Craig A Aspinwall

PMID: 16316200;Abstract:

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. Copyright © 2005 American Chemical Society.

Suspended planar lipid membranes (or black lipid membranes (BLMs)) are widely used for studying reconstituted ion channels, although they lack the chemical and mechanical stability needed for incorporation into high-throughput biosensors and biochips. Lipid polymerization enhances BLM stability but is incompatible with ion channel function when membrane fluidity is required. Here, we demonstrate the preparation of a highly stable BLM that retains significant fluidity by using a mixture of polymerizable and nonpolymerizable phospholipids. Alamethicin, a voltage-gated peptide channel for which membrane fluidity is required for activity, was reconstituted into mixed BLMs prepared using bis-dienoyl phosphatidylcholine (bis-DenPC) and diphytanoyl phosphatidylcholine (DPhPC). Polymerization yielded BLMs that retain the fluidity required for alamethicin activity yet are stable for several days as compared to a few hours prior to polymerization. Thus, these polymerized, binary composition BLMs feature both fluidity and long-term stability.

The ability to rapidly screen complex libraries of pharmacological modulators is paramount to modern drug discovery efforts. This task is particularly challenging for agents that interact with lipid bilayers or membrane proteins due to the limited chemical, physical, and temporal stability of conventional lipid-based chromatographic stationary phases. Here, we describe the preparation and characterization of a novel stationary phase material composed of highly stable, polymeric-phospholipid bilayers self-assembled onto silica microparticles. Polymer-lipid membranes were prepared by photochemical or redox initiated polymerization of 1,2-bis[10-(2',4'-hexadieoyloxy)decanoyl]-sn-glycero-2-phosphocholine (bis-SorbPC), a synthetic, polymerizable lipid. The resulting polymerized bis-SorbPC (poly(bis-SorbPC)) stationary phases exhibited enhanced stability compared to particles coated with 1,2-dioleoyl-sn-glycero-phosphocholine (unpolymerized) phospholipid bilayers when exposed to chemical (50mM triton X-100 or 50% acetonitrile) and physical (15min sonication) insults after 30 days of storage. Further, poly(bis-SorbPC)-coated particles survived slurry packing into fused silica capillaries, compared to unpolymerized lipid membranes, where the lipid bilayer was destroyed during packing. Frontal chromatographic analyses of the lipophilic small molecules acetylsalicylic acid, benzoic acid, and salicylic acid showed >44% increase in retention times (P0.0001) for all analytes on poly(bis-SorbPC)-functionalized stationary phase compared to bare silica microspheres, suggesting a lipophilic retention mechanism. Phospholipid membrane-functionalized stationary phases that withstand the chemical and physical rigors of capillary LC conditions can substantially increase the efficacy of lipid membrane affinity chromatography, and represents a key advance toward the development of robust membrane protein-functionalized chromatographic stationary phases.

PMID: 14740158;Abstract:

Aims/hypothesis. The ATP-regulated potassium (KATP) channel in the pancreatic beta cell couples the metabolic state to electrical activity. The primary regulator of the KATP channel is generally accepted to be changes in ATP/ADP ratio, where ATP inhibits and ADP activates channel activity. Recently, we showed that long-chain CoA (LC-CoA) esters form a new class of potent KATP channel activators in rodents, as studied in inside-out patches. Methods. In this study we have investigated the effects of LC-CoA esters in human pancreatic beta cells using the inside-out and whole-cell configurations of the patch clamp technique. Results. Human K ATP channels were potently activated by acyl-CoA esters with a chain length exceeding 12 carbons. Activation by LC-CoA esters did not require the presence of Mg²⁺ or adenine nucleotides. A detailed characterization of the concentration-dependent relationship showed an EC50 of 0.7±0.1 μmol/l. Furthermore, in the presence of an ATP/ADP ratio of 10 (1.1 mmol/l total adenine nucleotides), whole-cell KATP channel currents increased approximately six-fold following addition of 1 μmol/l LC-CoA ester. The presence of 1 μmol/l LC-CoA in the recording pipette solution increased beta-cell input conductance, from 0.5±0.2 nS to 2.5±1.3 nS. Conclusion/interpretation. Taken together, these results show that LC-CoA esters are potent activators of the KATP channel in human pancreatic beta cells. The fact that LC-CoA esters also stimulate K ATP channel activity recorded in the whole-cell configuration, points to the ability of these compounds to have an important modulatory role of human beta-cell electrical activity under both physiological and pathophysiological conditions.

β-particle emitting radionuclides are useful molecular labels due to their abundance in biomolecules. Detection of β-emission fromH,S, andP, important biological isotopes, is challenging due to the low energies (E≤ 300 keV) and short penetration depths (≤0.6 mm) in aqueous media. The activity of biologically relevant β-emitters is usually measured in liquid scintillation cocktail (LSC), a mixture of energy-absorbing organic solvents, surfactants, and scintillant fluorophores, which places significant limitations on the ability to acquire time-resolved measurements directly in aqueous biological systems. As an alternative to LSC, we developed polystyrene-core, silica-shell nanoparticle scintillators (referred to as nanoSCINT) for quantification of low-energy β-particle emitting radionuclides directly in aqueous solutions. The polystyrene acts as an absorber for energy from emitted β-particles and can be loaded with a range of hydrophobic scintillant fluorophores, leading to photon emission at visible wavelengths. The silica shell serves as a hydrophilic shield for the polystyrene core, enabling dispersion in aqueous media and providing better compatibility with water-soluble analytes. While polymer and inorganic scintillating microparticles are commercially available, their large size and/or high density complicates effective dispersion throughout the sample volume. In this work, nanoSCINT nanoparticles were prepared and characterized. nanoSCINT responds toH,S, andP directly in aqueous solutions, does not exhibit a change in scintillation response between pH 3.0 and 9.5 or with 100 mM NaCl, and can be recovered and reused for activity measurements in bulk aqueous samples, demonstrating the potential for reduced production of LSC waste and reduced total waste volume during radionuclide quantification. The limits of detection for 1 mg/mL nanoSCINT are 130 nCi/mL forH, 8 nCi/mL forS, and 1 nCi/mL forP.