Biomedical subjects

Craig A Aspinwall

Publications and source records attributed to Craig A Aspinwall.

8 recordsLinked to original sources

PubMed2006

Stabilized porous phospholipid nanoshells.

Chemically stabilized, porous phospholipid nanoshells (PPNs) were prepared via copolymerization of reactive monomers with unilamellar bis-Sorbyl phosphatidylcholine vesicles. The resulting PPN vesicular assemblies possess a highly porous membrane structure that allows passage of small molecules, which can react with encapsulated proteins and reporters. The unique combination of membrane stability and porosity will prove useful for preparing nanometer-sized sensor, container, and reactor platforms stable in harsh chemical and biological environments.

Genes, Reporter↗

PubMed2006

High-speed capillary zone electrophoresis with online photolytic optical injection.

We report an online, optical injection interface for capillary zone electrophoresis (CZE) based upon photophysical activation of a caged, fluorogenic label covalently attached to the target analyte. This injection interface allows online analysis of biomolecular systems with high temporal resolution and high sensitivity. Samples are injected onto the separation capillary by photolysis of a caged-fluorescein label using the 351-364 nm irradiation of an Ar+ laser. Following injection, the sample is separated and detected via laser-induced fluorescence detection at 488 nm. Detection limits for online analysis of arginine, glutamic acid, and aspartic acid were less than 1 nM with separation times less than 5 s and separation efficiencies exceeding 1,000,000 plates/m. Rapid injection of proteins was demonstrated with migration times less than 500 ms and 0.5 nM detection limits. Online monitoring was performed with response times less than 20 s, suggesting the feasibility of this approach for online, in vivo analysis for a range of biologically relevant analytes.

Amino Acids↗

PubMed2006

Fast Hadamard transform capillary electrophoresis for on-line, time-resolved chemical monitoring.

We report a new approach for collecting and deconvoluting the data in Hadamard transform capillary electrophoresis, referred to as fast Hadamard transform capillary electrophoresis (fHTCE). Using fHTCE, total analysis times can be reduced by up to 48% per multiplexed separation compared to conventional Hadamard transform capillary electrophoresis (cHTCE) while providing comparable signal-to-noise ratio enhancements. In fHTCE, the sample is injected following a pseudorandom pulsing sequence derived from the first row of a simplex matrix (S-matrix) in contrast to cHTCE, which utilizes a sequence of twice the length. In addition to the temporal savings provided by fHTCE, a 50% reduction in sample consumption is also realized due to the decreased number of sample injections. We have applied fHTCE to the analysis of mixtures of neurotransmitters and related compounds to yield improved signal-to-noise ratios with a total analysis time under 10 s. In addition, we demonstrate the capability of fHTCE to perform time-resolved monitoring of changes in the concentration of model neurochemical compounds.

Electrophoresis, Capillary↗

PubMed2006

Capillary electrophoresis with a UV light-emitting diode source for chemical monitoring of native and derivatized fluorescent compounds.

We report the utilization of a high power UV light-emitting diode for fluorescence detection (UV-LED-IF) in CE separations. CE-UV-LED-IF allows analysis of a range of environmentally and biologically important compounds, including PAHs and biogenic amines, including neurotransmitters, amino acids, proteins, and peptides, that have been derivatized with UV-excited fluorogenic labels, e.g., o-phthalic dicarboxaldehyde/beta-mercaptoethanol (OPA/beta-ME). The 365 nm UV-LED was used as a stable, low cost source for detection of UV-excited fluorescent compounds. UV-LED-IF was used with both zonal CE separations and MEKC. Native fluorescence detection of PAHs was accomplished with detection limits ranging from 10 nM to 1.3 microM. Detection limits for OPA/beta-ME-labeled glutamic acid and aspartic acid were 11 and 10 nM, respectively, for off-line labeling, and 47 and 47 nM, respectively, for on-line labeling, comparable to UV-laser-based systems. Analysis of OPA/beta-ME-labeled proteins and peptides was performed with 28 and 47 nM detection limits for BSA and myoglobin, respectively.

Amino Acids↗

PubMed2005

Nanometre-sized molecular oxygen sensors prepared from polymer stabilized phospholipid vesicles.

Nanometre-sized, chemically-stabilized phospholipid vesicle sensors have been developed for detection of dissolved molecular oxygen. Sensors were prepared by forming 150 nm phospholipid vesicles from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or DOPC doped with small (<1%) mole percentages of 1,2-dioleoyl-sn-glycero-3-phosphoethanol amine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE). Sensors were stabilized via cross-linking polymerization of hydrophobic methacrylate monomers partitioned into the hydrophobic interior of the DOPC bilayer. The resultant unilamellar, nanometre-sized, polymer-lipid vesicles are spherical, biocompatible and protect sensing components that are loaded into the aqueous interior of the vesicle from interfering species in the exterior environment. For O(2) detection, the oxygen-sensitive fluorescent dye, tris(1,10-phenanthroline)ruthenium(II) chloride (Ru(phen)(3)) was encapsulated into the aqueous interior of the polymerized phospholipid vesicle. NBD-PE was introduced into the phospholipid bilayer of the sensor as a reference dye, allowing ratiometric sensors to be constructed. The resultant sensors show high sensitivity, excellent reversibility and excellent linearity over a physiological range of dissolved oxygen concentrations. These results suggest that polymerized phospholipid vesicle sensors can be used for monitoring intracellular O(2) dynamics.

Biosensing Techniques↗

PubMed2005

In situ fabrication of three-dimensional chemical patterns in fused silica separation capillaries with polymerized phospholipids.

We report a new molecular approach for in situ generation of micron scale, chemically and biochemically functionalized patterns inside three-dimensional, completely enclosed fluidic channels. The formation of chemical patterns is based upon a combination of lipid bilayer self-assembly and UV photopolymerization of photoreactive, cross-linkable phospholipids. Using this approach, we have functionalized capillaries of varying inner diameters with a range of chemistries useful for protein and peptide immobilization. Here, we demonstrate the ability to produce small molecule and protein-based chemical patterns.

Lipid Bilayers↗

PubMed2004

Screening populations of individual cells for secretory heterogeneity.

Many common metabolic and neurological disorders are related to defective regulation of exocytosis at the level of single cells. In exocytosis, vesicles containing the secretory product of a given cell type fuse with the plasma membrane allowing release of the vesicular contents into the extracellular environment where the physiological action can be exerted. The typical secretory vesicle contains between 0.15 and 10 attomoles of material that is released on a millisecond timescale. Hence, detection of this process presents several chemical and analytical challenges. In this work, we utilize the native ATP, stored at high concentrations within the secretory vesicles of most neuroendocrine cells and co-released during exocytosis and during cell lysis, as a universal tracer of cellular secretion events. Organisms studied include pancreatic islets, mast cells, and Escherischia coli. Cellular processes investigated include exocytotic release, stimulated cell lysis, and programmed cell lysis.

Adenosine Triphosphate↗

PubMed2004

Removal of Ca2+ channel beta3 subunit enhances Ca2+ oscillation frequency and insulin exocytosis.

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.

Animals↗