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Paul S Cremer

Publications and source records attributed to Paul S Cremer.

At least 19 recordsLinked to original sources
PubMed2006

Single giant vesicle rupture events reveal multiple mechanisms of glass-supported bilayer formation.

The formation of supported lipid bilayers (SLBs) on glass from giant unilamellar vesicles (GUVs) was studied using fluorescence microscopy. We show that GUV rupture occurs by at least four mechanisms, including 1), spontaneous rupture of isolated GUVs yielding almost heart-shaped bilayer patches (asymmetric rupture); 2), spontaneous rupture of isolated GUVs yielding circular bilayer patches (symmetric rupture); 3), induced rupture of an incoming vesicle when it contacts a planar bilayer edge; and 4), induced rupture of an adsorbed GUV when a nearby GUV spontaneously ruptures. In pathway 1, the dominant rupture pathway for isolated GUVs, GUVs deformed upon adsorption to the glass surface, and planar bilayer patch formation was initiated by rupture pore formation near the rim of the glass-bilayer interface. Expanding rupture pores led to planar bilayer formation in approximately 10-20 ms. Rupture probability per unit time depended on the average intrinsic curvature of the component lipids. The membrane leaflet adsorbed to the glass surface in planar bilayer patches originated from the outer leaflet of GUVs. Pathway 2 was rarely observed. We surmise that SLB formation is predominantly initiated by pathway 1 rupture events, and that rupture events occurring by pathways 3 and 4 dominate during later stages of SLB formation.

Computer Simulation↗
PubMed2006

Ionic conductivity of the aqueous layer separating a lipid bilayer membrane and a glass support.

The in-plane ionic conductivity of the approximately 1-nm-thick aqueous layer separating a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer membrane and a glass support was investigated. The aqueous layer conductivity was measured by tip-dip deposition of a POPC bilayer onto the surface of a 20- to 75-microm-thick glass membrane containing a single conical-shaped nanopore and recording the current-voltage (i-V) behavior of the glass membrane nanopore/POPC bilayer structure. The steady-state current across the glass membrane passes through the nanopore (45-480 nm radius) and spreads radially outward within the aqueous layer between the glass support and bilayer. This aqueous layer corresponds to the dominant resistance of the glass membrane nanopore/POPC bilayer structure. Fluorescence recovery after photobleaching measurements using dye-labeled lipids verified that the POPC bilayer maintains a significant degree of fluidity on the glass membrane. The slopes of ohmic i-V curves yield an aqueous layer conductivity of (3 +/- 1) x 10(-3) Omega(-1) cm(-1) assuming a layer thickness of 1.0 nm. This conductivity is essentially independent of the concentration of KCl in the bulk solution (10-4 to 1 M) in contact with the membrane. The results indicate that the concentration and mobility of charge carriers in the aqueous layer between the glass support and bilayer are largely determined by the local structure of the glass/water/bilayer interface.

Electric Conductivity↗
PubMed2006

Interactions between macromolecules and ions: The Hofmeister series.

The Hofmeister series, first noted in 1888, ranks the relative influence of ions on the physical behavior of a wide variety of aqueous processes ranging from colloidal assembly to protein folding. Originally, it was thought that an ion's influence on macromolecular properties was caused at least in part by 'making' or 'breaking' bulk water structure. Recent time-resolved and thermodynamic studies of water molecules in salt solutions, however, demonstrate that bulk water structure is not central to the Hofmeister effect. Instead, models are being developed that depend upon direct ion-macromolecule interactions as well as interactions with water molecules in the first hydration shell of the macromolecule.

Acrylamides↗
PubMed2006

Supported lipopolymer membranes as nanoscale filters: simultaneous protein recognition and size-selection assays.

A technique for size-selective discrimination of protein analytes was developed by incorporating poly(ethylene glycol) (PEG) lipopolymers into supported lipid bilayers. The membranes also contained biotinylated lipids, which recognized both streptavidin and anti-biotin IgG. By employing various PEG lipopolymer concentrations, clear discrimination against anti-biotin (Mw = 150 000 Da) binding could be observed, which became more pronounced at higher polymer densities. On the other hand, streptavidin (Mw = 52 800) binding to the membrane remained unaffected even at PEG concentrations that were well into the mushroom-to-brush phase transition. These observations were exploited to create an on-chip ligand-receptor binding assay that favored streptavidin binding over anti-biotin by several orders of magnitude in the presence of the lipopolymer. Control experiments revealed that the two proteins are bound to similar extents from a multi-protein analyte solution in the absence of PEG.

Binding, Competitive↗
PubMed2006

Probing molecular structure at interfaces for comparison with bulk solution behavior: water/2-propanol mixtures monitored by vibrational sum frequency spectroscopy.

The orientation of the isopropyl group at the liquid/vapor interface in 2-propanol/water binary mixtures was studied by vibrational sum frequency spectroscopy. The CH(3) stretch modes of the two methyl groups were used to determine the molecule's orientation by employing a novel united atom approach to model the (CH(3))(2)X moiety. For this purpose, the changes in the molecular susceptibility of the isopropyl group stretches were derived in the laboratory frame as a function of the tilt and twist angles. The results indicated that the methyl groups lay down on the surface at low alcohol mole fraction and gradually twisted with increasing mole fraction. At the azeotrope, x(iso) = 0.68, one of the methyl groups aligned approximately parallel to the surface normal, whereas the other was nearly parallel with the liquid/vapor interface. When the mole fraction of 2-propanol was higher than 0.68, the orientation of 2-propanol remained almost constant. The change in the alcohol's orientation with 2-propanol mole fraction closely tracked changes in its bulk activity coefficient. Such results lead to a picture in which the surface structure and bulk properties of the system are closely linked.

Journal Article↗
PubMed2006

Direct writing of metal nanoparticle films inside sealed microfluidic channels.

Herein we demonstrate the ability to pattern Ag nanoparticle films of arbitrary geometry inside sealed PDMS/TiO2/glass microfluidic devices. The technique can be employed with aqueous solutions at room temperature under mild conditions. A 6 nm TiO2 film is first deposited onto a planar Pyrex or silica substrate, which is subsequently bonded to a PDMS mold. UV light is then exposed through the device to reduce Ag+ from an aqueous solution to create a monolayer-thick film of Ag nanoparticles. We demonstrate that this on-chip deposition method can be exploited in a parallel fashion to synthesize nanoparticles of varying size by independently controlling the solution conditions in each microchannel in which the film is formed. The film morphology was checked by atomic force microscopy, and the results showed that the size of the nanoparticles was sensitive to solution pH. Additionally, we illustrate the ability to biofunctionalize these films with ligands for protein capture. The results indicated that this could be done with good discrimination between addressed locations and background. The technique appears to be quite general, and films of Pd, Cu, and Au could also be patterned.

Coated Materials, Biocompatible↗
PubMed2006

Aqueous two-phase system formation kinetics for elastin-like polypeptides of varying chain length.

The kinetics of aqueous two-phase system (ATPS) formation for elastin-like polypeptides (ELP) with defined chemical composition and chain length was investigated by dark field microscopy in an on-chip format with a linear temperature gradient. Scattering intensities from peptide solutions in the presence and absence of sodium dodecyl sulfate (SDS) were recorded as a function of temperature and time, simultaneously. It was found that the formation of the ATPS for three ELPs of different molecular weights (36 075, 59 422, and 129 856 Da) in the absence of SDS followed a coalescence mechanism, and the rate constant and activation energy were independent of chain length. With the introduction of SDS into the ELP solutions, the rate constants were attenuated more strongly with increasing chain length. Moreover, the coalescence process in the presence of SDS showed non-Arrhenius kinetics as a function of temperature. For the two shorter ELPs, ATPS formation occurred via coalescence at all SDS concentrations and temperatures investigated. On the other hand, the coalescence process was greatly suppressed for the longest ELP at elevated temperatures and higher SDS concentrations. Under these circumstances, ATPS formation was forced to proceed via a mixed Ostwald ripening and coalescence mechanism.

Elastin↗
PubMed2005

Effect of average phospholipid curvature on supported bilayer formation on glass by vesicle fusion.

The adsorption of large unilamellar vesicles composed of various combinations of phosphatidylcholine, phosphatidylethanolamine (PE), monomethyl PE, and dimethyl PE (PE-Me2) onto a glass surface was studied using fluorescence microscopy. The average lipid geometry within the vesicles, described mathematically by the average intrinsic curvature, C(0,ave), was methodically altered by changing the lipid ratios to determine the effect of intrinsic curvature on the ability of vesicles to rupture and form a supported lipid bilayer. We show that the ability of vesicles to create fluid planar bilayers is dependent on C(0,ave) and independent of the identity of the component lipids. When the C(0,ave) was approximately -0.1 nm(-1), the vesicles readily formed supported lipid bilayers with almost full mobility. In contrast, when the C(0,ave) ranged from approximately -0.2 to approximately -0.3 nm(-1), the adsorbed vesicles remained intact upon the surface. The results indicate that the average shape of lipid molecules within a vesicle (C(0,ave)) is essential for determining kinetically viable reactions that are responsible for global geometric changes.

Absorption↗
PubMed2005

Specific ion effects on the water solubility of macromolecules: PNIPAM and the Hofmeister series.

Aqueous processes ranging from protein folding and enzyme turnover to colloidal ordering and macromolecular precipitation are sensitive to the nature and concentration of the ions present in solution. Herein, the effect of a series of sodium salts on the lower critical solution temperature (LCST) of poly(N-isopropylacrylamide), PNIPAM, was investigated with a temperature gradient microfluidic device under a dark-field microscope. While the ability of a particular anion to lower the LCST generally followed the Hofmeister series, analysis of solvent isotope effects and of the changes in LCST with ion concentration and identity showed multiple mechanisms were at work. In solutions containing sufficient concentrations of strongly hydrated anions, the phase transition of PNIPAM was directly correlated with the hydration entropy of the anion. On the other hand, weakly hydrated anions were salted-out through surface tension effects and displayed improved hydration by direct ion binding.

Acrylic Resins↗
PubMed2005

Fluid and air-stable lipopolymer membranes for biosensor applications.

The behavior of poly(ethylene glycol) (PEG) conjugated lipids was investigated in planar supported egg phosphatidylcholine bilayers as a function of lipopolymer density, chain length of the PEG moiety, and type of alkyl chains on the PEG lipid. Fluorescence recovery after photobleaching measurements verified that dye-labeled lipids in the membrane as well as the lipopolymer itself maintained a substantial degree of fluidity under most conditions that were investigated. PEG densities exceeding the onset of the mushroom-to-brush phase transition were found to confer air stability to the supported membrane. On the other hand, substantial damage or complete delamination of the lipid bilayer was observed at lower polymer densities. The presence of PEG in the membrane did not substantially hinder the binding of streptavidin to biotinylated lipids present in the bilayer. Furthermore, above the onset of the transition into the brush phase, the protein binding properties of these membranes were found to be very resilient upon removal of the system from water, rigorous drying, and rehydration. These results indicate that supported phospholipid bilayers containing lipopolymers show promise as rugged sensor platforms for ligand-receptor binding.

Biosensing Techniques↗
PubMed2005

Glycolipids as receptors for Bacillus thuringiensis crystal toxin.

The development of pest resistance threatens the effectiveness of Bacillus thuringiensis (Bt) toxins used in transgenic and organic farming. Here, we demonstrate that (i) the major mechanism for Bt toxin resistance in Caenorhabditis elegans entails a loss of glycolipid carbohydrates; (ii) Bt toxin directly and specifically binds glycolipids; and (iii) this binding is carbohydrate-dependent and relevant for toxin action in vivo. These carbohydrates contain the arthroseries core conserved in insects and nematodes but lacking in vertebrates. We present evidence that insect glycolipids are also receptors for Bt toxin.

Animals↗
PubMed2005

Microfluidic tools for studying the specific binding, adsorption, and displacement of proteins at interfaces.

A combination of temperature and concentration gradient microfluidic devices were employed to study the mechanistic details of biomacromolecule interactions at oxide interfaces. These lab-on-a-chip techniques allowed high-throughput, multiplexed data collection using only nanoliters of analyte. The three examples presented demonstrate rapid data acquisition relative to standard methods. First, we show ligand-receptor binding data for multivalent binding between membrane-bound ligands and incoming aqueous proteins with several binding pockets. A model is described for obtaining both the first and second dissociation constant for the reaction. The second example employs temperature gradient microfluidics to study the thermoresponsive properties of polymers and proteins. Both the folding mechanism and subsequent formation of an aqueous two-phase system were followed. Finally, these microfluidic techniques were combined with fluorescence microscopy and nonlinear optical spectroscopy to elucidate the mechanism of fibrinogen displacement from silica surfaces. This combination of methods enabled both direct and indirect observation of protein conformational changes.

Adsorption↗
PubMed2004

On the mechanism of the hofmeister effect.

Vibrational sum frequency spectroscopy was used to probe fatty amine monolayers spread on various electrolyte solutions. The spectra revealed ion specific changes in both monolayer ordering and water structure with the former following the Hofmeister series. Separate measurements of the surface potential as a function of ion tracked closely to changes in alkyl chain structure, but less closely to changes in water structure. The disruption of the monolayer ordering could be ascribed to the relative ability of the ions to penetrate past the hydrophilic surface of the monolayer's headgroups and into the more hydrophobic portion of the thin film. The corresponding trends observed in the surface water structure showed significant deviations from the Hofmeister series, leading to the conclusion that the changes in surface water structure, often credited with being the origin of Hofmeister effects, are probably not of primary importance. On the other hand, dispersion forces almost certainly play a large role in the order of the Hofmeister series.

Amines↗
PubMed2004

Creating fluid and air-stable solid supported lipid bilayers.

Solid supported lipid bilayers are rapidly delaminated when drawn through the air/water interface. We have discovered that a close packed monolayer of specifically bound protein prevents this process. The protection mechanism worked in two ways. First, when protein-protected bilayers were drawn through the air/water interface, a thin bulk water layer was visible over the entire bilayer region, thereby preventing air from contacting the surface. Second, a stream of nitrogen was used to remove all bulk water from a protected bilayer, which remained fully intact as determined by fluorescence microscopy. The condition of this dried bilayer was further probed by fluorescence recovery after photobleaching. It was found that lipids were not two-dimensionally mobile in dry air. However, when the bilayer was placed in a humid environment, 91% of the bleached fluorescence signal was recovered, indicating long-range two-dimensional mobility. The diffusion coefficient of lipids under humid conditions was an order of magnitude slower than the same bilayer under water. Protected bilayers could be rehydrated after drying, and their characteristic diffusion coefficient was reestablished. Insights into the mechanism of bilayer preservation were suggested.

Air↗
PubMed2004

Patterning enzymes inside microfluidic channels via photoattachment chemistry.

We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.

Animals↗