We report on the physical and optical characterization of liposomes formed by extrusion and sonication, two widely used methods for vesicle preparation. We also address the issue of whether the properties of bilayers formed from liposomes prepared by the two techniques differ at the molecular and mesoscopic levels. We used the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), with and without cholesterol, to form liposomes, incorporating 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (18:1-12:0 NBD-PC) as an optical probe of dynamics. We measured the physical morphology of liposomes by transmission electron microscopy (TEM) and dynamic light scattering (DLS), and the rotational and translational diffusion of 18:1-12:0 NBD-PC by time correlated single photon counting (TCSPC) and fluorescence recovery after pattern photobleaching (FRAPP), respectively. We find that, despite apparent differences in average size and size distribution, both methods of preparation produced liposomes that exhibit the same molecular scale environment. The translational diffusion behavior of the tethered chromophore in planar bilayer lipid membranes formed from the two types of liposomes also yielded similar results.

We have studied the interactions of the chromophore 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-7-nitro-2-1,3-benzoxadiazol-4-yl (18:1 NBD-PE) imbedded in the headgroup region of bilayer lipid membranes consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG). We have examined the molecular and mesoscale dynamics of the chromophore using time-correlated single photon counting (TCSPC) to measure rotational diffusion dynamics in lipid vesicles and fluorescence recovery after pattern photobleaching (FRAPP) to determine translational diffusion coefficients and mobile fractions in supported lipid bilayers. TCSPC data reveal that chromophore rotational diffusion rates in DOPG vesicles are statistically the same as in DOPC and mixed DOPC/DOPG vesicles, suggesting that the NBD-PE chromophore does not interact strongly with the headgroup region of these bilayers; however, FRAPP experiments show that lateral diffusion is statistically lower in mixed DOPC/DOPG-supported bilayers than in DOPC-supported bilayers. These results suggest that bilayers containing DOPG likely undergo interlipid headgroup hydrogen bonding interactions that suppress translational diffusion.

Neuropathy target esterase (NTE) is an integral membrane protein localized in the endoplasmic reticulum in neurons. Irreversible inhibition of NTE by certain organophosphorus compounds produces a paralysis known as organophosphorus compound-induced delayed neuropathy. In vitro, NTE has phospholipase/lysophospholipase activity that hydrolyses exogenously added single-chain lysophospholipids in preference to dual-chain phospholipids, and NTE mutations have been associated with motor neuron disease. NTE's physiological role is not well understood, although recent studies suggest that it may control the cytotoxic accumulation of lysophospholipids in membranes. We used the NTE catalytic domain (NEST) to hydrolyze palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (p-lysoPC) to palmitic acid in bilayer membranes comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and the fluorophore 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC). Translational diffusion coefficients (D(L)) in supported bilayer membranes were measured by fluorescence recovery after pattern photobleaching (FRAPP). The average D(L) for DOPC/p-lysoPC membranes without NEST was 2.44 microm(2)s(-1)+/-0.09; the D(L) for DOPC/p-lysoPC membranes containing NEST and diisopropylphosphorofluoridate, an inhibitor, was nearly identical at 2.45+/-0.08. By contrast, the D(L) for membranes comprising NEST, DOPC, and p-lysoPC was 2.28+/-0.07, significantly different from the system with inhibited NEST, due to NEST hydrolysis. Likewise, a system without NEST containing the amount of palmitic acid that would have been produced by NEST hydrolysis of p-lysoPC was identical at 2.26+/-0.06. These results indicate that NTE's catalytic activity can alter membrane fluidity.

Abstract-A multi-analyte electrochemical sensor array platform has been developed for protein-based biosensors utilizing post-CMOS compatible fabrication procedures that enables formation of single-chip biosensor array microsystems. The process was developed on a glass substrate to emulate the surface of a CMOS chip. A three-electrode system, including an array of gold working electrodes (WE) and an on-chip Ag/AgCl reference electrode (RE), was formed to facilitate electrochemical analysis protein-based interfaces. SU-8 epoxy resin was subsequently applied and patterned as an insulation and planerization layer before application of microfluidic channels used to self-assemble novel bio-interfaces on individual working electrodes. The protein-based biointerfaces provide selectivity, and the electrode floorplan maximizes sensitivity and minimizes solution resistance errors. The electrodes on the prototype platform can be easily scaled to form ~100 electrodes on the surface of a 10mm 2 CMOS chip.