Photo-sensitive lipid membrane perturbation by a single chain lipid having terminal spiropyran group

The synthesis a novel single chain lipid (SP-16A), containing a photo-sensitive spiropyran group at its hydrophobic terminus is described. The photoisomerization behavior of SP-16A has been studied by UV-Vis spectroscopy. The lipid has been incorporated into small unilamellar vesicles (SUVs) in quantities up to 10 mol% by mixing with dipalmitoylphosphatidylcholine (DPPC) and consequent sonication. The photo-induced membrane perturbation of the SP-16A/DPPC mixed SUV liposome was observed by monitoring of leakage of carboxyfluorescein(CF), a water soluble fluorescent dye, from the interior aqueous phase of the SUV. SUVs containing 5–10 mol% of SP-16A showed drastic leakage of CF by UV irradiation at 465 nm, however, neither liposome rupture nor release of the lipid from the SUV was observed after the UV irradiation. On/off switching of the leakage of interior material from the vesicle could also be demonstrated. In order to discuss the mechanism and kinetics of the photo-sensitive membrane perturbation behavior for the SP-16A/DPPC mixed SUV, the isomerization behavior of the SP-16A molecule and the leakage of CF from the SUV over time were investigated using a pulsed excimer laser. These experiments suggested that the main factor involved in membrane perturbation by SP-16A is a conformational change of the lipid in the membrane, which occurs after isomerization of the molecule. This new lipid molecule is thus a novel and useful photo-sensitive switching system, and may have future applications in drug delivery systems.     

Spontaneous membrane fusion induced by chemical formation of ceramides in a lipid bilayer

Mere chemical generation of ceramide and related double-chain lipids in the membrane of small unilamellar vesicles (SUVs) induces fusion of the vesicles. The lipids can be successfully prepared by dehydrocondensation between single-chain lipids (fatty acids and sphingosine or its analogues) in a lipid bilayer of the SUV by using a combination of 2-chloro-4,6-dimethoxy-1,3,5-triazine and amphiphilic tertiary amine catalysts, a process that can be compared to a successive enzyme model system for a fatty acyl-CoA synthetase followed by acyltransferase. The SUV spontaneously undergoes membrane fusion upon this internal chemical stimulation by the artificial enzyme system.     

Effect of lipid molecule headgroup mismatch on non steroidal anti-inflammatory drugs induced membrane fusion

Membrane fusion is an essential process guiding many important biological events, which most commonly requires the aid of proteins and peptides as fusogenic agents. Small drug induced fusion at low drug concentration is a rare event. Only three drugs, namely, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx), belonging to the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs) have been shown by us to induce membrane fusion successfully at low drug concentration. A better elucidation of the mechanism and the effect of different parameters in modulating the fusion process will allow the use of these common drugs to induce and control membrane fusion in various biochemical processes. In this study, we monitor the effect of lipid headgroup size mismatch in the bilayer on oxicam NSAIDs induced membrane fusion, by introducing dimyristoylphosphatidylethanolamine (DMPE) in dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUVs). Such headgroup mismatch affects various lipid parameters which includes inhibition of trans-bilayer motion, domain formation, decrease in curvature, etc. Changes in various lipidic parameters introduce defects in the membrane bilayer and thereby modulate membrane fusion. SUVs formed by DMPC with increasing DMPE content (10, 20, and 30 mol %) were used as simple model membranes. Transmission electron microscopy (TEM) and differential scanning calorimetry (DSC) were used to characterize the DMPC-DMPE mixed vesicles. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage and also used to determine the partitioning of the drugs in the membrane bilayer. How the inhibition of trans-bilayer motion, heterogeneous distribution of lipids, decrease in vesicle curvature, etc., arising due to headgroup mismatch affect the fusion process has been isolated and identified here. Mx amplifies these effects maximally followed by Px and Tx. This has been correlated to the enhanced partitioning of the hydrophobic Mx compared to the more hydrophilic Px and Tx in the mixed bilayer.     

Dissipation-enhanced quartz crystal microbalance studies on the experimental parameters controlling the formation of supported lipid bilayers

We report on the investigations of the transformation of spherically closed lipid bilayers to supported lipid bilayers in aqueous media in contact with SiO(2) surfaces. The adsorption kinetics of small unilamellar vesicles composed of dimyristoyl- (DMPC) and dipalmitoylphosphatidylcholine (DPPC) mixtures on SiO(2) surfaces were investigated using a dissipation-enhanced quartz crystal microbalance (QCM-D) as a function of buffer (composition and pH), lipid concentration (0.01-1.0 mg/mL), temperature (15-37 degrees C), and lipid composition (DMPC and DMPC/DPPC mixtures). The lipid mixtures used here possess a phase transition temperature (T(m)) of 24-33 degrees C, which is close to the ambient temperature or above and thus considerably higher than most other systems studied by QCM-D. With HEPES or Tris.HCl containing sodium chloride (150 mM) and/or calcium chloride (2 mM), intact vesicles adsorb on the surface until a critical density ((c)) is reached. At close vesicle contact the transformation from vesicles to supported phospholipid bilayers (SPBs) occurs. In absence of CaCl(2), the kinetics of the SPB formation process are slowed, but the passage through (c) is still observed. The latter disappears when buffers with low ionic strength were used. SPB formation was studied in a pH range of 3-10, yet the passage through (c) is obtained only for pH values above to the physiological pH (7.4-10). With an increasing vesicle concentration, (c) is reached after shorter exposure times. At a vesicle concentration of 0.01-1 mg/mL, vesicle fusion on SiO(2) proceeds with the same pathway and accelerates roughly proportionally. In contrast, the pathway of vesicle fusion is strongly influenced by the temperature in the vicinity of T(m). Above and around the T(m), transformation of vesicles to SPB proceeds smoothly, while below, a large number of nonruptured vesicles coexist with SPB. As expected, the physical state of the membrane controls the interaction with both surface and neighboring vesicles.     

Hypelcin A,an α-aminoisobutyric acid containing antibiotic peptide,induced fusion of egg yolk-l-α-phosphatidylcholine small unilamellar vesicles

Hypelcin A, an α-aminoisobutyric acid-containing antibiotic peptide inducing fusion of egg yolk-l-α-phosphatidylcholine (egg PC) small unilamellar vesicles (SUVs), was investigated by lipid-mixing assay based on resonanceenergy transfer between fluorescent probes, electron microscopy, light scattering, and¹H-nuclear magnetic-resonance spectroscopy. At a high peptide-to-lipid ratio of approximately 1:5, the peptide fuses several SUVs of 20–30 nm in diameter into a 40–100 nm vesicle. Under mild conditions where the permeability enhancement (leakage of a trapped fluorescent dye, calcein) of lipid bilayers are observed (peptide to lipid ratios around 1/100), the fusion of the SUVs also occurs, although the fusion requires a somewhat larger amount of the peptide than the leakage does. Furthermore, at higher lipid concentrations, where the aggregation step is sufficiently rapid, the fusion rate is determined by the amount of the membrane bound peptide per lipid molecule, as is the leakage rate. In contrast, for egg PC large unilamellar vesicles (110 nm), hypelcin A induces the leakage, but not the fusion. We conclude that the leakage is not due to the fusion.     

Experimental evidence to support a theory of lipid membrane fusion

Membrane fusion between two lipid membranes with different curvatures was measured by using a fluorescence fusion assay for lipid vesicle systems and was also obtained by measuring lipid monolayer surface tension upon the fusion of vesicles to monolayer membranes. For such membrane systems, it was found that when lysolipid was incorporated only in the membrane with a greater curvature, membrane fusion was more suppressed than those for the case where the same amount (molar ratio of lysolipid to non-lysolipids) of lysolipid was incorporated only in the membrane with a lower curvature. When lysolipid was incorporated only in a flat membrane (e.g., monolayer) and the fusion of small vesicles (SUV) to the monolayer was measured, suppression of membrane fusion by lysolipid was minimal. It is known that lysolipid lowers the surface energy of curved membranes, which stabilizes energetically such membrane surfaces, and thus suppresses membrane fusion. Our results support our theory of lipid membrane fusion where the membrane fusion occurs through the most curved membrane region at the contact area of two interacting membranes.     

Effect of increase in orientational order of lipid chains and head group spacing on non steroidal anti-inflammatory drug induced membrane fusion

Membrane fusion is a key event in many biological processes. The fusion process, both in vivo and in vitro, is induced by different agents which include mainly proteins and peptides. For protein- and peptide-mediated membrane fusion, conformational reorganization serves as a driving force. Small drug molecules do not share this advantage; hence, drug induced membrane fusion occurring in absence of any other fusogenic agent and at physiologically relevant concentration of the drugs is a very rare event. To date, only three drugs, namely, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx), belonging to the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs), have been shown by us to induce fusion at very low drug to lipid ratio without the aid of any other fusogenic agent. In our continued effort to understand the interplay of different physical and chemical parameters of both the participating drugs and the membrane on the mechanism of this drug induced membrane fusion, we present here the effect of increase in orientational order of the lipid chains and increase in head group spacing. This is achieved by studying the effect of low concentration cholesterol (<10 mol %) at temperatures above the chain-melting transition. Low concentration cholesterol (<10 mol %), above the gel to fluid transition temperature, is mainly known to increase orientational order of the lipid chains and increase head group spacing. To isolate the effect of these parameters, small unilameller vesicles (SUVs) formed by dimyristoylphosphatidylcholine (DMPC) with an average diameter of 50-60 nm were used as simple model membranes. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage and also used to determine the partitioning of the drugs in the membrane bilayer. Differential scanning calorimetry (DSC) was used to study the effect of drugs in the presence of cholesterol on the chain-melting temperature which reflects the fluidization effect of the hydrophobic tail region of the bilayer. Our results show contradictory effect of low concentration cholesterol on the fusion induced by the three drugs, which has been explained by parsing the effect of orientational order and increase in head group spacing on the fusion process.     

Formation of substrate-supported membranes from mixtures of long- and short-chain phospholipids

We studied the formation of substrate-supported planar phospholipid bilayers (SPBs) on glass and silica from mixtures of long- and short-chain phospholipids to assess the effects of detergent additives on SPB formation. 1,2-Hexyanoyl-sn-glycero-3-phosphocholine (DHPC-C6) and 1,2-heptanoyl-sn-glycero-3-phosphocholine (DHPC-C7) were chosen as short-chain phospholipids. 1-Palmitoyl-2-oleol-sn-glycero-3-phosphocholine (POPC) was used as a model long-chain phospholipid. Kinetic studies by quartz crystal microbalance with dissipation monitoring (QCM-D) showed that the presence of short-chain phospholipids significantly accelerated the formation of SPBs. Rapid rinsing with a buffer solution did not change the adsorbed mass on the surface if POPC/DHPC-C6 mixtures were used below the critical micelle concentration (cmc) of DHPC-C6, indicating that an SPB composed of POPC molecules remained on the surface. Fluorescence microscopy observation showed homogeneous SPBs, and the fluorescence recovery after photobleaching (FRAP) measurements gave a diffusion coefficient comparable to that for SPBs formed from POPC vesicles. However, mixtures of POPC/DHPC-C7 resulted in a smaller mass of lipid adsorption on the substrate. FRAP measurements also yielded significantly smaller diffusion coefficients, suggesting the presence of defects. The different behaviors for DHPC-C6 and DHPC-C7 point to the dual roles of detergents to enhance the formation of SPBs and to destabilize them, depending on their structures and aggregation properties.     

Lateral organization of a membrane protein in a supported binary lipid domain: direct observation of the organization of bacterial light-harvesting complex 2 by total internal reflection fluorescence microscopy

A unique method is described for directly observing the lateral organization of a membrane protein (bacterial light-harvesting complex LH2) in a supported lipid bilayer using total internal reflection fluorescence (TIRF) microscopy. The supported lipid bilayer consisted of anionic 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1'-glycerol)] (DOPG) and 1,2-distearoly-sn-3-[phospho-rac-(1'-glycerol)] (DSPG) and was formed through the rupture of a giant vesicle on a positively charged coverslip. TIRF microscopy revealed that the bilayer was composed of phase-separated domains. When a suspension of cationic phospholipid (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine: EDOPC) vesicles (approximately 400 nm in diameter), containing LH2 complexes (EDOPC/LH2 = 1000/1), was put into contact with the supported lipid bilayer, the cationic vesicles immediately began to fuse and did so specifically with the fluid phase (DOPG-rich domain) of the supported bilayer. Fluorescence from the incorporated LH2 complexes gradually (over approximately 20 min) spread from the domain boundary into the gel domain (DSPG-rich domain). Similar diffusion into the domain-structured supported lipid membrane was observed when the fluorescent lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine-rhodamine B sulfonyl: N-Rh-DOPE) was incorporated into the vesicles instead of LH2. These results indicate that vesicles containing LH2 and lipids preferentially fuse with the fluid domain, after which they laterally diffuse into the gel domain. This report describes for first time the lateral organization of a membrane protein, LH2, via vesicle fusion and subsequent lateral diffusion of the LH2 from the fluid to the gel domains in the supported lipid bilayer. The biological implications and applications of the present study are briefly discussed.     

Investigation of interaction of Leu-enkephalin with lipid membranes

Enkephalins are peptides with morphine-like activity. To achieve their biological function, they must be transported from an aqueous phase to the lipid-rich environment of their membrane bound receptor proteins. In our study, zeta potential (ZP) method was used to detect the association of Leu-enkephalin and Leu-enkephalinamide with phospholipid liposomes constituted from egg-phosphatidylcholine (EPC), dioleoyl-phosphatidylethanolamine (DOPE), cholesterol (Chol), sphingomyelin (SM) as well as soybean phospholipid (SBPL). Transfer of the peptides over lipid membranes was examined by electrophysiology technique (ET) and fluorescence spectroscopy (FS), and further confirmed using 4-fluoro-7-nitrobenzofurazan (NBD-F) labeled Leu-enkephalin (NBD-F-enkephalin) with confocal laser scanning microscopy method (CLSM). Results of zeta potential showed that enkephalinamide associated with lipid membranes and gradually saturated on the membranes either hydrophobically or electrostatically or both. Data from electrophysiology technique indicated that Leu-enkephalin could cause transmembrane currents, suggesting the transfer of peptides across lipid membranes. Transfer examined by fluorescence spectroscopy implied that it could be separated into three steps, adsorption, transportation and desorption, which was afterward reaffirmed by confocal laser scanning microscopy. Transfer efficiencies of enkephalin across SBPL, EPC/DOPE, EPC/DOPE/SM, EPC/SM and EPC/Chol lipid bilayer membranes were evaluated with ET and CLSM experiments. Results showed that the addition of either sphingomyelin or cholesterol, or negatively charged lipid in lipid membrane composition could lower the transfer efficiency.     

A PDMS-based biochip with integrated sub-micrometre position control for TIRF microscopy of the apical cell membrane

A poly(dimethylsiloxane) (PDMS)-based biochip with an integrated pressure controlled positioning system with sub-micrometre precision was realized. The biochip was easy and cheap to manufacture and enabled positioning in a wet environment. It allowed the application of total internal reflection fluorescence (TIRF) microscopy at the dorsal cell membrane, which is not adhering to a support. Specifically, the chip enabled TIRF microscopy at the apical membrane of polarized epithelial cells. Thereby, the device allowed us for the first time to monitor individual fusion events of GPI-GFP bearing vesicles at the apical membrane in live Madin-Darby canine kidney II (MDCK II) cells. Moreover, a mapping of fusion sites became feasible and revealed that the whole apical membrane is fusion competent. In total, the biochip offers an all-in-one solution for apical TIRF microscopy and contributes a novel tool to study trafficking processes close to the apical plasma membrane in polarized epithelial cells.     

Visualizing the solubilization of supported lipid bilayers by an amphiphilic peptide

The effect of the presequence peptide of cytochrome c oxidase subunit IV (p25) on supported phospholipid bilayers (SPBs) was visualized using atomic force microscopy (AFM). The presequence was found to cause the complete disruption of supported bilayers containing neutral lipids. At relatively low concentrations of presequence, the peptide was found to bind to the membrane, coalescing to form microdomains within the liquid-crystalline bilayer that were located predominantly at bilayer-mica boundaries. Further increases in peptide concentration resulted in the formation of holes within the SPB that were spanned by an interpenetrating network of narrower regions of the bilayer, which, at higher applied peptide concentrations, were observed to disappear through a budding process, ultimately leading to the formation of spherical structures at yet higher peptide concentrations. Within this paper, the impact the presequence has upon the structure and order of the membrane is discussed, as is the potential implication of this apparent solubilization process on the translocation of cytochrome c oxidase into the inner mitochondrial membrane.     

Surface planar bilayers of phospholipids used in protein membrane reconstitution: an atomic force microscopy study

In this work, using atomic force microscopy (AFM), we have studied the influence of the temperature on the properties of the surface planar bilayers (SPBs) formed with: (i) the total lipid extract of Escherichia coli; (ii) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPC) (1:1, mol/mol); and, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol-amine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). According to the height profile analysis we performed, the height of the SPBs of DMPC:POPC were temperature dependent. Separated domains were observed in the SPBs of the POPE:POPG mixture and the E. coli lipid extract. The implication of those domains for the correct insertion of membrane proteins into proteoliposomes is discussed.     

Membrane affinity of the amphiphilic marinobactin siderophores

Marinobactins are a class of newly discovered marine bacterial siderophores with a unique amphiphilic structure, suggesting that their functions relate to interactions with cell membranes. Here we use small and large unilamellar L-alpha-dimyristoylphosphatidylcholine vesicles (SUVs and LUVs) as model membranes to examine the thermodynamics and kinetics of the membrane binding of marinobactins, particularly marinobactin E (apo-M(E)) and its iron(III) complex, Fe-M(E). Siderophore-membrane interactions are characterized by NMR line broadening, stopped-flow spectrophotometry, fluorescence quenching, and ultracentrifugation. It is determined that apo-M(E) has a strong affinity for lipid membranes with molar fraction partition coefficients K(x)()(apo)(-)(M)E = 6.3 x 10(5) for SUVs and 3.6 x 10(5) for LUVs. This membrane association is shown to cause only a 2-fold decrease in the rate of iron(III) binding by apo-M(E). However, upon the formation of the iron(III) complex Fe-M(E), the membrane affinity of the siderophore decreased substantially (K(x)()(Fe)(-)(M)E = 1.3 x 10(4) for SUVs and 9.6 x 10(3) for LUVs). The kinetics of membrane binding and dissociation by Fe-M(E) were also determined (k(on)(Fe)(-)(M)E = 1.01 M(-)(1) s(-)(1); k(off)(Fe)(-)(M)E = 4.4 x 10(-)(3) s(-)(1)). The suite of marinobactins with different fatty acid chain lengths and degrees of chain unsaturation showed a range of membrane affinities (5.8 x 10(3) to 36 M(-)(1)). The affinity that marinobactins exhibit for membranes and the changes observed upon iron binding could provide unique biological advantages in a receptor-assisted iron acquisition process in which loss of the iron-free siderophore by diffusion is limited by the strong association with the lipid phase.     

Phospholipids as an alternative to direct covalent coupling: surface functionalization of nanoporous alumina for protein recognition and purification

Anodic aluminum oxide (AAO) substrates with aligned, cylindrical, non-intersecting pores with diameters of 75 nm and depths of 3.5 or 10 μm were functionalized with lipid monolayers harboring different receptor lipids. AAO was first functionalized with dodecyl-trichlorosilane, followed by fusion of small unilamellar vesicles (SUVs) forming a lipid monolayer. The SUVs' lipid composition was transferred onto the AAO surface, allowing us to control the surface receptor density. Owing to the optical transparency of the AAO, the overall vesicle spreading process and subsequent protein binding to the receptor-doped lipid monolayers could be investigated in situ by optical waveguide spectroscopy (OWS). SUV spreading occurred at the pore-rim interface, followed by lateral diffusion of lipids within the pore-interior surface until homogeneous coverage was achieved with a lipid monolayer. The functionality of the system was demonstrated through streptavidin binding onto a biotin-DOPE containing POPC membrane, showing maximum protein coverage at 10 mol% of biotin-DOPE. The system enabled us to monitor in real-time the selective extraction of two histidine-tagged proteins, PIGEA14 (14 kDa) and ezrin (70 kDa), directly from cell lysate solutions using a DOGS-NTA(Ni)/DOPC (1:9) membrane. The purification process including protein binding and elution was monitored by OWS and confirmed by SDS-PAGE.     

Biosensing using lipid bilayers suspended on porous silicon

We demonstrate for the first time the formation of a fluid lipid bilayer membrane on mesoporous silicon substrates for bioapplications. Using fluorescence recovery after photobleaching, the diffusion coefficients for the bilayers supported on oxidized, amino-, and biotin-functionalized mesoporous silicon were determined. The biodetection of a single human umbilical vein endothelial cell was accomplished using confocal microscopy and exploiting Foerster resonance energy transfer effects after the incorporation of RGD covalently linked lipid soluble dyes, with fluorescence donor and acceptor components, within the fluid membrane. A signal response of greater than 100% was achieved via the clustering of RGD peptides binding with areas of high integrin density on the surface of a single cell. These results are a testament to the usefulness of such functional molecular assemblies, based on mobile receptors, mimicking the cell membrane in the development of a new generation of biosensors.     

Phase behavior and the partitioning of caveolin-1 scaffolding domain peptides in model lipid bilayers

The membrane binding and model lipid raft interaction of synthetic peptides derived from the caveolin scaffolding domain (CSD) of the protein caveolin-1 have been investigated. CSD peptides bind preferentially to liquid-disordered domains in model lipid bilayers composed of cholesterol and an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and brain sphingomyelin. Three caveolin-1 peptides were studied: the scaffolding domain (residues 83-101), a water-insoluble construct containing residues 89-101, and a water-soluble construct containing residues 89-101. Confocal and fluorescence microscopy investigation shows that the caveolin-1 peptides bind to the more fluid cholesterol-poor phase. The binding of the water-soluble peptide to lipid bilayers was measured using fluorescence correlation spectroscopy (FCS). We measured molar partition coefficients of 10(4) M(-1) between the soluble peptide and phase-separated lipid bilayers and 10(3) M(-1) between the soluble peptide and bilayers with a single liquid phase. Partial phase diagrams for our phase-separating lipid mixture with added caveolin-1 peptides were measured using fluorescence microscopy. The water-soluble peptide did not change the phase morphology or the miscibility transition in giant unilamellar vesicles (GUVs); however, the water-insoluble and full-length CSD peptides lowered the liquid-liquid melting temperature.     

Atomic force microscopy of model lipid membranes

Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir–Blodgett or Langmuir–Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles.

Analysis of spherical polyelectrolyte brushes by small angle X-ray scattering

Spherical polyelectrolyte brushes （SPBs） with PS core and poly（acrylic acid） （PAA） brushes were prepared and analyzed by SAXS in this article. A radial electron density profile of SPB was brought up, which fits well with the SAXS result and shows a core-shell structure. The effect of pH on SPB form was represented by SAXS and it proves that the chains of SPB will stretch in response to increased pH owning to the increased electrostatic repulsion. SPBs immobilized with magnetic nanoparticles or bovine serum albumin （BSA） were prepared and analyzed by SAXS as well. SAXS could characterize the changes of electron density inside brushes of SPBs due to the immobilization of magnetic nanoparticles or BSA. This provides significant supports for further application of immobilized metal nanoparticles or proteins.     

Cholesterol modulated antibody binding in supported lipid membranes as determined by total internal reflectance microscopy on a microfabricated high-throughput glass chip

A high-throughput microfabricated all-glass microchip, lipid biochip, was created and used to measure fluorescently tagged antibody binding to dinitrophenol (DNP) haptens in planar supported phospholipid/cholesterol lipid bilayers as a function of cholesterol-to-lipid molar ratio (X(CHOL)). Multiple parallel microchannels etched in the lipid biochip allowed simultaneous measurement of antibody binding to hapten-containing and hapten-free lipid bilayers, for a range of aqueous antibody concentrations. Specific and nonspecific antibody binding to the supported lipid bilayers was determined from the internally calibrated intensity of the surface fluorescence using total internal reflectance fluorescence (TIRF) microscopy. The TIRF intensity data of the specific antibody binding were fitted to the Langmuir isotherm and Hill equation models to determine the apparent dissociation constant K(d), the maximum fluorescence parameter F(infinity), and binding cooperativity n. As X(CHOL) increased from 0 to 0.50, K(d) exhibited a minimum of approximately 4 microM and n reached a maximum of approximately 2.2 at X(CHOL) approximately 0.20. However, F(infinity) appeared to be insensitive to the cholesterol content. The nonspecific binding fraction (NS), defined as the ratio of the TIRF intensity for hapten-free bilayers to that with hapten, showed a minimum of approximately 0.08 also at X(CHOL) approximately 0.20. The results suggest that cholesterol regulates the specific binding affinity and cooperativity, as well as suppresses nonspecific binding of aqueous antibody to a planar supported lipid bilayer surface at an optimal cholesterol content of X(CHOL) approximately 0.20. Interestingly, for X(CHOL) approximately 0.40, NS reached a maximum of approximately 0.57, suggesting significant packing defects in the lipid bilayer surface, possibly as a result of lipid domain formation as predicted by the lipid superlattice model. We conclude that cholesterol plays a significant role in regulating both specific and nonspecific antibody/antigen binding events on the lipid bilayer surface and that our lipid biochip represents a new and useful high-resolution microfluidic device for measuring lipid/protein surface binding activities in a parallel and high-throughput fashion.