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Biomedical subjects

Andrew G Ewing

Publications and source records attributed to Andrew G Ewing.

10 recordsLinked to original sources
PubMed2003

Seven cDNAs enriched following hippocampal lesion: possible roles in neuronal responses to injury.

Synaptic plasticity is important for formation of long-term memories and in re-establishment of function following injury. Seven cDNAs enriched following lesion in the hippocampus of the rat have been isolated using a PCR-based cDNA suppression subtraction hybridization. Sequence analysis resulted in the identification of two genes with known roles in synaptic development and neuronal activities: astrotactin and calcineurin. These two neuron-specific genes have established roles in development or synaptogenesis. Sequence analysis of the other five additional genes shows that two are likely to be involved in G-protein signaling pathways, one is a WD repeat protein, and the remaining two are entirely novel. All seven candidates are expressed in the hippocampus and, in some cases, cortical layers of adult brains. RT-PCR data show that expression increases following synaptogenic lesion. Immunocytochemical analysis in primary hippocampal neurons showed that Calcineurin immunoreactivity was redistributed in neurons during 2 weeks in culture. This redistribution suggests that Calcineurin's role changes during neurite outgrowth immediately prior to synapse formation in vitro. In addition, inhibiting Calcineurin activity with cyclosporin A enhanced neurite outgrowth, suggesting that Calcineurin has a regulatory role in axon sprouting. The discovery of previously unknown genes involved in the response to neurodegeneration will contribute to our understanding of neural development, responses to CNS trauma, and neurodegenerative diseases.

Animals↗
PubMed2003

Rapid serial analysis of multiple oligonucleotide samples on a microchip using optically-gated injection.

Optically-gated injection of fluorescently-labeled DNA has been accomplished for the first time. Rapid, serial analysis of oligonucleotide ladders has been shown on a microchip using this injection technique. Separations of five- and six-component samples have been completed in 60 s or less with a capability to carry out serial injections of these samples every 15 s. The technique has been shown to have better than five base resolution for small oligonucleotides and excellent reproducibility in migration times (< or = 0.75% RSD). Currently, the limit of detection for the system is 0.23 microM. Additionally, multiple unique samples of DNA have been consecutively analyzed in a single separation lane using optical gating. Six consecutive injections of three different samples have been achieved with no sample carryover and a total analysis time of approximately 10 min. These results show the potential of optical gating as an alternative injection technique for high-throughput DNA applications, such as genotyping and monitoring dynamic processes.

Fluorescent Dyes↗
PubMed2003

Etched electrochemical detection for electrophoresis in nanometer inner diameter capillaries.

Capillary electrophoresis in nanometer inner diameter capillaries allows for the analysis of extremely small volume samples, such as the contents of single cells. However, the utilization of these ultrasmall capillaries requires a very sensitive and low volume detector. An improved method for end-column amperometric detection for capillary electrophoresis in nanometer inner diameter (i.d.) capillaries is presented. This new method involves etching both the electrode and the detection end of the capillary. These design improvements allow for better alignment between the capillary bore and the electrode. As a result, dead volume in the detector is minimized. The etched method for electrochemical detection in ultrasmall capillaries provides average coulometric efficiencies of 70 +/- 10% for dopamine and 40 +/- 20% for catechol in 770 nm i.d. capillaries. Furthermore, this technique provides peak efficiencies as high as 100,000 theoretical plates and detection limits as low as 340 zmol for both dopamine and catechol.

Catechols↗
PubMed2002

Parallel analysis with optically gated sample introduction on a multichannel microchip.

As an alternative to the T-type injection on microchips, optically gated sample introduction previously has been demonstrated to provide fast, serial, and reproducible injections on a single-channel microchip. Here, the ability to perform high throughput, multichannel analysis with optically gated sample introduction is described using a voice coil actuator. The microchip is fixed on a stage, which moves back and forth via the voice coil actuator, scanning two laser beams across the channels on the microchip. For parallel analysis on a multichannel microchip, both the gating beam and the probe beam are scanned at 10 Hz to perform multiple injections and parallel detection. Simultaneous, fast separations of 4-choloro-7-nitrobenzofurazan (NBD)-labeled amino acids are demonstrated in multiple channels on a microchip. Serial separations of different samples in multiple channels are also reported. Optically gated sample introduction on multiple, parallel channels shows the potential for high-speed, high-throughput separations that are easily automated by using a single electronic shutter.

4-Chloro-7-nitrobenzofurazan↗
PubMed2002

Imaging of freeze-fractured cells with in situ fluorescence and time-of-flight secondary ion mass spectrometry.

Bioanalytical imaging techniques have been employed to investigate cellular composition at the single-cell and subcellular regimes. Four imaging modes have been performed sequentially in situ to demonstrate the utility of a more integrated approach to imaging cells. The combination of bright-field, scanning ion, and fluorescence microscopy complements TOF-SIMS imaging of native biomolecules. Bright-field microscopy provides a blurred visualization of cells in frozen-hydrated samples, while scanning ion imaging provides a morphological view of freeze-fractured cells after TOF-SIMS analysis is completed. With the use of selective fluorescent labels, fluorescence microscopy allows single mammalian cells to be located in the complex ice matrix of freeze-fractured samples, a task that has not been routine with either bright-field or TOF-SIMS. A fluorescent label, DiI (m/z 834), that does not interfere with the mass spectra of membrane phosphatidylcholine, has been chosen for fluorescence and TOF-SIMS imaging of membrane phospholipids. In this paper, in situ fluorescence microscopy allows the distinction of single cells from ice and other sample debris, previously not possible with bright-field or scanning ion imaging. Once cells are located, TOF-SIMS imaging reveals the localization of membrane lipids, even in the membrane of a single 15-microm rat pheochromocytoma cell. The utility of mapping lipids in the membranes of single cells using this integrated approach will provide more understanding of the functional role of specific lipids in functions of cellular membranes.

Adrenal Gland Neoplasms↗
PubMed2002

Identification of cellular sections with imaging mass spectrometry following freeze fracture.

Freeze-fracture techniques have been used to maintain chemical heterogeneity of frozen-hydrated mammalian cells for static TOF-SIMS imaging. The effects the fracture plane has on scanning electron microscopy and dynamic SIMS images of cells have been studied, but the implications this preparation method has on static SIMS have not been addressed to date. Interestingly, the chemical specificity and surface sensitivity of TOF-SIMS have allowed the identification of unique sections of rat pheochromocytoma cells exposed to the sample surface during freeze fracture. Using the extensive chemical information of the fractured surface, cellular sections have been determined using TOF-SIMS images of water, sodium, potassium, hydrocarbons, phosphocholine, and DiI, a fluorescent dye that remains in the outer leaflet of the cell membrane. Higher amounts of potassium have been imaged inside a cell versus the surrounding matrix in a cross-fractured cell. In other fractures exposing the cell membrane, phosphocholine and DiI have been imaged on the outer leaflet of the cell membrane, while phosphocholine alone has been imaged on the inner leaflet. In this paper, we discuss how imaging mass spectrometry isused to uniquely distinguish three possible sections of cells obtained during freeze fracture. The identification of these sections is important in choosing cells with a region of interest, like the cell membrane, exposed to the surface for a more thorough investigation with imaging static TOF-SIMS.

Adrenal Gland Neoplasms↗
PubMed2002

Dual fluorescence and electrochemical detection on an electrophoresis microchip.

Simultaneous amperometric and fluorescence detection in a microfabricated electrophoresis chip is reported. Detection limits of 448 nM and 1.52, 16, and 28 microM have been achieved for dopamine, catechol, NBD-arginine, and NBD-phenylalanine, respectively. These two orthogonal detection schemes allow analysis of a wider spectrum of compounds per separation, leading to higher throughput and enabling resolution of two neutral analytes, NBD-arginine and catechol. In addition, insight into the detection and separation mechanisms is realized. Differences in migration time and peak widths between the two detectors are compared, providing a better understanding of detector alignment. A common problem encountered in electrophoresis is run-to-run migration time irreproducibility for certain samples. This novel microchip dual detection system has been utilized to reduce this irreproducibility. An unknown sample is monitored with one detector while a standard (i.e., ladder) is added to the sample and monitored simultaneously with the other detector. This dual detection method is demonstrated to normalize unknown peak mobilities in a cerebral spinal fluid sample.

4-Chloro-7-nitrobenzofurazan↗
PubMed2002

Prototyping disposable electrophoresis microchips with electrochemical detection using rapid marker masking and laminar flow etching.

Two novel methods are described for the fabrication of components for microchip capillary electrophoresis with electrochemical detection (microchip CEEC) on glass substrates. First, rapid marker masking is introduced as a completely nonphotolithographic method of patterning and fabricating integrated thin-film metal electrodes onto a glass substrate. The process involves applying the pattern directly onto the metal layer with a permanent marker that masks the ensuing chemical etch. The method is characterized, and the performance of the resulting electrode is evaluated using catecholamines. The response compares well with photolithographically defined electrodes and exhibits detection limits of 648 nM and 1.02 microM for dopamine and catechol, respectively. Second, laminar flow etching is introduced as a partially nonphotolithographic method of replicating channel networks onto glass substrates. The replication process involves applying a poly(dimethylsiloxane) (PDMS) mold of the channel network onto a slide coated with a sacrificial metal layer and then pulling solutions of metal etchants through the channels to transfer the pattern onto the sacrificial layer. The method is tested, and prototype channel networks are shown. These methods serve to overcome the time and cost involved in fabricating glass-based microchips, thereby making the goal of a disposable high performance lab-on-a-chip more attainable.

Catechols↗