Biomedical subjects

Michael T Bowser

Publications and source records attributed to Michael T Bowser.

15 recordsLinked to original sources

PubMed2006

Optimizing band width and resolution in micro-free flow electrophoresis.

The broadening mechanisms for micro-free flow electrophoresis (micro-FFE) have been investigated using a van Deemter analysis. Separation power, the product of electric field and residence time, is presented as a parameter for predicting the position of sample streams and for comparing separations under different conditions. Band broadening in micro-FFE is governed by diffusion at lower linear velocities and a migration distance-dependent mechanism at higher linear velocities. At higher linear velocities, the parabolic flow profile is elongated, generating a distribution of analyte residence times in the separation channel. This distribution of residence times gives rise to a distribution of migration distances in the lateral direction since analytes spend different amounts of time in the electric field. Equations were derived to predict the effect of electric field and buffer flow rate on broadening. Experimental data were collected to determine whether the derived equations were useful in explaining broadening caused by diffusion and hydrodynamic flow at different linear velocities and electric fields. Overall there was an excellent correlation between the predicted and experimentally observed values allowing linear velocity and electric field to be optimized. Suppression of electroosmotic flow is proposed as a means of reducing micro-FFE band broadening due to hydrodynamic effects and maximizing resolution and peak capacity.

Algorithms↗

PubMed2006

Using channel depth to isolate and control flow in a micro free-flow electrophoresis device.

A multiple-depth micro free-flow electrophoresis chip (mu-FFE) has been fabricated with a 20-microm-deep separation channel and 78-microm-deep electrode channels. Due to the difference in channel heights, the linear velocity of buffer in the electrode channels is approximately 15 times that of the buffer in the separation channel. Previous mu-FFE devices have been limited by electrolysis product formation at the electrodes. These electrolysis products, manifested as bubbles, decreased the electric field and disrupted the buffer flow profile, limiting performance and preventing continuous operation. Using channel depth to control buffer flow over the electrodes and in the separation channel effectively removes electrolysis products, allowing continuous operation. The linear velocities in the channels were confirmed using particle velocimetry and compared well with values predicted using lubrication theory. A separation potential of 645 V could be applied before significant Joule heating was observed. This corresponded to an electric field of 586 V/cm in the separation channel, a 4-fold increase over our previous design. A separation of fluorescent standards was demonstrated using the new mu-FFE device. Resolution increased by a factor of 1.3 over our previous design, even when operated under similar conditions, suggesting that effective removal of electrolysis products is more important than originally thought.

Electrodes↗

PubMed2006

Measuring D-serine efflux from mouse cortical brain slices using online microdialysis-capillary electrophoresis.

Efflux of a number of important neurochemicals, including D-serine, L-serine, taurine, glutamate, and gamma-aminobutyric acid (GABA), from mouse cortical brain slices housed in a 7 microL perfusion chamber was monitored using online microdialysis-CE (MD-CE). Analyte concentrations could be measured every 20-27 s using the MD-CE instrument. Stimulation with high potassium induced increased release of D-serine. Kainic acid (KA) induced D-serine release, but this release was not blocked by 6-cyano-7-nitroquinoxaline-2,3-dione, suggesting that alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid/KA receptors do not mediate D-serine release. Application of L-serine, the precursor of D-serine, resulted in increased extracellular D-serine concentrations. L-Cysteine also increased extracellular D-serine levels in a partially Na+-dependent manner. The observed effects upon application of L-serine and L-cysteine support the involvement of ASC neutral amino acid transporters in regulating the extracellular concentration of D-serine concentration through competitive inhibition of uptake or increased release through heteroexchange.

Animals↗

PubMed2005

Measuring the effect of glutamate receptor agonists on extracellular D-serine concentrations in the rat striatum using online microdialysis-capillary electrophoresis.

Online microdialysis-capillary electrophoresis (CE) was used to measure the changes in extracellular D-serine concentration in response to the application of glutamate agonists and antagonists in the rat striatum. The microdialysis-CE assay was capable of measuring concentration changes as small as 8% with a sampling rate of 12-15s. Kainic acid (KA) induced increases in D- and L-serine concentrations. Application of the non-NMDA receptor antagonist CNQX did not affect the increases observed for D- or L-serine, suggesting a nonspecific effect. NMDA also induced increases in D-serine, L-serine, glutamate and GABA concentrations. These increases were attenuated by the NMDA receptor antagonist MK-801.

Animals↗

PubMed2005

Capillary electrophoresis-SELEX selection of aptamers with affinity for HIV-1 reverse transcriptase.

Capillary electrophoresis-SELEX (CE-SELEX) was used to select ssDNA aptamers with affinity for HIV reverse transcriptase (HIVRT). A library of ssDNA was incubated with HIVRT. Sequences bound to HIVRT were isolated using CE, PCR amplified, and purified, yielding an enriched ssDNA pool suitable for further rounds of selection. Aptamers with dissociation constants as low as 180 pM were isolated after four rounds of selection. This is the first report of aptamers isolated by CE-SELEX with higher affinity than those obtained for the same target using conventional selection techniques. No sequence motifs were identified in the 27 clones sequenced, suggesting that there are many sequences that can bind HIVRT with low picomolar dissociation constants.

Aptamers, Nucleotide↗

PubMed2005

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 Ringer's 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.

4-Chloro-7-nitrobenzofurazan↗

PubMed2005

Free-flow electrophoresis on an anodic bonded glass microchip.

A micro-free-flow electrophoresis chip has been fabricated into a glass wafer etched with 20-microm-deep channels. Wafers were bonded anodically using an intermediate amorphous silicon film. Electric fields as high as 283 V/cm were applied across the separation channel to obtain baseline resolution of fluorescent standards in 4.8 s. The effect of electric fields ranging from 0 to 283 V/cm on the separations and resulting resolutions were examined. Resolution was shown to increase linearly with the applied electric field. Joule heating was not significant under the conditions tested. Instead, the generation of electrolysis products at higher currents proved to be the limiting factor preventing higher separation potentials from being used.

Electrophoresis↗

PubMed2005

In vitro selection of aptamers with affinity for neuropeptide Y using capillary electrophoresis.

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) was used to select aptamers for neuropeptide Y (NPY). This is the first example of a CE-SELEX selection for aptamers that bind a target molecule smaller than itself. One of the limitations of CE-SELEX is that the aptamer must exhibit a significant mobility shift when it binds the target to facilitate fraction collection. Before this study, it was not clear if smaller targets would be capable of inducing a large enough shift in mobility for CE-SELEX to be successful. NPY is a 36-amino acid peptide (MW = 4272 g/mol), much smaller than the 80-base ssDNA used in the selection ( approximately 25 kDa). NPY binding aptamers with 300-1000 nM dissociation constants were obtained after only four rounds of selection. The specificity of the aptamers was tested using human pancreatic polypeptide (hPP). hPP is a 36-amino acid peptide with approximately 50% homology with NPY. Aptamers with up to 42-fold selectivity for NPY over hPP were observed.

Biosensing Techniques↗

PubMed2005

Effect of anionic additive type on ion pair formation constants of basic pharmaceuticals.

Due to their beneficial effect on selectivity, peak shape, and sample loading, the use of mobile phase anionic additives, such as formate (HCOO-), chloride (Cl-), and trifluoroacetate (CF3COO-), is increasing in both reversed-phase chromatography (RPLC) and liquid chromatography-mass spectrometry (LC/MS). Similarly, perchlorate is a common "ion pair" agent in reversed-phase separation of peptides. Although many studies have suggested that anions effect in chromatography is due to the formation of ion pairs in the mobile phase between the anions and cationic analytes, there has been no independent verification that ion pairs are, in fact, responsible for these observations. In order to understand the mechanisms by which anionic additives influence retention in chromatography and ionization efficiency in electrospray mass spectrometry, we studied the formation of ion pairs between a number of prototypical basic drugs and various additives by measuring the effect of anionic additives on the electrophoretic mobility of the probe drugs under solvent conditions commonly used in chromatography. For the first time, ion pair formation between basic drugs and anionic additives under conditions commonly used in reversed-phase liquid chromatography has been confirmed independently with all anions (i.e. hexafluorophosphate, perchlorate, trifluoroacetate, and chloride) used in this study. We measured ion pair formation constants (Kip) for different anionic additives using capillary electrophoresis (CE) and obtained quantitative estimates for the extent of ion pairing in buffered acetonitrile-water. The data clearly indicate that different anionic additives ion pair with cationic drugs to quite different extents. The ion pair formation constants show a clear trend with the order being: PF6- > ClO4- > CF3COO- > Cl-. However, the extent of ion pairing is not large. At a typical RPLC mobile phase additive concentration of 20mM, the percentages of the analytes that are present as ion pairs are about 15%, 6%, and 3% for hexafluorophosphate, perchlorate, and trifluoroacetate, respectively. The fraction of the analytes present as a chloride pair is even smaller.

Amitriptyline↗

PubMed2005

SELEX: just another separation?

The potential for quickly isolating high affinity, highly selective ligands generated much excitement when SELEX was first described. Fifteen years later, SELEX has still not achieved widespread acceptance due to limitations in aptamer affinity, stability and throughput. Recent research is beginning to address these limitations though and SELEX is quickly regaining its standing as a hot topic in areas as diverse as drug discovery, chemical analysis, and even genomics and proteomics.

Animals↗

PubMed2004

Monitoring D-serine dynamics in the rat brain using online microdialysis-capillary electrophoresis.

D-Serine was detected in dialysate collected from the rat striatum using an online microdialysis-CE-LIF instrument. Dialysate can be analyzed every 12.5 s using the online instrument, giving much better temporal resolution than previously possible for D-serine. Basal concentrations of D-serine (8 +/- 2 microM), glutamate (0.8 +/- 0.2 microM), GABA (0.11 +/- 0.04 microM), and L-serine (23 +/- 4 microM) were measured. Increases in the concentrations of these neurochemicals induced by the introduction of high-K+ aCSF were quantitated. Notably, an increase in D-serine concentration in response to high-K+ aCSF was observed for the first time. The identity of the D-serine peak was confirmed unambiguously using D-amino acid oxidase to selectively remove D-serine from a dialysate sample. The microdialysis-CE-LIF instrument was able to monitor this enzymatic reaction as it proceeded over a period of 60 min, demonstrating that online microdialysis-CE-LIF is not only useful in monitoring in vivo dynamics but can also be used to monitor other chemical systems.

Animals↗

PubMed2004

In vitro selection of high-affinity DNA ligands for human IgE using capillary electrophoresis.

Aptamers with high affinity for IgE were selected using capillary electrophoresis to demonstrate the compatibility of this technique with SELEX. The high selectivity and efficiency of CE gave rise to a very high rate of enrichment, allowing high-affinity, high-selectivity aptamers to be obtained in only four rounds of selection. Decreasing the number of rounds shortens the selection procedure from the 4-6 weeks typical of SELEX to several days. The use of "bulk" dissociation constant measurements was introduced as a method for assessing the DNA pool after each round of selection. The average dissociation constant of the sequences in the DNA pool for IgE after four rounds of selection was 29 nM. The distribution of the dissociation constants for the sequences in the pool was very narrow with a standard deviation of only 6 nM. All of the sequences assessed exhibited high specificity for human IgE when compared with human IgG or mouse IgE.

Animals↗

PubMed2004

Monitoring neurotransmitter release from isolated retinas using online microdialysis-capillary electrophoresis.

Release of neurotransmitters and other primary amine-containing analytes from intact, isolated larval salamander (Ambystoma tigrinum) retinas maintained in a 6.5-microL perfusion chamber was monitored using online microdialysis-capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Primary amines were derivatized online with o-phthaldialdehyde (OPA) and beta-mercaptoethanol. With the use of overlapping injections, the perfusate was sampled every approximately 10 s. Although separation conditions were optimized using 20 mM hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) for a number of important neuromessengers including D- and L-serine, D- and L-asparate, glutamate, GABA, serotonin, dopamine, norepinephrine, and taurine, only glutamate (0.48 +/- 0.27 microM), GABA (0.25 +/- 0.12 microM), taurine (5.5 +/- 2.1 microM), and l-serine (2.8 +/- 1.0 microM) were identified in the perfusate. Elevated levels of glutamate, GABA, and taurine were detected during stimulation with 60 mM K+. This method is the first to directly sample multiple neurotransmitters from perfused, isolated retinas and to observe changes in efflux of these neurotransmitters as a result of pharmacological stimulation.

Ambystoma↗

PubMed2004

In vitro evolution of functional DNA using capillary electrophoresis.

Electrophoretic selection with capillary electrophoresis (CE) is used, for the first time, to isolate functional nucleic acid sequences using SELEX (systematic evolution of ligands by exponential enrichment). SELEX uses molecular evolution to select functional sequences (aptamers) from random RNA or DNA libraries. Conventional SELEX is usually performed with affinity chromatography, which may introduce significant bias into the selection step. Important biases include the slow kinetics involved in the elution of strongly bound sequences and performing the selection with the target molecule tethered to the stationary support, not in free solution. In this novel CE-SELEX approach, selection occurs in free solution. The nucleic acid sequences that bind the target undergo a mobility shift, migrating at a different rate, allowing them to be separated from the inactive sequences. Thus, there is no need to wash the active sequences off a column as in conventional SELEX, eliminating any kinetic bias. In this work, the viability of CE-SELEX was demonstrated by performing selections against immunoglobulin E (IgE). Anti-IgE aptamers with dissociation constants as low as 40 nM were obtained in only two rounds of selection.

Animals↗

PubMed2003

A high-throughput on-line microdialysis-capillary assay for D-serine.

A high-throughput method is described for the analysis of D-serine and other neurotransmitters in tissue homogenates. Analysis is performed by microdialysis-capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection in a sheath flow detection cell. Sample pretreatment is not required as microdialysis sampling excludes proteins and cell fragments. Primary amines are derivatized on-line with o-phthaldialdehyde (OPA) in the presence of beta-mercaptoethanol followed by on-line CE-LIF analysis. Under the separation conditions described here, D-serine is resolved from L-serine and other primary amines commonly found in biological samples. Each separation requires less than 22 s. Eliminating the need for sample pretreatment and performing the high-speed CE analysis on-line significantly reduces the time required for D-serine analysis when compared with traditional methods. This method has been used to quantify D-serine levels in larval tiger salamander retinal homogenates, as well as dopamine, gamma-amino-n-butyric acid (GABA), glutamate and L-aspartate. D-serine release from an intact retina was also detected.

Animals↗