time and complexity, are summarized and compared.

Micro free-flow electrophoresis: theory and applications

Free-flow electrophoresis (FFE) is a technique that performs an electrophoretic separation on a continuous stream of analyte as it flows through a planar flow channel. The electric field is applied perpendicularly to the flow to deflect analytes laterally according to their mobility as they flow through the separation channel. Miniaturization of FFE (μFFE) over the past 15xa0years has allowed analytical and preparative separation of small volume samples. Advances in chip design have improved separations by reducing interference from bubbles generated by electrolysis. Mechanisms of band broadening have been examined theoretically and experimentally to improve resolution in μFFE. Separations using various modes such as zone electrophoresis, isoelectric focusing, isotachophoresis, and field-step electrophoresis have been demonstrated.

Analyte–additive interactions in nonaqueous capillary electrophoresis: a critical review

Abstract This article reviews the recent developments in nonaqueous capillary electrophoresis and discusses the effect of the solvent on analyte–additive interactions. When nonaqueous solvents are used, solvophobic interactions are generally weaker than those in water; therefore, those additives that can change the analyte mobility through electrostatic or donor–acceptor interactions should be further explored.

Higher order equilibria and their effect on analyte migration behavior in capillary electrophoresis.

This paper presents a quantitative investigation into the effect of analyte-additive interactions on analyte migration behavior in capillary electrophoresis (CE) when both 1:1 and 1:2 stoichiometries are present. Equations based on the individual capacity factors for each interaction are derived to account for the effect of both first- and second-order equilibria. The analyte migration behavior is described using these equations with a full account of how the microscopic equilibrium constants and microscopic mobilities are combined to give the macroscopic values. The binding isotherms of interactions with both 1:1 and 1:2 stoichiometries are compared with those of a 1:1 stoichiometry. 4,4-Biphenol and 4-phenylphenol were chosen as analytes that undergo complexation with one and two hydroxypropyl-β-cyclodextrin (HP-β-CD) molecules; phenol was used as an analyte that interacts with only one HP-β-CD molecule. The process of calculating higher order equilibrium constants and complex mobilities from the binding isotherms is demonstrated. The effects of experimental conditions, such as the additive concentration range and the number of data points, on the error in the calculated constants and the ability of the equations to accurately describe the experimental data are discussed. A comparison of the linear transformations of the binding isotherm with respect to their ability to detect higher order equilibria is made, and the advantage of using the capacity factor in CE is illustrated.

D-Serine uptake by isolated retinas is consistent with ASCT-mediated transport.

Uptake of the neuromodulator D-serine by isolated larval tiger salamander (Ambystoma tigrinum) retinas was measured using capillary electrophoresis (CE). Excised retinas were incubated in Ringers solution in the presence of 5 microM D-serine. The supernatant was removed after 30 min, mixed with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD-F) to fluorescently label amines and analyzed using CE. Significant D-serine uptake was observed over a period of 1.5h. This is the first observation of D-serine uptake by an intact retinal tissue. D-Serine uptake in the retina was Na(+)-dependent and blocked by l-alanine, l-threonine, and l-cysteine. This pharmacology is consistent with the sodium dependent heteroexchange expected of system ASC-type transporters.

Capillary electrophoresis-SELEX selection of catalytic DNA aptamers for a small-molecule porphyrin target

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has previously been used to select aptamers for large-molecule targets such as proteins, lipopolysaccharides, and peptides. For the first time, we have performed CE-SELEX selection for a small-molecule target, N-methyl mesoporphyrin (NMM), with a molecular weight of only 580 g/mol. DNA aptamers with high-nanomolar to low-micromolar dissociation constants were achieved after only three rounds of selection. This corresponds to an >50-fold improvement in affinity over the random library. Two out of eight randomly chosen aptamers were found to catalyze the metal insertion reaction of mesoporphyrin with 1.7- and 2.0-fold rate enhancements, respectively.

Fast Determination of Mitochondria Electrophoretic Mobility Using Micro Free-Flow Electrophoresis

Fast, continuous separation of mitochondria from rat myoblasts using micro free-flow electrophoresis (muFFE) with online laser-induced fluorescence detection (LIF) is reported. Mitochondrial electrophoretic profiles were acquired in less than 30 s. In comparison to macroscale FFE instruments, muFFE devices consumed approximately 100-fold less sample, used 10-fold less buffer, and required a 15-fold lower electric field. Mitochondrial electrophoretic mobility distributions measured using muFFE were compared to those measured with a capillary electrophoresis instrument with laser-induced fluorescence detection (CE-LIF). There was high similarity between the two distributions with CE-LIF distribution being offset by 1.8 x 10(-4) cm(2) V(-1) s(-1) with respect to the microFFE distribution. We hypothesize that this offset results from the differences in electric field strength used in the techniques. In comparison to CE-LIF, analysis of mitochondria using muFFE greatly decreased separation time and required less separation voltage, while maintaining low sample (125 nL) and buffer (250 microL) volumes. These features together with the potential for collecting separated organelle fractions for further characterization make microFFE a very attractive tool for the high-throughput analysis of organelle subpopulations as well as investigating the fundamentals of the electrophoretic mobility of biological particles.

Isolation of DNA Aptamers Using Micro Free Flow Electrophoresis

A micro free flow electrophoresis (μFFE) device was used to select DNA aptamers for human immunoglobulin E (IgE). The continuous nature of μFFE allowed 1.8 × 10(14) sequences to be introduced over a period of 30 min, a 300-fold improvement in library size over capillary electrophoresis based selections (CE-SELEX). Four rounds of selection were performed within four days. Aptamers with low nM dissociation constants for IgE were identified after a single round of μFFE selection.