WO2002071944A1 - Stabilized, biocompatible supported lipid membrane - Google Patents
Stabilized, biocompatible supported lipid membrane Download PDFInfo
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- WO2002071944A1 WO2002071944A1 PCT/US2002/007369 US0207369W WO02071944A1 WO 2002071944 A1 WO2002071944 A1 WO 2002071944A1 US 0207369 W US0207369 W US 0207369W WO 02071944 A1 WO02071944 A1 WO 02071944A1
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- B—PERFORMING OPERATIONS; TRANSPORTING
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Definitions
- the present invention relates to a self-assembled lipid membrane, in the form of a " monolayer, bilayer, or multilayer, that is stabilized on a solid support.
- One example is the design of a biosensor surface at which a ligand binding event must be detected in the presence of numerous other non-target proteins (Wisniewski, N.; Reichert, M., Coll. Surf. B: Biointerfaces 2000, 18, 197-219; Stelzle, M.; Weissmuller, G.; Sackman, E., J. Phys. Chem., 1993, 97, 2974; Duschl, C; Liley, M.; Corradin, G.; Nogel, H., Biophys. J., 1994, 67, 1229; Song, X.D.; Swanson, B. I., Anal. Chem., 1999, 71, 2097; Parikh, A.
- the transducer is an oxide or noble metal surface to which dissolved proteins can irreversibly adsorb, "fouling" the sample/transducer interface.
- Planar lipid monolayer, bilayer, and multilayer structures have been used to coat such surfaces (Sackman, E., Science, 1996, 271, 43; Plant, A.L., Langmuir, 1999, 15, 5128; Song, X.D.; Swanson, B.I., Anal. Chem., 1999, 71, 2097; Parikh, A.N.; Beers, J.D.; Shreve, A.P.; Swanson, B. I., Langmuir, 1999, 15, 5369; Fischer, B.; Heyn, S.
- Such lipid monolayers, bilayers, or multilayers offer the ability to minimize sensor "fouling", i.e., the undesirable adsorption of non-target proteins and biomolecules invariably present in complex biological matrices, by exploiting the characteristic protein adsorption resistance associated with the phosphorylcholine (PC) lipid headgroup (Hay ward, J.; Chapman, D., Biomaterials, 1984, 5, 135; Chapman, D., Langmuir, 1993, 9, 39; Malmsten, M. J., Colloid Interface Sci., 1995, 171, 106; Murphy, I. F.; Lu, J. R; Lewis, L.
- PC phosphorylcholine
- Supported lipid membrane structures also provide the necessary environment for transmembrane receptor incorporation, which has been demonstrated by several authors through the fabrication of proteo-lipid structures with retained protein activity (Salafsky, J.; Groves, J. T.; Boxer, S.G., Biochem., 1996, 35, 14773-14781; Schmidt, E.
- Supported lipid monolayers, bilayers and multilayers can be self-assembled by fusion of fluid, unilamellar vesicles, an important issue for commercial application, onto a variety of optically or electrically active substrates.
- micro-patterned techniques to modify planar, substrate supported thin films, including supported lipid bilayers, adds promise to the potential of biochips with parallel arrays of sensing elements for high throughput biological or pharmaceutical screening or sensing (Ho vis, J. S.; Boxer, S. B., Langmuir, 2000, 16(3), 894-897; Hovis, J. S.; Boxer, S.
- SAMS self-assembled monolayers
- oligo(ethylene glycol) Yang, Z.; Galloway, J. A.; Yu, H., Langmuir, 1999, 15
- headgroups Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R. E.; Yan, L.; Whitesides, G.M., J. Am.
- Chem. Soc, 2000, 122, 8303-8304 address the stability issue of biosensor coatings but are not without shortcomings, including an increased difficulty in functionalizing these films with water-soluble proteins in a well-defined manner, and not providing a suitable environment for transmembrane receptor proteins. Therefore, interest in stabilizing lipid films on solid supports continues to receive scientific attention.
- unilamellar vesicles composed of bis-substituted lipids can be polymerized to form cross-linked vesicles that are insoluble in surfactant solutions and organic solvents (Sisson, T. M.; Lamparski, H. G.; Kolchens, S.; Elyadi, A.; O'Brien, D. F., Macromolecules, 1996, 29, 8321).
- At least two research groups have used the polymerization strategy to stabilize lipid mono- and bilayers on solid supports.
- Regen and coworkers adsorbed films of mono- and di-acrylate functionalized lipids on poly(ethylene), followed by UN-photo-polymerization to form a supported polymerized lipid film of near monolayer thickness (Regen, S. L.: Kirszensztejn, P.; Singh, A., Macromolecules, 1983, 16, 338; Foltynowicz, Z.; Yamaguchi, K.; Czajka, B,. Regen, S. L., Macromolecules, 1985, 18, 1394).
- Chaikof and coworkers formed a hybrid bilayer by fusing vesicles (Marra, K. G.; Winger, T. M.; Hanson, S. R; Chaikof, E. L., Macromolecules, 1997; 30, 6483; Orban, J. M.; Faucher, K. M.: Dluhy, R. A.; Chaikof, E. L., Macromolecules, 2000, 33, 4205) composed of mono-acrylate lipids onto a support coated with an alkylsilane monolayer; in situ polymerization produced linear polymers in the upper leaflet of this structure. Although enhanced stability during extended incubation in water was observed, significant lipid desorption occurred when the assembly was exposed to surfactant.
- the first embodiment which includes a method for the self-assembly and stabilization of a lipid membrane at a solid surface, comprising: depositing a lipid monolayer or a lipid multilayer on a substrate, thereby obtaining a supported lipid monolayer or a supported lipid multilayer; in situ polymerizing said supported lipid monolayer or said supported lipid multilayer, thereby obtaining a polymerized membrane.
- Figure 1 shows types of polymerizable groups that can be used in polymerizable lipids.
- Figure 2 shows examples of mono-substituted polymerizable lipids.
- Figure 3 shows examples of bis-substituted polymerizable lipids.
- Figure 4 shows examples of heterobifunctional polymerizable lipids.
- Figure 5 shows examples of polymerizable lipids that differ in the length of the lipid tail (can be 14 to 22 atoms) and the extent and location of unsaruration and/or branching in the lipid tail(s).
- Figure 6 shows some examples of the different types of head groups for polymerizable lipids.
- Figure 7 shows a schematic of the vesicle fusion process, forming a fluid supported lipid bilayer (1,2), followed by redox-initiated, radical polymerization (3) to produce a cross- linked bilayer (4).
- Figure 8 shows AFM images and linescans of a polymerized bis-SorbPC (redox) bilayer in air (left) and under water (center). On the right is an image of a region of the film that was deliberately damaged by repeated high force scanning.
- redox polymerized bis-SorbPC
- Figure 9 shows a bar graph of relative bovine serum albumin (BSA) adsorption to various films.
- the diagram illustrates the principle of total internal reflectance fluorescence (TIRF), which is used to measure adsorption of rhodamine labeled BSA molecules to the various films.
- TIRF total internal reflectance fluorescence
- Figure 10 shows TIRF generated BSA adsorption isotherms for various films on quartz substrates.
- the dried and rehydrated polymerized bis-SorbPC (redox) film demonstrates equivalent adsorption resistance at a BSA solution concentration of 1.5 lO "5 M.
- Figure 11 shows AFM images and linescans of a blank silicon substrate and a polymerized bis-SorbPC (redox) supported bilayer before and after exposure to a 15 ⁇ M BSA solution.
- Figure 12 shows an AFM image and a linescan of a dried, poly-diacetylenic PC lipid bilayer deposited by the Langmuir-Schaefer technique and polymerized by direct UN irradiation.
- Figure 13 shows show an AFM image and linescan of a dried, polymerized bis- SorbPC bilayer deposited by vesicle fusion and polymerized by direct UN irradiation.
- Figure 14 shows an AFM image of a dried, redox polymerized bilayer deposited by vesicle fusion and composed of 10% bis-SorbPC monomer and 30% non-polymerizable lipid DOPC.
- Figure 15 shows an AFM image and linescan of a dried, redox polymerized mono- SorbPC bilayer deposited by vesicle fusion.
- Figure 16 shows an AFM image and linescan of a dried, redox polymerized bis- DenPC bilayer deposited by vesicle fusion.
- Figure 17 shows an AFM image and linescan of a dried, redox polymerized DenSorbPC bilayer deposited by vesicle fusion.
- Figure 18 shows an AFM image (left) of biotin-BSA microcontact printed on a polymerized bis-SorbPC (redox) bilayer.
- the schematic on the right depicts binding of rhodamine labeled avidin to the patterned regions of biotin-BSA.
- Figure 19 shows an AFM image (left) of a UN polymerized, bis-SorbPC film patterned by microcontact printing. Printing removed portions of the supported fluid bilayer (dark stripes); UN polymerization then stabilized the remaining regions (light stripes). The illustration on the right depicts the procedure graphically.
- Figure 20 shows schematic of TIRF spectroscopy instrumentation, a) fused silica slide, b) quartz prism, c) TeflonTM block and NitonTM o-ring, d) 4X microscope objective, e) long pass filter, f) PMT, g) lock-in amplifier, h) frequency generator, i) data acquisition computer, and j) reference photo diode.
- Figure 21 shows kinetic data for the UV polymerization of bis-SorbPC bilayers which was obtained by measuring the depletion of the monomer absorbance as a function of time.
- Inset absorbance spectrum of the monomeric bis-SorbPC prior to polymerization.
- Figure 22 shows AFM images for (a) a dried bis-SorbPC bilayer film, and (b) the same film imaged under water to Example for UV polymerized filer.
- the film was deposited using the Langmuir-Schaefer method and polymerized with UV light.
- Figure 23 shows adsorption isotherms of FITC labeled BSA to a POPC monolayer, (a hydrophobic surface, solid line), a dehydrated bis-SorbPC bilayer (dashed line), and a POPC bilayer (dash-dot line).
- the lines through the data in each case represent the fitting the data to a Langmuir adsorption isotherm.
- Figure 24 shows the structures of several cyanine dyes that can be used for photosensitized polymerization of supported lipid films.
- the present inventors have found a novel and successful strategy for the self- assembly and stabilization of a lipid bilayer, particularly a phospholipid bilayer, at a solid surface. After deposition of a lipid bilayer on a substrate, in situ polymerization of the supported bilayer produces a cross-linked membrane that is stable to transfer into air and exposure to surfactant solutions and organic solvents, yet retains the protein resistance characteristic of a fluid phosphatidylcholine (PC) bilayer.
- PC fluid phosphatidylcholine
- a self-assembled, supported fluid membrane is formed by fusion of fluid, small unilamellar vesicles (SUVs) composed of a polymerizable lipid to a clean surface in a buffered aqueous solution or deionized water.
- the buffer solution or water used may also include added mono-, di-, or trivalent metal salts.
- fluid bilayer SUVs spontaneously unroll to produce an extended, continuous lipid monolayer or bilayer ( Figure 7).
- pre-polymerized phospholipid vesicles do not fuse to surfaces.
- the supported lipid film is then transferred to a redox polymerization medium to initiate polymerization without exposing the film to air.
- the film is removed, cleaned, and dried under an inert gas atmosphere.
- Polymerizable lipids that are useful for this invention include those which contain at least one of the polymerizable groups shown in Figure 1, e.g. styryl, dienyl, dienoyl, sorbyl, acryloyl, methacryloyl, vinyl ester, among others. These groups can be located anywhere along the lipid tails as indicated by the examples shown in the following Figures 2-6. These examples include mono- and bis-substituted lipids, shown in Figures 2 and 3 respectively as phosphatidylcholines, which are ester lipids based on a glycerol backbone.
- the lipid backbone is not limited to glycerol, but could also be l-aminopropane-2,3-diol, glutamic acid, aspartic acid, among others.
- the lipid tail is linked to the glycerol backbone through an ester bond. It is also possible to prepare similar polymerizable lipids with an ether bond.
- the polymerizable lipid can have two identical reactive groups in each lipid tail, or two different reactive groups in the same lipid tail, which are heterobifunctional lipids ( Figure 4).
- the main phase transition temperature of the lipid can be controlled through the choice of the length of the lipid tail from 14 to 22 atoms, and the extent and location of unsaturation and/or branching in the lipid tail(s) as shown in Figure 5.
- the lipid head group can vary widely from phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylserine (PS), to PE-like lipids with associated groups such as succinate or chelating groups for the conjugation of functional compounds and metals to the lipid membrane surface (Figure 6).
- lipid headgroups include headgroups terminated with thioethanol, maleimido, pyridyldithio, biotinyl, succinimidyl ester, sulfo succinimidyl ester, alkyl halide, or haloacetamide groups, as well as lipids functionalized with ethylene glycol-based oligomers and polymers.
- the lipid solutions are prepared as follows: Lipids from stock chloroform or benzene solutions or any other organic solvent in which the lipid is soluble are dried under a flowing inert gas such as Ar or N 2 to remove storage solvents. The lipids are then resuspended in deionized water (18 M ⁇ ) or aqueous buffer.
- the lipid concentration is in the range of from 0.01 mg/1 to 5 mg/1, and preferably in the order of 0.5mg/ml.
- the lipid concentration includes all values and subvalues therebetween, especially including 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 and 4.5 mg/1.
- the lipid suspension is then mechanically treated, for example, vortexed and sonicated to clarity, forming SUVs (eg., Barenholz, Y.; Gibbes, D.; Litman, B; Goll, J.; Thomson, T.; Carlson, F., Biochemistry, 1977, 16, 2806).
- Temperature control is preferably maintained at more than 10 degrees above the reported lipid transition temperature.
- the SUVS are preferably used within 30 minutes of preparation, more preferably within 20 minutes after preparation and most preferably within 10 minutes after preparation.
- unilamellar vesicles include, but are not limited to, extrusion of lipids through porous membranes (eg., MacDonald, R.; MacDonald, R. I.; Menco, B.; Takeshita, K.; Subbarao, N.; Lan-rong, H., Biochimica et Biophysica Acta, 1991, 1061, 297) and surfactant dialysis (eg., Minims, L. T.; Zampighi, G.; Nozaki, Y.; Tanford, C; Reynolds, J. A., Biochemsitry, 1981, 20, 833).
- porous membranes eg., MacDonald, R.; MacDonald, R. I.; Menco, B.; Takeshita, K.; Subbarao, N.; Lan-rong, H., Biochimica et Biophysica Acta, 1991, 1061, 29
- surfactant dialysis eg
- extrusion involves resuspension of dried lipids in appropriate solutions, as described above.
- a repeated freeze, thaw cycle may or may not be applied to produce multilamellar vesicles before the suspension is repeatedly passed through a porous size exclusion membrane.
- Unilamellar vesicles with a mean diameter ranging from 50 to 1000 nm are created depending on the size of the pores in the membrane used. The diameter includes all values and subvalues therebetween, especially including 100, 200, 300, 400, 500, 600, 700, 800 and 900 nm.
- Surfactant dialysis also known as detergent depletion, occurs when a suspension of lipid and detergent, (present together in aqueous solution at a concentration above the detergent critical micelle concentration) is dialyzed against another aqueous solution.
- the detergent passes through the dialysis membrane and is removed from the compartment containing the lipid, whereupon the remaining lipid spontaneously forms unilamellar vesicles.
- Supported lipid films are prepared by vesicle fusion ( Figure 7), while avoiding exposure of the unpolymerized films to air, or excessive mechanical shocks. Care must be taken to avoid light exposure to polymerizable lipids or lipid films. Thus, they are handled under yellow light.
- Vesicle fusion to solid supports is a well documented, and commonly used practice to form substrate supported fluid lipid bilayers. The rate of fusion and bilayer spreading is controlled by a subtle balance ' of van der Waals, electrostatic, hydration, and steric forces, but it is of yet, poorly understood what relation these forces play in the process. (Cremer, P. S.; Boxer, S. G., J. Phys. Chem. B, 1999, 103, 2554).
- Vesicle fusion of liposomes containing no net charge (eg., phosphorylcholine headgroups) to glass supports has no observable pH dependence over a range of 2.5-12.3, nor a dependency upon ionic strength. (Cremer, P. S.; Boxer, S. G., J. Phys. Chem. B 1999, 103, 2554)
- the concentration of suspended vesicles in the aqueous solution plays a role in the kinetics of bilayer formation, but not in the physical structure of the final supported film.
- a concentration is used that will allow timely formation of the bilayer, for example, on oxidized silicon, this is a lipid concentration of typically greater than 0.1 mg/ml, but it is noted that lower and higher concentrations will produce supported films.
- the lipid concentration is greater than 0.5 mg/ml, particularly preferably greater than 1 mg/ml.
- lipid films can be formed using standard Langmuir-Schaefer techniques according to reference procedures (Morigaki, K.; Baumgart, T.; Offenatorir, A.; Knoll, W., Angew. Chem., Int. Ed., 2001, 40, 172).
- the substrate surface is preferably cleaned using a plasma cleaner, a sonicator, UV light, an organic solvent such as alcohol or chloroform, a strong acid solution such as a pirhana solution, an aqueous or alcoholic solution of H 2 O 2 , or an aqueous or alcoholic solution of a hydroxide of an alkali earth metal, such as NaOH or KOH.
- a plasma cleaner a sonicator, UV light
- an organic solvent such as alcohol or chloroform
- a strong acid solution such as a pirhana solution
- an aqueous or alcoholic solution of H 2 O 2 an aqueous or alcoholic solution of a hydroxide of an alkali earth metal, such as NaOH or KOH.
- Surfaces are preferably used within 1 hours of cleansing, preferably within 30 minutes, more preferably within 20 minutes and most preferably within 10 minutes.
- Preferred surfaces of the solid support are silicon dioxide (SiO 2 ), silicon oxide (SiO x ), a noble metal such as gold, silver, platinum; mica, a polymer surface, a thin polymer film coated substrate, indium-tin oxide (ITO), tin oxide, indium oxide and silicon.
- the surface can be planar or non-planar.
- a preferred buffer solution is phosphate.
- a preferred pH of the buffer solution is 7.4.
- the pH of the solution can be any value from pH 5.6 to pH 8.
- the buffer can be prepared with any chemical compound having a pK a between 5 and 9.
- the solution can also contain added metal salts, including monovalent, divalent, and trivalent metal salts. Preferred concentrations are from 0 up to and including 500 mM. The concentration includes all values and subvalues therebetween, especially including 1, 10, 50, 100, 150, 200, 250, 300, 350, 400 and 450 mM.
- the redox initiator system is preferably K 2 S 2 O s /NaHSO 3 ( Figure 7).
- a preferred concentration of the persulfate is 1 mM to 1 M.
- the concentration of the persulfate includes all values and subvalues therebetween, especially including 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800 and 900 mM.
- a preferred oxidant to reductant ratio is from 1:1 to 1:10.
- the oxidant to reductant ratio includes all values and subvalues therebetween, especially including 1: 2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 and 1:9.
- polymerized lipid films are indistinguishable by AFM and ellipsometry.
- redox initiator systems can also be used.
- suitable oxidants include H 2 O 2 , KrBrO 3 , CuCl, Cs(SO 4 ) 2 .
- suitable reductants include L-cysteine, H 2 N 2 H 2 , ascorbic acid, HCOOH, R 3 N (where R is hydrogen or any group that contains carbon), and salts of Fe +2 , Ag + , SO 3 ⁇ .
- a preferred concentration of the oxidant is 1 mM to 1 M.
- the oxidant concentration includes all values and subvalues therebetween, especially including 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800 and 900 mM.
- a preferred oxidant to reductant ratio is from 1 : 1 and 1:10.
- the oxidant to reductant ratio includes all values and subvalues therebetween, especially including 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 and 1:9.
- oxygen is excluded by deoxygenating the reaction solutions with a flowing inert gas such as Ar or N 2 .
- a flowing inert gas such as Ar or N 2 .
- the gas flow can occur before the polymerization and can continue throughout the polymerization.
- the film is preferably incubated in the redox polymerization medium for 1 minute to five hours.
- the incubation time includes all values and subvalues therebetween, especially including 5 min, 10 min., 20 min., 40 min., 60 min., 80 min., 100 min., 120 min., 140 min., 160 min., 180 min., 200 min., 220 min., 240 min., 260 min. and 280 min.
- the film is preferably rinsed with water or an aqueous solution of an organic solvent, such as a lower alcohol in water.
- Water is preferably purified to 18 MOhms and made organic free.
- the inert gas for drying is preferably Ar or N 2 .
- bis-SorbPC(redox) with a thickness of about 45 A and a sessile water contact angle of about 32 degrees.
- the contact angle of 32 degrees for the bis- SorbPC(redox) film is very similar to the value of 28 degrees reported by Cooper et al. (Tegoulia, V.; Rao, W.; Kalamber, A.; Rabolt, J.; Cooper, S., Langmuir, 2001, 17, 4396), for a phosphorylcholine terminated SAM film on gold.
- the images in Figure 8, acquired using tapping mode atomic force microscopy (AFM), show that the polymerized bilayer surface is very smooth. The root mean square roughness of the image acquired in air
- Polymerized bilayers can be deliberately damaged by repeated, high force scanning (right image in Figure 8); a line scan across a film containing such a 'trough' yielded an apparent film thickness of 39-47 A, consistent with the ellipsometry data.
- the phospholipid bilayer of the present invention is stable in organic solvents, particularly to chlorinated hydrocarbons such as chloroform, ethers such as tetrahydrofuran, alcohols such as methanol and ethanol, sulfur-containing solvents such as DMSO, ketones such as acetone, and aromatic solvents such as toluene, benzene. It is also stable when exposed to solutions of anionic, cationic, non-ionic, or polymeric surfactants. Exposure to organic solvents or surfactant solutions does not alter the ellipsometric thickness or the AFM images of the stabilized bilayers.
- the polymerized phospholipid bilayer according to the first embodiment of the present invention exhibits resistance to nonspecific protein adsorption even after polymerization of the hydrophobic tails of the lipid monomers, which provides evidence that the "headgroup out" structure of the bilayer is preserved after drying and rehydration.
- the resistance of the bis-SorbPC bilayer of the present invention for BSA is comparable to that of a fluid l-palmitoyl-2-oleolyl-PC (POPC) bilayer as demonstrated by the comparative data shown in Figures 9-11.
- Lipids in addition to bis-SorbPC have also been used in the present invention.
- the above described vesicle fusion, Langmuir-Schaefer, redox-initiated polymerization, or the UV polymerization methods may be used as described above.
- Supported lipid bilayers have been prepared using both bis-DenPC ( Figure 3) and DenSorbPC ( Figure 4).
- a DenSorbPC lipid bilayer formed by vesicle fusion and redox polymerization was indistinguishable from a bilayer of bis-SorbPC (redox) as judged by AFM ( Figure 17). Ellipsometric thickness were nearly equivalent as well, and upon bath sonication in surfactant, only a minute thickness change was observed.
- FIG. 15 Another example, shown in Figure 15, is an AFM image of a dried, redox- polymerized bilayer composed of mono-SorbPC ( Figure 2) that was deposited by vesicle fusion.
- Figure 2 Another example, shown in Figure 15, is an AFM image of a dried, redox- polymerized bilayer composed of mono-SorbPC ( Figure 2) that was deposited by vesicle fusion.
- the incomplete structure of the film is ascribed to the absence of cross-linking, which is precluded when using mono-functionalized lipid at a mole fraction of 1.
- the lipid bilayers are prepared by the vesicle fusion method, or using Langmuir-Blodgett and/or Langmuir-Schaefer technique, and polymerized by direct photo-irradiation with UV, visible or near infrared light or ⁇ -rays.
- the rays can be polarized or unpolarized.
- Preferred polymerizable lipids are those described above in the first embodiment and shown in Figures 1-6.
- Direct UN polymerization is performed by exposing the lipid bilayer films to UN radiation at a wavelength of between 230 and 350 nm, preferably at 260 nm and more preferably at 254 nm.
- the wavelength includes all values and subvalues therebetween, especially including 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, and 340 nm.
- the UV light may be polarized or unpolarized.
- Both, direct UV-photoinitiation and redox-initiated radical polymerization stabilize films of the lipid to surfactant dissolution suggesting the formation of a cross-linked polymeric network.
- a difference in the degrees of polymerization occurs for the two initiation methods.
- redox initiated polymers of bis-SorbPC are larger (Xn approx 50+) than UV photopolymerized polymers (Xn ⁇ 10), which suggests different propagation mechanisms for the polymerizations.
- the UN-irradiation proceeds for 1 second to 1 hour at photon fluxes ranging from 1 x 10 13 to 1 x 10 17 photons/second.
- the irradiation time includes all values and subvalues therebetween, especially including 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260 and 280 seconds, 10 min., 15 min., 20 min., 25 min., 30 min., 35 min., 40 min., 45 min., 50 min., and 55 min.
- the photon flux includes all values and subvalues therebetween, especially including 5 x 10 13 , 1 x 10 14 , 5 x 10 14 , 1 x 10 15 , 5 x 10 15 , 1 x 10 16 and 5 x 10 16 photons/second.
- the thickness of the bis-SorbPC (UN) films deposited by vesicle fusion and UN polymerized are about 29 A and the surface is usually more hydrophobic than redox polymerized bis-SorbPC films (redox) with a contact angle of 52 degrees.
- the ellipsometric thickness combined with the depth of the features suggest that they are likely regions of film where the surrounding lipid-polymer has been removed upon drying and rinsing and likely do not extend to the substrate because partial coverage of a 1.5 to 2 mm film would be inconsistent with the ellipsometric thickness of 29 A.
- the UN-polymerizations are usually not sensitive to the presence of oxygen, nor has the rate of polymerization a noticeable effect on the film properties.
- the rate of polymerization can be affected by altering the intensity of the light used to photopolymerize the film.
- UN-Nis spectroscopy of polymerized bilayers reveals an equivalent degree of conversion for both UV and redox-initiated polymerizations. The degree of conversion is >90%, preferably >95% and most preferably >99%>. Because the polymerization by redox initiators and UV light produce the same polymer product, the difference in acyl-chain structure is not likely the reason for the difference in film properties.
- AFM images of surfaces of unpolymerized fluid lipid films on silica were basically indistinguishable from images of a blank silica surface. This is consistent with the observation of several authors that lipid film loss and/or disruption to the lamellar structure occurs upon drying fluid supported phospholipid bilayers (Cremer, P.S., Boxer, S.G., J. Phys. Chem. B, 1999, 103, 2554).
- Lipid polymerization can also be initiated by a dye-sensitized process (Clapp, P. J.; B. A. Armitage, B.A.; O'Brien, D.F. Macromolecules, 1997, 29, 32).
- a membrane-bound cyanine dye that absorbs in the visible or near-infrared spectral regions is incorporated into the membrane. Irradiation at a wavelength at which the dye absorbs, in the presence of oxygen, is thought to generate hydroxyl radicals which initiate lipid polymerization.
- the dye can be added to the lipid solution either before or after formation of SUNs, prior to using the vesicles to perform vesicle fusion.
- the dye is added to the lipid before it is spread as a monolayer film on a Langmuir trough.
- the preferred molar ratio of lipid to dye ranges from 5:1 up to 30:1.
- the preferred pH range is 6.0 to 9.5.
- the preferred temperature is 15 °C to 45 °C.
- the preferred wavelength of incident light is 350-800 nm.
- the irradiation proceeds for 1 second to 5 hours at preferred incident photon flux ranges from 0.036 x l0 18 to 2 x l0 18 photons/second.
- ambient oxygen is present in the solution and the gas surrounding the solution.
- a number of different dyes can be used to initiate the polymerization of the types of lipids shown in Figures 1-6, including but not limited to the cyanine dyes shown in Figure 24.
- Supported lipid membranes polymerized using the dye-sensitized process have been prepared in our laboratories, with results similar to that obtained using direct UN photopolymerization (described above). Further optimization of the dye-sensitized process is anticipated by systematically varying the numerous variables involved, including dye: lipid ratio, irradiation time and photon flux, type of dye used, type of lipid used, temperature, oxygen concentration, lipid film deposition method.
- a third embodiment relates to the incorporation of non-polymerizable amphiphiles (e.g. surfactants or lipids) or any other molecule that will insert in the stabilized lipid membranes.
- non-polymerizable amphiphiles e.g. surfactants or lipids
- Phospholipid bilayer films according to the present invention may be formed using a mixture of polymerizable lipid and non-polymerizable lipid by the above described methods.
- the amount of non-polymerizable lipid in the mixture is in the range of from 0.01 to 50%, preferably not more than 30%, more preferably not more than 10% and most preferably not more than 2%.
- the amount of non-polymerizable lipid in the mixtures includes all values and subvalues therebetween, especially including 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40 and 45%.
- the polymerizable lipid in these films can be any of the lipids or lipid types shown in Figures 1-6, and mixtures thereof, in any molar desired ratio.
- the above described vesicle fusion, Langmuir-Schaefer, redox-initiated polymerization, and light-driven (UV, visible, or near-infrared) methods may be used as described above to deposit and polymerize the polymerizable lipids in the membrane.
- the non- polymerizable molecules are mixed with the polymerizable lipids prior to vesicle preparation.
- Vesicles composed of non-polymerizable molecules and polymerizable lipids are prepared and fused to appropriate substrates (as described above) to form supported lipid membranes that are subsequently polymerized.
- the polymerized, supported lipid membrane is prepared and then a solution of non-polymerizable molecules is brought into contact with the membrane, which causes the non-polymerizable molecules to bind to and insert into the membrane.
- the non-polymerizable molecules incorporated into the membrane are typically amphiphilic, e.g. a single-chain surfactant or a non-polymerizable lipid, that will bind to and associate with a membrane.
- the nature of the association reaction can be either non-covalent or covalent.
- Preferred non-polymerizable molecules are those which impart a functional property to the membrane, i.e. a surfactant or lipid bearing a headgroup that is functionally distinct from the headgroups on the polymerizable lipids in the membrane.
- Examples include single-chain and double-chain surfactants having anionic or cationic headgroups, headgroups functionalized with ethylene glycol-based oligomers and polymers, headgroups designed to chelate metal ions, headgroups functionalized with dyes that absorb light and/or emit fluorescence in the UN, visible, and/or near-infrared spectral regions, and headgroups designed to react with other molecules.
- Examples of the latter category include headgroups terminated with thioethanol, maleimido, pyridyldithio, biotinyl, succinimidyl ester, sulfo succinimidyl ester, alkyl halide, or haloacetamide groups.
- Non-polymerizable lipid e.g. DOPC
- a non-polymerizable lipid e.g. DOPC
- the non-polymerizable lipid will spatially segregate from the domains of polymerized lipid, forming a lipid membrane that contains spatially defined and distinct fluid and polymerized regions.
- Figure 14 shows an AFM image of a dried, redox polymerized, supported lipid bilayer deposited by vesicle fusion and composed of 70% bis-SorbPC and 30% non-polymerizable lipid DOPC.
- supported phospholipid membranes are produced from mixtures of polymerizable lipids.
- Phospholipid bilayer films according to the present invention may be formed using a mixture of different types of polymerizable lipids by the above described methods.
- the amount of each different type of lipid in the mixture is in the range of from 0.01 to 99.99%, including all values and subvalues therebetween.
- the polymerizable lipids can be any of the lipids or lipid types shown in Figures 1-6, and mixtures thereof, in any molar desired ratio.
- the above described vesicle fusion, Langmuir-Schaefer, redox-initiated polymerization, and light-driven (UN, visible, or near- infrared) methods may be used to deposit and polymerize the membrane.
- the different types of polymerizable lipids are mixed prior to vesicle preparation. Vesicles are then prepared and fused to appropriate substrates (as described above) to form supported lipid membranes that are subsequently polymerized.
- two types of polymerizable lipid molecules are present in the membrane.
- One type of lipid molecule present in an amount less than 50%, preferably less than 30%, imparts a functional property to the membrane, i.e. it bears a headgroup that is functionally distinct from the headgroups on the other type of lipid in the membrane.
- Examples include functional lipids having anionic or cationic headgroups, having headgroups functionalized with ethylene glycol-based oligomers and polymers, having headgroups designed to chelate metal ions, or having headgroups designed to react with other molecules.
- the second type of lipid molecule in the membrane is selected to be protein resistant, e.g. bis-SorbPC.
- the lipid membrane is composed of a mixture of complementary mono- and bisfunctionalized polymerizable lipids, e.g. mono-SorbPC and bis-SorbPC. Prior to polymerization, such lipids mix homogeneously in a fluid supported lipid membrane. Thus by varying the percentage of each, the density of cross-links in the polymerized bilayer is systematically adjusted. A lower cross-link density generates a more flexible yet still polymeric membrane. As long as the mole fraction of bis-substituted lipid exceeds 0.30 ⁇ 0.05, the polymerized bilayer will be still be cross-linked (Sisson, T. M.; Lamparski, H. G.; Kolchens, S.; Elyadi, A.; O'Brien, D. F., Macromolecules, 1996, 29, 8321).
- the fifth embodiment of the present invention relates to the incorporation of membrane proteins into polymerized, supported lipid membranes. Incorporating protein receptors into a lipid membrane confers a biorecognition function to the membrane. Any membrane-associated protein can be incorporated into a polymerized, supported lipid membrane. In all cases, a preferred surface coverage of receptors is 0.1% to 50% of the coverage equivalent to a one monolayer of receptor. Receptor incorporation in an appropriate manner and orientation that maintains receptor activity can be assayed by the observation of the specific binding to complementary partners.
- Membrane proteins especially transmembrane proteins, require a lipid bilayer environment to preserve their structure and support their specific bioactivity. Reconstitution of transmembrane receptors into fluid, supported lipid membranes has been described (Z. Salamon, S. Cowell, E. Narga, H.I. Yamamura, N. J. Hruby and G. Tollin, Biophys. J., 2000, 79, 2463; J. D. Burgess, M. C. Rhoten and F. M. Hawkridge, Langmuir, 1998, 14, 2467; Heyse, S.; Ernst, O.P.; Dienes, Z.; Hofmann, K.P.; Nogel, H.
- the receptor is solubilized in an aqueous buffer containing a surfactant above its critical micelle concentration (cmc).
- a surfactant above its critical micelle concentration (cmc).
- cmc critical micelle concentration
- removal of the surfactant from the solution causes spontaneous insertion of the receptor into the bilayer, forming proteo-liposomes. Fusion of the proteo-liposomes to a solid support results in formation of a fluid, supported membrane containing receptor molecules (Salafsky, J.; Groves, J.T.; Boxer, S.G. Biochemistry 1996, 35, 14773).
- a fluid lipid bilayer lacks the required physical and chemical stability, such as removal from water. Polymerization of the lipid monomers to create a stabilized membrane, as described above, is a logical solution to this problem.
- CcO cytochrome c oxidase
- d-OR human delta opioid receptor
- the receptor is incorporated into the fluid, supported lipid membrane prior to carrying out polymerization step.
- the receptor is incorporated into the fluid, supported lipid membrane prior to carrying out polymerization step.
- surfactant dialysis followed by vesicle fusion (described briefly above and extensively in the literature; e.g. Salafsky, J.; Groves, J.T.; Boxer, S.G. Biochemistry 1996, 35, 14773), and insertion into a pre-formed supported membrane (Z. Salamon, S. Cowell, E. Narga, H.I. Yamamura, N.J. Hruby and G. Tollin, Biophys. J., 2000, 79, 2463).
- SUNs composed of polymerizable lipids are fused on a support to form a fluid, supported lipid membrane that does not contain protein (as described above).
- Small aliquots of a concentrated solution of the receptor solubilized in a surfactant, e.g. octylglucoside, present above its cmc are added to the aqueous buffer solution in contact with the supported membrane. This dilutes the surfactant to a final concentration below its cmc, which results in spontaneous transfer of the receptor from the surfactant micelles to the supported membrane.
- the present invention is also advantageous for biofunctional presentation of water-soluble protein receptors.
- Attaching water-soluble proteins to the surface of a polymerized, supported lipid membrane confers a biorecognition function to the membrane.
- Any water-soluble protein can be attached to the supported membranes described herein, either before or after polymerization has been effected, but preferably after.
- a preferred surface coverage of receptors is 0.1% to 100% of the coverage equivalent to a one monolayer of receptor. Attachment in an appropriate manner and orientation that maintains receptor activity can be assayed by the observation of the specific binding to complementary partners.
- yeast cytochrome c can be attached to a supported lipid membrane that contains pyridyldithio-conjugated lipids (Edmiston, P. L.; Saavedra, S. S., Biophys. J., 1998, 74, 999).
- a functional group on the protein e.g an amino or a thiol group
- a lipid bearing a reactive headgroup e.g. a maleimido, pyridyldithio, or succinimidyl ester group.
- yeast cytochrome c can be attached to a supported lipid membrane that contains pyridyldithio-conjugated lipids (Edmiston, P. L.; Saavedra, S. S., Biophys. J., 1998, 74, 999).
- horse cytochrome c which is positively charged at neutral pH, can be adsorbed to the surface of a lipid membrane that contains lipids having negatively charged headgroups such as phosphatidic acid and/or phosphatidylserine (Pachence, J.M.; Amador, S.; Maniara, G.; Vanderkooi, J.; Dutton, P.L.; Blasie, J.K., Biophys. J., 1990, 58, 379).
- headgroups such as phosphatidic acid and/or phosphatidylserine
- the above described redox-initiated or light-driven (UV, visible, or near-infrared) methods may then be used to polymerize the membrane.
- a preferred strategy to preserve receptor activity during the polymerization step is pre-incubation of receptors with a solution of their respective ligand or agonist or antagonist at a concentration sufficiently high to saturate the binding sites on the receptors. Occupancy of the binding sites before polymerization provides a degree of steric 'protection' during the subsequent polymerization step.
- the bound ligands can be dissociated from the receptors by standard methods to generate a membrane with unoccupied ligand binding sites.
- the sixth embodiment of the present invention relates to the fabrication of spatially addressable, planar arrays of biomolecules.
- Techniques for such processes are currently being developed in numerous laboratories, based on projected applications for these arrays in rapid screening assays and multianalyte biosensors.
- a method to generate an array of protein molecules adsorbed to a substrate using microcontact printing is disclosed in Bernard, A.; Renault, J.R.; Michel, B.; Bosshard, H.R.; Delamarche, E., Adv. Mater,. 2000, 12, 1067-1070. Boxer and coworkers have pioneered the development of methods to generate micro-patterned fluid lipid bilayers.
- the present invention relates to a) an array of protein molecules deposited on a uniformly polymerized lipid membrane; and b) an array of fluid (or partially polymerized) lipid domains in the membrane, separated by a regular array of domains in which the lipids are highly cross-linked.
- microcontact printing can be used to generate arrays of protein molecules attached to membrane surfaces in a manner designed to maximize specific activity.
- a poly(dimethylsiloxane) (PDMS) stamp is linked with the molecule of interest, which is then transferred to a planar substrate by stamping.
- PDMS poly(dimethylsiloxane)
- Two recent reviews of ⁇ CP and related soft lithography techniques are: (a) Xia, Y.; Whitesides, G.M.; Annu. Rev. Mater. Sci., 1998, 28, 153-184 and (b) Xia, Y.; Rogers, J.A.; Paul, K.E.; Whitesides, G.M., Chem.
- ⁇ CP of proteins has been performed on high energy substrate surfaces (e.g. silica), to which the proteins bind by strong nonspecific interactions (St. John, P.M.; Davis, R.; Cady, N.; Czajka, J.; Batt, C.A.; Craighead, H.G., Anal. Chem., 1998, 70, 1108- 1111; Bernard, A; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H.R.; Biebuyck, H., Langmuir, 1998, 14, 2225-2229; Bernard, A.; Renault, J. R; Michel, B.; Bosshard, H.
- a spatial array of protein molecules is deposited on a uniformly polymerized lipid membrane.
- the present inventors have found that arrays of proteins can be deposited by ⁇ CP on a uniformly polymerized, supported lipid bilayer when the bilayer surface is dried. Upon subsequent immersion into aqueous solution, the printed proteins remain adhered to the printed areas on the bilayer. Furthermore, the printed proteins retain the capability to bind to other dissolved proteins that are subsequently incubated with the patterned surface.
- An example is shown in Figure 18.
- a bis-SorbPC (redox) bilayer was prepared on a SiO 2 substrate as described above and dried under Ar.
- a PDMS stamp was inked with a solution of biotin-BSA (BSA molecules bearing covalently attached biotin groups. Stamping was used to create a pattern of biotin-BSA on the lipid bilayer.
- FIG. 18 An AFM image of the biotin-BSA stripes on the dried lipid bilayer is shown on the left side of Figure 18.
- the printed bilayer was then immersed in a solution of rhodamine-conjugated avidin (schematic on right side of Figure 18).
- the avidin bound to the exposed biotin groups in the regions where biotin-BSA had been printed, but did not adsorb to the non-printed regions, as expected since the bare bis-SorbPC (redox) bilayer is highly protein resistant.
- An epifluorescence micrograph shows the emission pattern of rhodamine-conjugated avidin bound to the lipid bilayer, and confirms that binding occurred only in the regions where biotin-BSA had been printed. At this time, it is not known why proteins adsorb strongly and nonspecifically to a dried bilayer, whereas a hydrated bilayer is protein resistant.
- microcontact printed protein adheres strongly to the printed regions, remains so when the membrane is rehydrated, and retains the capability to specifically bind other ligands, including other proteins. Furthermore, the remaining regions of the membrane retain their characteristic protein resistance.
- This implementation can also be performed on polymerized lipid membranes containing any of the types of lipids shown in Figures 1-6, or mixtures thereof.
- uCP of proteins can be performed on membranes containing functional lipids having anionic or cationic headgroups, headgroups designed to chelate metal ions, or headgroups designed to covalently react with other molecules.
- Examples of the latter category include headgroups terminated with thioethanol, maleimido, pyridyldithio, biotinyl, succinimidyl ester, sulfo succinimidyl ester, alkyl halide, or haloacetamide groups.
- the second type of lipid molecule is usually selected to be protein resistant, e.g. bis-SorbPC.
- the first lipid type is usually selected so that it reacts with the protein molecules that are being printed on the membrane; the objective being to maximize the adherence of the protein to the printed regions on the membrane.
- uCP of a protein onto a polymerized lipid bilayer containing succinimidyl ester-conjugated lipids will result in formation of a covalent bond between these lipids and the lysine groups on the surface of the protein, thereby firmly attaching the protein to the bilayer surface.
- a supported lipid membrane is composed of an array of fluid (or partially polymerized) lipid domains that are separated by a regular array of domains in which the lipids are highly cross-linked.
- Transmembrane proteins can be reconstituted into polymerized bilayers as described above. However, to maintain bioactivity, some transmembrane proteins require a fluid membrane environment. Thus, it may be necessary to preserve a domain of fluid lipids in the immediate vicinity of an incorporated protein, while the remainder of the bilayer is polymerized to generate a stabilized membrane.
- membrane proteins can be reconstituted into microfluid domains within a supported lipid membrane that has undergone patterned polymerization to effect overall stability.
- a patterned polymerized supported lipid bilayer is shown in Figure 19.
- the pattern was obtained using a uCP method developed by Boxer's group ( Hovis, J.S.; Boxer, S.G., Langmuir, 2000, 16, 894-897, Kung, L.A.; Hovis, J.S.; Boxer, S.G., Langmuir, 2000, 16, 6773-6776).
- a PDMS stamp was pressed against and then removed from a fluid bis-SorbPC lipid bilayer on SiO 2 under water; the contacted regions of the bilayer adhered to the stamp and were removed, leaving the underlying glass surface exposed (shown on the right in Figure 19). UN polymerization of the remaining regions of the bilayer then yielded an air-stable structure from which the AFM image shown on the left in Figure 19 was acquired.
- Patterned polymerization is achievable using UN exposure to initiate cross-linking, either through an optical mask or using holography.
- the UN-light may be polarized or unpolarized.
- the unreacted lipids can be dissolved away from the substrate, yielding a pattern of substrate exposed and polymeric bilayer-coated regions. Vesicle fusion can then be used to form fluid bilayer domains between the polymerized regions.
- "incompletely" polymerized domains of lipids can be created between the highly cross-linked domains. Incomplete polymerization can be achieved, for example, using an appropriate molar ratio of a non-polymerizable lipid and mono-SorbPC and/or bis-SorbPC.
- patterned bilayers composed of polymerized and fluid domains can be obtained by uCP printing and UV lithography.
- the seventh embodiment relates to the use in sensors.
- polymerized, supported lipid membranes, with and without associated proteins are used as nonfouling coatings for chemical sensing and biosensing devices.
- biosensing device the characteristic selectivity of biorecognition is exploited in the form of an integrated device that couples a biological binding element, e.g. a protein receptor, to a physical transducer, to perform highly selective analysis of one component (or class of components) in a complex sample matrix
- a biological binding element e.g. a protein receptor
- a physical transducer to perform highly selective analysis of one component (or class of components) in a complex sample matrix
- a biochip is a biosensor that presents a spatially defined array of different recognition elements to a sample, permitting parallel analysis of multiple analytes in a single sample (Vo-Dinh, T.; Cullum, B.M.; Stokes, D.L., Sensors and Actuators B, 2001, 74, 2-11).
- Supported lipid membranes are useful as transducer coatings for biosensing devices because: a) they preserve the bioactivity of incorporated and/or attached proteins (e.g. P. L. Edmiston and S. S. Saavedra, Biophys. J., 1998, 74, 999-1006; P. L.
- Polymerized, supported lipid membranes can be used in many types of biosensing devices, including devices based on electrochemical, spectro-electrochemical, or optical (absorbance, luminescence, reflectivity, or scattering) transduction methods.
- a polymerized, supported lipid membrane containing receptors either water-soluble or membrane-associated receptor proteins or nucleic acids, is present between the physical transducer and the sample solution.
- the sample solution contains the analyte of interest. Binding of the analyte molecules to the membrane-incorporated receptors is detected at the transducer using, for example, electrochemical, spectro-electrochemical, or optical (absorbance, luminescence, reflectivity, or scattering) methods.
- the protein resistant properties of the lipid membrane prevent binding of other molecules present in the sample matrix, especially other proteins.
- a supported, lipid membrane that contains transmembrane protein receptors is deposited on the transducer surface and polymerized using the preparation methods described above. Binding of ligands to receptors, where the ligands are also analytes, is detected optically as a change in absorbance, luminescence, reflectivity, or scattering at the transducer surface. More preferably the binding is detected using fluorescence methods or surface plasmon resonance methods.
- a self-assembled, supported lipid bilayer formed from the types of lipids shown in Figures 1-6 can be stabilized to surfactants, organic solvents, and transfer across the water/air interface by cross-linking polymerization of moieties in the acyl chains.
- the self- assembled, supported lipid membrane of the present invention can be utilized as a protein- resistant coating for molecular devices.
- the stabilized lipid membrane of the present invention are suitable as a non-fouling coating for medical implant materials or analytical fluid handling instruments or biomedical devices requiring a non-fouling coating.
- they find application as a cell-membrane mimetic for supporting surface-associated and transmembrane proteins in their native state in various biological detection devices (e.g. biosensors).
- the stabilized phospholipid bilayers of the present invention can also be used as a general non-fouling coating for mass produced commercial items, for example razor blades.
- Bis-sorbyl phosphatidylcholine (bis-SorbPC) was prepared by a modification of the procedure reported by Lamparski, H.; Liman, U.; Frankel, D. A.; Barry, J. A.; Ramaswami, V.; Brown, M. F.; O'Brien, D. F., Biochemistry, 1992, 31, 685-694.
- the synthesis of bis- dienoyl phosphatidylcholine (bis-DenPC) was adapted from that reported by Dorn, K.; Klingbiel, R. T.; Specht, D. P.; Tyminski, P. N.; Ringsdorf, H.; O'Brien, D. F., J. Am. Chem.
- Potassium persulfate and sodium bisulfate were purchased from Aldrich and used as received.
- Bovine serum albumin labeled with fluorescein (FITC-BSA, labeling ratio of 11.2:1) and tetramethylrhodamine (TMR-BSA, ratio of 1:0.9) were obtained from Sigma and used without any further purification.
- Fluorescein labeled dextran (10,000 MW, 2.9: 1 labeling ratio) and rhodamine labeled dextran were purchased from Molecular Probes. All other chemicals and solvents were purchased from standard commercial suppliers and used without further purification.
- Single crystal (111) silicon wafers having a 20 ⁇ 5 A thick native oxide layer were purchased from Wacker.
- Fused silica slides were purchased from Dynasil Corp.
- Deionized water (18 MOhms and made organic free ( ⁇ 10 ppb)) was obtained from a Barnstead Nanopure water system.
- Substrate preparation Si wafers and fused silica slides were soaked for 30 minutes in pirhana solution (70% H 2 SO 4 /30% H 2 O 2 ), followed by extensive rinsing and sonication in deionized water. Unless otherwise noted, substrates were stored in deionized water until used, within 1 hour of cleansing.
- Clean Si substrates were dried by ⁇ 2 immediately prior to fusion.
- a few drops of lipid vesicle solution (SUVs) were deposited on the Si substrate (or fused silica). Lipids were fused at a temperature equal to or greater than their respective main phase transition temperature for at least ten minutes.
- the surfaces were then either transferred to test tubes for redox polymerization, or to shallow crystallization dishes to be polymerized by direct UN irradiation. Care was taken to not expose the unpolymerized films to air, or excessive mechanical shocks.
- the first layer of the bilayer was deposited vertically. Langmuir lipid monolayers were spread on a Nima Model 61 ID Langmuir-Blodgett trough using benzene as the spreading solvent and deionized water as the subphase. Film depositions were performed at a surface pressure of 35-40 rriN/m, corresponding to approximately 6 ⁇ A 2 /molecule. The inner leaflet of the bilayer was deposited by withdrawing the substrate from the subphase at a rate of 10 mm/min. Transfer ratios of approximately 98.5% were repeatedly obtained.
- the second leaflet of the bilayer structure was deposited using the Langmuir- Schaefer horizontal transfer technique.
- the substrate with the previously deposited lipid monolayer was passed horizontally through the air- water interface at constant pressure (35- 40 mN/m). After formation, the unpolymerized bilayer was maintained in an aqueous environment at all times. All depositions were carried out at 25 °C.
- Redox polymerization Redox initiated, radical polymerization was performed with deoxygenated solutions of potassium persulfate and sodium bisulfate. The concentration ratio was 100 mM K 2 S 2 O 8 /10 mM NaHSO 3 . Polymerizations were also performed at other K 2 S 2 O 8 / NaHSO 3 concentrations, ranging from 0.001 to 1.0 M. After deposition by vesicle fusion or Langmuir-Blodgett-Schaefer techniques, the supported lipid bilayer is transferred to the Ar-saturated polymerization solution without exposing the bilayer to air, incubated for two hours under flowing Ar, then rinsed extensively with deionized water, and dried under a stream of N 2 .
- a two hour incubation period was determined to be sufficient to achieve near quantitative polymerization of bis-SorbPC bilayers, based on the near quantitative disappearance of the monomer absorbance band at 260 nm during the incubation period.
- the disappearance of the band was monitored by UN transmission spectroscopy performed (as described in Example 4) on 4 bis-SorbPC bilayers prepared by vesicle fusion as described above.
- UN polymerization UN-induced polymerization of supported lipid films was performed by exposure to UN radiation from a low-pressure mercury pen lamp (Fisher Scientific) with a rated intensity of 4500 mW/cm 2 at 254 nm. A 1.0 mm thick UN band pass filter from Scott Glass (UG5) was used to remove the short wavelength UN ( ⁇ 230 nm) that can fragment polymer chains into oligomers. In cases where oxygen was to be excluded, the solution in contact with the lipid film was deoxygenated with flowing Ar for at least 30 minutes prior to and throughout the polymerization.
- a low-pressure mercury pen lamp (Fisher Scientific) with a rated intensity of 4500 mW/cm 2 at 254 nm.
- a 1.0 mm thick UN band pass filter from Scott Glass (UG5) was used to remove the short wavelength UN ( ⁇ 230 nm) that can fragment polymer chains into oligomers.
- the solution in contact with the lipid film was deoxygenated with flowing Ar for at least 30 minutes prior to and
- Ellipsometry The thickness of dried lipid films deposited on Si substrates was determined by ellipsometry. Measurements were made with a Rudolph Research model 43603-200E ellipsometer using a 632.8 nm He-Ne laser at an incident angle of 70 degrees. Initial readings were taken on the bare Si substrates to establish the substrate optical constants and oxide layer thickness prior to any film formation. A refractive index of 1.46 was assumed for all lipid layers. The ellipsometry data were used to calculate the corresponding thickness values using DaflBM version 2.0, a computer program supplied by Rudolph Research and implemented on a DOS-based PC system.
- Contact angle measurements Contact angles of deionized water deposited on supported lipid films were measured using the sessile drop method. In some case, images of multiple 3 mL water droplets on each surface were taken using a Pulnex TM-7CN video camera and Video Snapshot Snappy and were the average of at least three samples. Images were converted into tagged image format using corresponding software, and angles were measured using Image-Pro Plus 1.3 software (Media Cybernetics). In other cases, water droplets on surfaces were photographed using a TE-cooled CCD camera (Princeton Instruments Model 512TK) and the contact angle retrieved via imaging analysis software (Scion Image). Both methods gave equivalent results.
- Atomic force microscopy The surface morphology of supported lipid films was examined by atomic force microscopy (AFM), performed in tapping mode on a Digital Instruments Multimode III microscope. Oxide sharpened silicon nitride tips (TESP-7) were purchased from Digital Instruments, and were tuned to between 300 and 400kHz. For water immersion studies, measurements were performed in a fluid cell (Digital Instruments) in tapping mode with contact tips tuned to 33kHz as per supplemental Digital Instrument instructions. Samples were immersed in deionized water for 0.5-1.5 hours before image acquisition commenced. Images were acquired at several areas on each substrate, and images presented in this document are representative of scans from different locations on each sample, different samples, and with different tips used to image the surfaces.
- the optical arrangement ( Figure 20) consists of two right-angle quartz prisms mounted in a TIRF flow cell.
- One prism is used to couple the excitation light from an Ar-ion laser into the cell; the light then propagates by total internal reflection down the fused silica slide.
- the other prism is used to outcouple the excitation light, thereby reducing scattered light in the cell volume.
- Index matching fluid (1.463 n d , Cargille) was used to allow for efficient incoupling and outcoupling of the incident laser light.
- the flow cell was mounted on a Nikon Diaphot inverted microscope. Excitation wavelengths were 488 nm (for measuring fluorescein emission) or 514 nm (for measuring rhodamine emission). Fluorescence emission was back-collected through the quartz slide with a 4X or 10X objective, optically filtered, and detected with a photomultiplier tube. The incident excitation light was modulated at a frequency of 2.5kHz. Phase sensitive detection was used to retrieve the fluorescence intensity. The experiment was interfaced to a PC for
- XPS X-ray photoelectron spectroscopy
- Integration times were 0.25 s, co-added four times, for a total of 1.0 s at an interval of 0.1 eN.
- the areas under the XPS peaks were measured by numerical integration after baseline correction.
- Relative peak area ratios were calculated using previously published photoionization cross-sections (Schofield, J. J. Electron Spectrosc. Relat. Phenom. 1976, 8, 129-137) after accounting for the transmission properties of the analyzer.
- lipid bilayer films composed of bis-SorbPC were self-assembled by vesicle fusion and polymerized by redox initiation as described above. Assuming an index of refraction of 1.46 for the lipid film, the ellipsometric thickness of the dried, polymerized bis- SorbPC bilayer was found to be 46 ⁇ 3 A.
- X-ray reflectometry was used to measure the electron density of a dried, polymerized bis-SorbPC bilayer supported on a quartz substrate along the axis normal to the bilayer plane. X-ray reflectivity measurements (kindly perfomed at the National Institute for Standards and Technology by Dr. Jarek Majewski of Los Alamos National Laboratory) yielded a thickness of 45 ⁇ 1.4 A.
- the contact angle of a sessile water drop on a polymerized bis-SorbPC bilayer was 31 ⁇ 4 degrees, consistent with a surface composed of outward facing phosphorylcholine headgroups.
- the water contact angle measurements on a freshly cleaned Si wafer and on a Langmuir-Blodgett transferred monolayer of bis-SorbPC were ⁇ 5 and 63 ⁇ 5 degrees, respectively.
- the contact angle for the bis-SorbPC monolayer is lower than that expected for a surface composed of saturated alkyl chains, and reflects the presence of ester groups near the chain termini.
- Evidence for extensive cross-linking in polymerized, supported bis-SorbPC bilayers is given by the insolubility of these structures in surfactant solution.
- the ellipsometric thickness did not change upon bath sonication in a 1% solution of Triton X-100 for ten minutes or immersion in chloroform or acetone for 10 seconds (both conditions at room temperature), which suggests that the polymer size in these films is sufficiently large to render them insoluble.
- the image in Figure 8 acquired using tapping mode atomic force microscopy (AFM) in air, shows that the surface of a polymerized, supported bis-SorbPC bilayer is very smooth.
- the rms of the image in Figure 8 (left) is 1.25 A, which is comparable to the bare silicon substrate (rms roughness of 1.1 to 1.3 A).
- the bilayer surface morphology was surprisingly uniform; the image shown in Figure 8 is representative of images acquired at numerous locations over a ca. 1 cm 2 sample area. No topographical features greater than 1 mn in height (peak-to-peak) were detected. Thus any defects at which bare substrate was exposed were too narrow to be detected by AFM.
- Supported bilayers composed of a mixture of bis-SorbPC and the non-polymerizable lipid DOPC at a molar ratio of 7 to 3 were also prepared by vesicle fusion and polymerized by redox initiation as described above. These films are observed to contain numerous defects as revealed by AFM ( Figure 14). This result shows that polymerized bilayer films that contain appreciable amounts of unpolymerized lipids are not stable to removal from water.
- Supported bis-SorbPC bilayers self-assembled by vesicle fusion as described above, were also polymerized by direct UV irradiation, as described above, and characterized by ellipsometry, AFM, and contact angle measurements, etc.
- the UV polymerized bis-SorbPC films were thinner and less hydrophilic. Specifically, the film thickness was approximately 29 A and the water contact angle was 52 degrees.
- supported lipid bilayers were also prepared using a commercially available, polymerizable diacetylenic PC lipid (l,2-bis(10,12-tricosadionyl)sn- glycero-3-phosphocholine (DAPC); Avanti Polar Lipids).
- DAPC diacetylenic PC lipid
- Supported bilayers of DAPC were prepared by Langmuir-Blodgett-Schaefer deposition, as described above, and photopolymerized using UN light (procedures for this lipid are described in detail in Morigaki, K; Baumgart, T.; Offenhausser, A.; Knoll, W., Angew. Chem., Int. Ed., 2001, 40, 172).
- Supported bilayers composed of mono-SorbPC were also self-assembled by vesicle fusion and polymerized by redox initiation as described above.
- the quality of the resulting films was generally poorer that the corresponding bis-SorbPC films.
- the ellipsometric thickness was measured to be 31 A, and the AFM images (e.g. Figure 15) revealed domainlike features similar to those observed for UV polymerized bis-SorbPC films. This result is consistent with the observation in vesicle studies that a cross-linked lipid polymer is more stable to solvent and surfactant dissolution than a linearly polymerized lipid polymer.
- the redox polymerization of supported bis-DenPC lipid bilayers self-assembled by vesicle fusion also produced relatively thick films.
- the ellipsometric thickness measured after drying the film was 52 A; however upon bath sonication in the surfactant Triton-X-100, a significant decrease in film thickness was observed.
- AFM images of the film after sonication in surfactant reveal the surface to contain defects located uniformly throughout the film (e.g. Figure 16). Linescans across the defects indicate that they do not reach the substrate (depth less than 3 nm); this is consistent with lipid loss from only the outer leaflet of bilayer.
- Redox polymerized and UN polymerized bis-SorbPC bilayers were self-assembled by vesicle fusion on fused silica substrates according to Example 1, rinsed and dried under nitrogen, mounted in the TIRF flow cell ( Figure 20), and rehydrated.
- POPC bilayers were fused to silica substrates that were preassembled in the cell, to avoid exposure of the fluid bilayer to air.
- AA arachidic acid
- TMR-BSA bovine serum albumin labeled with tetramethylrhodamine isothiocyanate
- TIRF emission from the adsorbed protein film was measured.
- Relative TMR-BSA surface coverages were determined using the calibration procedures described above (Hlady, N.; Reinecke, D. R; Andrade, J. D. J. Colloid Interface Sci. 1986, 111, 555-569; Conboy, J. C; McReynolds, K. D.; Gervay-Hague, J.; Saavedra, S. S., J. Amer. Chem. Soc, 2002; 124, 968-977); the calibration solutions had known concentrations of dissolved (i.e. non- adsorbed) TMR-BSA.
- the bar graph in Figure 9 shows the relative TMR-BSA adsorption to all surfaces listed above.
- the BSA surface coverages on the redox polymerized bis-SorbPC and fluid POPC bilayers were 6 ⁇ 3% and 6 ⁇ 6%, respectively, of that obtained on the hydrophobic AA monolayer (100 ⁇ 24%; estimated to be ca. one monolayer).
- TMR-BSA adsorbed to the bis-SorbPC (redox) bilayer could be removed by flushing the cell with a 1% Triton X-100 solution.
- No increase in the amount of adsorbed TMR-BSA was observed when the surface was re- exposed to 1 mg/ml TMR-BSA, which demonstrates the stability of the polymeric bis- SorbPC bilayer to surfactant solutions.
- the relative protein adsorption on the DAPC bilayer (40%) was slightly less than the 41% measured on clean fused silica (which is labeled as quartz in Figure 9).
- the relative adsorption on UN photopolymerized bis-SorbPC bilayers was 24%, intermediate between bis-SorbPC (redox) and DAPC.
- TIRF isotherms for TMR-BSA adsorption to several of these surfaces were measured over a protein concentration range of 5.0 x 10 "9 M to 1.5 x 10 " ⁇ M and are plotted in Figure 10.
- the raw data were calibrated as described above, allowing the magnitude of the normalized fluorescence intensities plotted in Figure 10 to be directly compared.
- the shape of the adsorption isotherms and relative measured intensities show that the BSA interacts most strongly with the hydrophobic AA monolayer surface.
- Relative protein adsorption to the bis-SorbPC (redox) and POPC bilayers is very similar.
- AFM images and line scans of a silicon wafer and a wafer coated with a bis-SorbPC (redox) bilayer are shown in Figure 11.
- the surfaces were imaged both before and after incubation in a 1 ml/mg BSA solution (conditions given above). Consistent with the TIRF data, a significant increase in measured roughness occurs on the SiO 2 surface, which is due to the considerable protein adsorption that occurs on clean SiO 2 . In contrast, a negligible change is observed for the bis-SorbPC (redox) bilayer, consistent with its demonstrated protein resistance.
- Example 4 bis-SorbPC and bis-DenPC Bilayers formed by Langmuir-Blodgett techniques and UN Polymerized.
- Preparation of supported lipid films Substrates (either Si wafers or fused silica slides) were first sonicated in 50% isopropyl alcohol/ 50%) water (v/v), rinsed in deionized water, and then cleaned in piranha solution as described above. The cleaned substrates were then sonicated in a 0.1 M solution of A1C1 3 for 30 minutes, rinsed repeatedly with deionized water, sonicated for 15 minutes in deionized water, and then rinsed again. This procedure resulted in hydrophilic substrates having with a sessile water contact angle of 10 ⁇ 3.5 degrees. Planar supported lipid bilayers (PSLBs) were deposited on substrates using Langmuir-Blodgett-Schaefer techniques and maintained under water until after polymerization was performed..
- PSLBs Planar supported lipid bilayers
- UV Polymerization The low-pressure mercury pen lamp was held 7.5 cm from the PSLB-coated substrate and illuminated for 4 minutes. The water solution contacting the PSLB was purged with Ar for 30 minutes prior to polymerization. After UN exposure, the PSLB was removed from solution, rinsed several times with deionized water and dried with a stream of nitrogen.
- the decay of the integrated monomer absorbance occurs at a rate of 18.9 ⁇ 0.96 per second.
- Irradiation of bilayer bis-SorbPC films for times greater than 2 minutes was found not to alter the film structure or morphology as observed by AFM and ellipsometry (described below). However, irradiation times below 2 minutes result in substantially reduced degrees of polymerization.
- Static water contact angle and ellipsometry measurements made on bilayers of UN polymerized bis-SorbPC are listed in Table 1. Also tabulated for comparison are the contact angle and ellipsometric thickness of a bis-SorbPC monolayer polymerized under the same conditions as the bis-SorbPC bilayers as well as a bis-DenPC bilayer. The measured thickness of 48.4 A for bis-SorbPC is consistent with a fully extended lipid bilayer structure. A static water contact angle of 41.973.1 degrees is indicative of a hydrophilicity intermediate between SiO 2 (about 10 degrees) and bis-SorbPC monolayer (60.4 degrees), which has a "tail group out" orientation.
- AFM was used to characterize the morphology of polymerized bis-SorbPC bilayers.
- AFM images of a dehydrated and hydrated (i.e. immersed in deionized water) polymerized bis-SorbPC bilayer are displayed in Figure 22a and Figure 22b respectively.
- Surprisingly different morphologies are seen for the water-immersed surface versus the same film in air.
- the surface of the bilayer appears as a uniformly coated surface with small irregularly shaped circular domains roughly 10-50 A in diameter, ranging in height from 5- 10 A. Larger voids are also apparent on the surface, 60715 nm in diameter with depths ranging from 15-25 nm.
- the rms roughness for the dehydrated surface, Figure 22a is 5.2 ⁇ 1.4 A.
- the roughness of the underlining silicon substrate was measured as 2.1 ⁇ 1.6 A.
- the topographical depth determined by AFM is 48-52 A, which is comparable to the thickness determined by ellipsometry.
- the surface morphology changes considerably, as shown in Figure 22b.
- the previously "cracked” surface becomes much more uniform and the calculated surface roughness declines to 3.5 ⁇ 0.8 A.
- the large voids, which were present in the dried sample are still apparent although the mean size decreases to roughly 40 nm in diameter with the void depth remaining constant at 20 ⁇ 5 nm.
- Analysis of the hydrated AFM image shows that approximately 36 ⁇ 8% of the surface area corresponds to large and smaller voids within film which extend to a depth of 15-20 A.
- UV polymerized lipid bilayers were prepared as described, dried, and then rehydrated after mounting in the TIRF flow cell.
- POPC bilayers were deposited on fused silica slides using Langmuir-Blodgett-Schaefer techniques and mounted in the TIRF flow cell without exposure to air. Measurements were also made on a hydrophobic reference surface, which was a Langmuir-Blodgett deposited, "tail group out" POPC monolayer. Representative adsorption isotherms are plotted in Figure 23. The binding affinities were extracted from the adsorption isotherms using a Langmuir model and are summarized in Table 2. The surface coverage data were normalized by assuming that protein adsorption was minimal on POPC bilayers and that monolayer coverage occurred on POPC monolayers.
- Both the binding affinity and surface coverage data show that the protein resistance of a UN polymerized bis-Sorb Bilayer falls in between that of a POPC monolayer and a POPC bilayer.
- the TIRF adsorption isotherm for a POPC bilayer and a re-hydrated bis- SorbPC bilayer are similar in shape, with an increase in protein adsorption observed for the polymer bilayer, as indicated by an increase in total fluorescence and an increase in the binding affinity. Both effects are attributed to the non-uniformity of the polymer films which have exposed hydrophobic domains as seen in the AFM images.
- a more quantitative examination of protein adsorption reveals a direct correlation between exposed hydrophobic domains on the polymer surface and the amount of adsorbed BSA.
- the relative percent surface concentrations of adsorbed BSA were determined by using the fluorescent intensity, F max , obtained by the nonlinear least squares fit to the adsorption data for each case.
- F max fluorescent intensity
- this example shows that air-stable, supported lipid bilayers can be formed by Langmuir-Blodgett-Schaefer deposition and UN-induced polymerization.
- the performance of these films is better than that of films formed from commercially available polymerizable lipids (i.e. DAPC) but is less optimal as compared to bis-SorbPC (redox) bilayers formed by vesicle fusion (see example 1).
- transmembrane protein activity can be supported in the polymerized bis-SorbPC films.
- Two transmembrane proteins, cytochrome c oxidase (CcO), and human delta opiod receptor (d-OR) were used in these experiments.
- CcO was isolated from fresh beef hearts and purified according to published procedures (T. Soulimane and G. Buse, Eur. J. Biochem., 227(1995) 588-595).
- d-OR was expressed, isolated, and purified from a transfected cell line, also according to published procedures (Salamon, S. Cowell, E. Narga, H.I. Yamamura, N.J. Hruby and G. Tollin, Biophys. J., 2000, 79, 2463).
- Detergent dialysis (described above) was used to insert each of these proteins into bilayer vesicles, forming proteo-vesicles, following standard procedures (Mimms, L. T.; Zampighi, G.; ⁇ ozaki, Y.; Tanford, C; Reynolds, J. A. Biochemistry 1981, 20, 833-840).
- the surfactant used was octyl glucoside.
- the surfactant concentration was initially 40 mM, well above the reported cmc of about 20 mM, and the lipid to protein ratio was 1000:1.
- Proteo-vesicles were formed using either pure bis-SorbPC or pure DOPC, and then fused to silica substrates to form planar supported proteo-lipid bilayers.
- CcO binds cytochrome c (Cyt c) in low ionic strength solutions; raising the ionic strength dissociates the complex.
- Cyt c nonspecifically adsorbs to lipid bilayers to some extent, this can be distinguished from specific binding to membrane-bound CcO by the difference in ionic strength dependence (i.e. rinsing with a high ionic strength buffer solution dissociates specifically bound Cyt c).
- the surface coverage of the specifically bound TMR-Cyt c was determined to be 9.8 ⁇ 4.5 x 10 "14 mol/cm 2 .
- a comparable value, 8.1 ⁇ 3.8 x 10 "14 mol/cm 2 was measured for CcO- functionalized fluid DOPC bilayers. Both of these surface coverages are within reasonable range of the theoretically calculated CcO surface coverage of 5.5 x 10 "13 mol/cm 2 , assuming a 1000 to 1 lipid to protein ratio in the film.
- d-OR selectively binds many opioid peptides, among them the ligand enkephalin analogue [D-Pen2, D-Pen2] enkephalin (DPDPE) (Mosberg, H. I.; Hurst, R.; Hruby, V. J.; Gee, K.; Yamamura, H.I.; Galligan, J.J.; Burks, T.F. Proc. Natl. Acad. Sci. U. S.A.
- d-OR was incorporated into fluid DOPC and polymerized bis-SorbPC lipid films and assayed for binding activity using a fluorescently labeled ligand, TMR-DPDPE. After the labeled ligand was incubated with each type of d-OR functionalized lipid film, the film was rinsed. Competitive desorption was effected by subsequent incubation with unlabeled ligand, DPDPE, and revealed the fraction of the TMR-DPDPE that was specifically associated with each proteo-lipid film.
- results are presented for two types of patterned arrays created by microcontact printing ( ⁇ CP): (a) Protein films are patterned on polymerized bis-SorbPC bilayers. (b) Patterned regions of polymerized bis-SorbPC are created by selective removal of portions of the fluid bilayer prior to the polymerization step.
- Poly(dimethylsiloxane) (PDMS) stamps were made by curing Sylgard 184 (Dow Corning) on a silicon master with line features (i.e. stripes) approximately 10 microns wide separated by 15 micron wide spaces. The PDMS stamp was then removed from the master, rinsed in deionized water.
- Sylgard 184 Dow Corning
- the stamp was immersed in an aqueous solution of 0.05 mg/ml BSA in 50 mM, pH 7.4, phosphate buffer for 30 minutes.
- the protein-coated stamp was rinsed with buffer and water, and then placed upon a dried, bis-SorbPC (redox) bilayer supported on a Si wafer.
- Light pressure 50-100 g over a 1cm 2 area was applied to the stamp; then it was removed after 20 seconds.
- the bilayer was then rinsed with deionized water, dried, and imaged by atomic force microscopy.
- Figure 18 shows an AFM image of the pattern of protein "stripes" that was transferred to the bilayer surface from the stamp.
- a ⁇ CP printing technique developed to create patterns in hydrated, fluid lipid bilayers (Hovis, J. S.; Boxer, S. B., Langmuir, 2000, 16(3), 894-897; Hovis, J. S.; Boxer, S. B., Langmuir, 2001, 17(11), 3400-3405) was adapted to create polymerized lipid bilayer patterns.
- a schematic of the process is shown in Figure 19 (right).
- a fluid bilayer of bis-SorbPC was formed by vesicle fusion on a clean Si wafer according to the procedures described above.
- the PDMS stamp was made to briefly (5 seconds) contact the bilayer while both are immersed in water.
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WO2005060330A2 (en) * | 2003-12-22 | 2005-07-07 | Statens Serum Institut | Freeze-dried vaccine adjuvant |
EP1732621B1 (en) * | 2004-03-22 | 2009-12-09 | Abbott Cardiovascular Systems, Inc. | Phospholipid and non-fouling coating compositions |
US8465758B2 (en) | 2001-01-11 | 2013-06-18 | Abbott Laboratories | Drug delivery from stents |
WO2015041608A1 (en) * | 2013-09-19 | 2015-03-26 | Nanyang Technological University | Methods for controlling assembly of lipids on a solid support |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4560599A (en) * | 1984-02-13 | 1985-12-24 | Marquette University | Assembling multilayers of polymerizable surfactant on a surface of a solid material |
US5260004A (en) * | 1991-12-02 | 1993-11-09 | The United States Of America As Represented By The Secretary Of The Army | Process of making Langmuir-Blodgett films having photo-electronic properties |
US5427915A (en) * | 1989-06-15 | 1995-06-27 | Biocircuits Corporation | Multi-optical detection system |
US5656211A (en) * | 1989-12-22 | 1997-08-12 | Imarx Pharmaceutical Corp. | Apparatus and method for making gas-filled vesicles of optimal size |
US6019998A (en) * | 1993-05-18 | 2000-02-01 | Canon Kabushiki Kaisha | Membrane structure |
-
2002
- 2002-03-11 WO PCT/US2002/007369 patent/WO2002071944A1/en not_active Application Discontinuation
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4560599A (en) * | 1984-02-13 | 1985-12-24 | Marquette University | Assembling multilayers of polymerizable surfactant on a surface of a solid material |
US5427915A (en) * | 1989-06-15 | 1995-06-27 | Biocircuits Corporation | Multi-optical detection system |
US5656211A (en) * | 1989-12-22 | 1997-08-12 | Imarx Pharmaceutical Corp. | Apparatus and method for making gas-filled vesicles of optimal size |
US5260004A (en) * | 1991-12-02 | 1993-11-09 | The United States Of America As Represented By The Secretary Of The Army | Process of making Langmuir-Blodgett films having photo-electronic properties |
US6019998A (en) * | 1993-05-18 | 2000-02-01 | Canon Kabushiki Kaisha | Membrane structure |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8465758B2 (en) | 2001-01-11 | 2013-06-18 | Abbott Laboratories | Drug delivery from stents |
WO2005060330A2 (en) * | 2003-12-22 | 2005-07-07 | Statens Serum Institut | Freeze-dried vaccine adjuvant |
WO2005060330A3 (en) * | 2003-12-22 | 2005-08-04 | Statens Seruminstitut | Freeze-dried vaccine adjuvant |
EP1732621B1 (en) * | 2004-03-22 | 2009-12-09 | Abbott Cardiovascular Systems, Inc. | Phospholipid and non-fouling coating compositions |
US9468706B2 (en) | 2004-03-22 | 2016-10-18 | Abbott Cardiovascular Systems Inc. | Phosphoryl choline coating compositions |
WO2015041608A1 (en) * | 2013-09-19 | 2015-03-26 | Nanyang Technological University | Methods for controlling assembly of lipids on a solid support |
US10427124B2 (en) | 2013-09-19 | 2019-10-01 | Nanyang Technological University | Methods for controlling assembly of lipids on a solid support |
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