Craig A Aspinwall

PMID: 9395458;Abstract:

The effects of extracellular Zn²⁺ and pH and intravesicular pH on insulin and 5-hydroxytryptamine (5-HT) secretion from pancreatic beta cells were investigated. Insulin and 5-HT secretion from single cells was detected by amperometry as a series of current spikes corresponding to detection of multimolecular packets secreted by exocytosis. Spike width was used as a measure of the kinetics of clearance from the cell and the area of spikes as a measure of amount released. Changes in extracellular pH from 6.9 to 7.9 caused insulin spikes to become narrower with no change in area, whereas the same treatments had no effect on 5-HT secretion. Treatment of cells with Bafilomycin A1 or N-ethylmaleimide, both of which are expected to increase intravesicular pH by inhibiting V-type H⁺-ATPase, had no effect on 5-HT secretion but caused insulin spikes to become more narrow. These results indicate that exposure to high pH, whether intravesicular or extracellular, accelerates release of insulin during exocytosis without affecting the amount of insulin released. Increasing extracellular Zn²⁺ concentration from 0 to 25 μM increased the width and decreased the area of insulin spikes without affecting 5-HT secretion. Zn²⁺ effects were likely exerted through a common-ion effect on Zn²⁺-insulin dissociation. It was concluded that intravesicular storage conditions and extracellular ions can affect free insulin concentration in the vicinity of beta cells during secretion.

Analysis of protein and peptide mixtures via capillary electrophoresis is hindered by nonspecific adsorption of analytes to the capillary walls, resulting in poor separations and quantitative reproducibility. Phospholipid bilayer (PLB) coatings are very promising for improving protein and peptide separations due to the native resistance to nonspecific protein adsorption offered by PLBs; however, these coatings display limited chemical and temporal stability. Here, we show the preparation and characterization of a highly cross-linked, polymerized phospholipid capillary coating prepared using bis-SorbPC. Poly(bis-SorbPC) PLB coatings are prepared in situ within fully enclosed fused silica capillaries via self-assembly and radical polymerization. Polymerization of the PLB coating stabilizes the membrane against desorption from the surface and migration in an electric field, improves the temporal and chemical stability, and allows for the separation of both cationic and anionic proteins, while preserving the native resistance to nonspecific protein adsorption of natural PLBs.

PMID: 23540253;PMCID: PMC3670586;Abstract:

Phosphorylcholine (PC) based phospholipid bilayers have proven useful as capillary coating materials due to their inherent resistance to non-specific protein adsorption. The primary limitation of this important class of capillary coatings remains the limited long-term chemical and physical stability of the coatings. Recently, a method for increasing phospholipid coating stability in fused silica capillaries via utilization of polymerized, synthetic phospholipids was reported. Here, we expand upon these studies by investigating polymerized lipid bilayer capillary coatings with respect to separation performance including run-to-run, day-to-day and column-to-column reproducibility and long-term stability. In addition, the effects of pH and capillary inner diameter on polymerized phospholipid coated capillaries were investigated to identify optimized coating conditions. The coatings are stabilized for protein separations across a wide range of pH values (4.0-9.3), a unique property for capillary coating materials. Additionally, smaller inner diameter capillaries (≤50. μm) were found to yield marked enhancements in coating stability and reproducibility compared to wider bore capillaries, demonstrating the importance of capillary size for separations employing polymerized phospholipid coatings. © 2013 Elsevier B.V.

Abstract:

The development of next-generation transmembrane protein-based biosensors relies heavily on the use of black lipid membranes (BLMs); however, electrical, mechanical, and temporal instability of BLMs poses a limiting challenge to biosensor development. In this work, micrometer-sized glass apertures were modified with silanes of different chain length and fluorine composition, including 3-cyanopropyldimethychlorosilane (CPDCS), ethyldimethylchlorosilane (EDCS), n-octyldimethylchlorosilane (ODCS), (tridecafluoro-1, 1, 2, 2-tetrahydrooctyl)dimethylchlorosilane (PFDCS), or (heptadecafluoro-1,1,2,2- tetrahydrodecyl)dimethylchlorosilane (PFDDCS), to explore the effect of substrate surface energy on BLM stability. Low energy silane-modified surfaces promoted enhanced lipid-substrate interactions that facilitate the formation of low-leakage, stable BLMs. The surface energies of silane-modified substrates were 30 ± 3, 16 ± 1, 14 ± 2, 11 ± 1, and 7.1 ± 2 mJ m^-2 for CDCS, EDCS, ODCS, PFDCS, and PFDDCS, respectively. Decreased surface energy directly correlated to improved electrical, mechanical, and temporal BLM stability. Amphiphobic perfluorinated surface modifiers yielded superior performance compared to traditional hydrocarbon modifiers in terms of stability and BLM formation, with only marginal effects on BLM membrane permeability. Leakage currents obtained for PFDCS and PFDDCS BLMs were elevated only 10-30%, though PFDDCS modification yielded >5-fold increase in electrical stability as indicated by breakdown voltage (> 2000 mV vs 418 ± 73 mV), and >25-fold increase in mechanical stability as indicated by air-water transfers (> 50 vs 2 ± 0.2) when compared to previously reported CPDCS modification. Importantly, the dramatically improved membrane stabilities were achieved with no deleterious effects on reconstituted ion channel function, as evidenced by α-hemolysin activity. Thus, this approach provides a simple, low cost, and broadly applicable alternative for BLM stabilization and should contribute significantly toward the development of next-generation ion-channel-functionalized biosensors. © 2013 American Chemical Society.