WO2005069004A2 - Membranes a support solide disposees a l'interieur de substrats poreux, et leur utilisation dans des capteurs biologiques - Google Patents

Membranes a support solide disposees a l'interieur de substrats poreux, et leur utilisation dans des capteurs biologiques Download PDF

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WO2005069004A2
WO2005069004A2 PCT/US2005/000069 US2005000069W WO2005069004A2 WO 2005069004 A2 WO2005069004 A2 WO 2005069004A2 US 2005000069 W US2005000069 W US 2005000069W WO 2005069004 A2 WO2005069004 A2 WO 2005069004A2
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lipid
receptor
membrane
protein
molecule
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PCT/US2005/000069
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WO2005069004A3 (fr
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Klaus Gawrisch
Holly C. Gaede
Keith M. Luckett
Ivan V. Polozov
Alexei Yeliseev
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The United States Of America As Represented By The Secretary Department Of Health And Human Services
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

  • This invention relates to reagents and methods for forming membranes that may be deposited into porous solid supports for use in biosensors.
  • Immobilized Membranes and Biosensors Immobilized membranes may be employed in a wide range of applications, including enabling biofunctionalization of inorganic solids (semiconductors, gold- covered surfaces, and optoelectronic devices) and polymeric materials; providing a natural, non-denaturing, and defined environment for the immobilization of biomolecules; and allowing the preparation of ultrathin, high-electric-resistance layers on conductors and the incorporation of receptors into insulating layers for the design of biosensors based on electrical and optical detection of ligand binding (Ho, W.S.
  • Biosensors are analytical devices that are used to measure the presence and/or concentration of desired biological molecules in a sample.
  • biomolecules such as antigens, hormone receptors, enzymes, cytokine receptors, antibodies, etc.
  • biosensors can be analyzed using biosensors (see, e.g., Keusgen M. (2002) “BIOSENSORS: NEW APPROACHES IN DRUG DISCOVERY,” Naturwissenschaften. 89(10):433-44. Epub 2002 Sep 11; Albers, J. et al. (2003) "ELECTRICAL BIOCHIP TECHNOLOGY ⁇ A TOOL FOR MICROARRAYS AND
  • Biosensors function by generating a detectable physical signal from the sensor's physical transducer component in response to the binding of a target biological molecule to the sensor's biological component.
  • the physical transducer component is typically an optical or electrical signal, e.g. a fluorescence signal elicited by the ligand binding event, a modulation of a current in response to binding, a pH change in response to binding, a modulation of electrical resistance in response to binding, etc.
  • Care must be taken to ensure that the target biological molecule of the biological component of the biosensor is immobilized, and that the immobilization procedure forms a stable layer of biomolecules.
  • the immobilization procedure must also not undesirably diminish the activity or structure of the target biological molecule of the biological component, or change its substrate reactivity (i.e., perturbations of the biological component is optimally minimized).
  • Integrated bilayers are characterized by an inner monolayer that is either covalently or ionically bonded to the support surface.
  • Freely supported bilayers are characterized as having an inner monolayer that is separated from the support by an ultrathin water layer ( ⁇ 10 A).
  • the third type of supported membrane consists of a bilayer membrane that rests on an ultrathin polymer film (see, Sackmann, E. (1996) "SUPPORTED MEMBRANES: SCIENTIFIC AND PRACTICAL APPLICATIONS,” Science 271 :43-48).
  • Such supported membranes can be associated with silicon microchips, or with beads (as in a column, etc.).
  • Immobilization of the biological molecule can be achieved by adsorbing the biological molecule onto the surface of a solid support, integrating the molecule within a gel or other matrix, or covalently coupling it to a solid support.
  • Some approaches achieve immobilization by adsorbing the biological molecule onto the surface of a solid support, integrating the molecule within a gel or other matrix, or covalently coupling it to a solid support.
  • Other approaches interpose a polymer cushion between the lipid bilayer and the polymer support. Supported lipid-protein bilayers separated from the solid surface by nanometer-thick water layers or ultrathin soft polymer cushions maintain the thermodynamic and structural properties of free bilayers.
  • Flat membrane supports have the advantage of being able to physically separate membranes (and thus provide protection against their disruption). They suffer from the disadvantage of low surface area. Beaded surfaces provide much greater surface area than flat membranes, however, the membranes can touch one another and are not protected. Porous supports provide very large surface area, but their application has suffered from issues related to uniformity and homogeneity of membrane preparation as well as from limited membrane accessibility for ligands that are delivered via the water phase.
  • AAO filters have a 60 ⁇ m thick support layer with pore diameters of 0.2 ⁇ m that is capped by a thin, ⁇ 1 ⁇ m layer with nominal pore diameters of 0.02, 0.1, or 0.2 ⁇ m.
  • AAO filters have a variety of other applications, including support for cell cultures and microscopy, sample preparation for HPLC, IC, and electrophoresis, and liposome extrusion.
  • Lipids have previously been added to AAO as monolayers to change liquid crystal director orientation (Crawford, G.P. et al. (1991) "SURFACE-INDUCED ORIENTATIONAL ORDER IN THE ISOTROPIC PHASE OF A LIQUID-CRYSTAL MATERIAL,” Phys.Rev.A 44:2558-2569), as pore-spanning bilayers (Hennesthal, C. et al. (2000) “PORE-SPANNING LIPID BILAYERS VISUALIZED BY SCANNING FORCE MICROSCOPY,” J.Am.Chem.Soc. 122:8085-8086), as the upper leaflet of hybrid bilayers (Marchal, D. et al.
  • Lipid monolayers have been used to make inner pore surfaces hydrophobic so that they may be used for thermotropic liquid crystal display applications (Marchal, D. et al. (1998) "ELECTROCHEMICAL MEASUREMENT OF LATERAL DIFFUSION COEFFICIENTS OF UBIQUINONES AND PLASTOQUINONES OF VARIOUS ISOPRENOID CHAIN LENGTHS INCORPORATED IN MODEL BILAYERS," Biophys.J. 74: 1937-1948). Multilamellar lipid bilayers have been deposited in AnoporeTM filters by the diffusion of small liposomes (Smirnov, A.I. et al. (2003) “SUBSTRATE-SUPPORTED LIPID NANOTUBE ARRAYS,” JAm.Chem.Soc. 125:8434-8435).
  • G-Protein Coupled Receptors G-Protein Coupled Receptors
  • G-Protein Coupled Receptors convey signals from extracellular hormones and neurotransmitters to intracellular effectors and linked signaling pathways.
  • G-Protein Coupled Receptors are a class of integral membrane proteins belonging to the "7TM" superfamily of transmembrane receptors. The GPCRs are characterized by the possession of seven intramembrane helices, and by domains that extend both into the extracellular environments and the cytosol (Wojcikiewicz, R.J. (2004) "REGULATED UBIQUITINATION OF PROTEINS IN GPCR-INITIATED SIGNALING PATHWAYS," Trends Pharmacol Sci.
  • the GPCRs use an amazing number of different domains both to bind their ligand and to activate G proteins. More than 150 GPCRs have been identified, in at least six families of proteins that show no sequence similarity. The fine-tuning of their coupling to G proteins is regulated by splicing, RNA editing and phosphorylation.
  • the G-Protein Coupled Receptor trigger cellular processes through a conformational shift that is caused by the binding of the GPCR to a ligand molecule (Gether, U. et al. (2002) "STRUCTURAL BASIS FOR ACTIVATION OF G-PROTEIN-COUPLED RECEPTORS," Pharmacol Toxicol. 91(6):304-12). As a consequence of the conformational shift, the GPCR acquires the capacity to bind and cleave intracellular proteins ("G-proteins"). The release of the cleavage products into the cytosol triggers cellular processes to occur.
  • GPCRs include the receptors of the olfactory sensory epithelium that bind odorants and neurotransmitter receptors (e.g., the serotonin receptor), the cannabinoid receptor (Picone, R.P. et al. (2002) “LigAND BASED STRUCTURAL STUDIES OF THE CB1 CANNABINOID RECEPTOR,” J Pept Res. 60(6):348-56; Onaivi, E.S. et al. (2002) “ENDOCANNABINOIDS AND CANNABINOID RECEPTOR GENETICS,” Prog Neurobiol. 66(5):307-44), and the rhodopsin receptor (Ballesteros, J. et al.
  • G protein-coupled receptors mediate the perception of smell, light, taste, and pain (Ahmad, S. et al. (2004) “NOVEL G PROTEIN-COUPLED RECEPTORS AS PAIN TARGETS,” Curr Opin Investig Drugs.
  • the microstructure of the membrane significantly impacts upon the properties of the biosensor.
  • Single lipid bilayers that are directly immobilized to a solid support, or immobilized via a polymer or "hairy rod” cushion see,
  • Figure 1 shows a schematic representation of the lipid bilayer adsorbed to the inner surface of an AAO pore.
  • the z-axis defines the direction of the pore, and the x-y axes define the plane of the filter.
  • the bilayer normal of the membrane is given by D
  • the vector of the external magnetic field is B
  • the angle between these vectors is given by ⁇ .
  • the angle between the pore axis and the magnetic field is defined by ⁇ .
  • the orientation of the bilayer normal in the x-y plane is defined by the angle ⁇ .
  • Panel A Unilamellar samples prepared by flushing the pores after lipid loading.
  • the angular distribution function used in the fit, displaying the mosaic spread of the lipid, is shown below the spectra.
  • the order parameters used in the simulation were obtained from the POPC-d 3 ⁇ MLV 2 H NMR spectrum and the profile is shown in Figure 2, Panel C, Multilamellar samples prepared with no flushing step after lipid loading.
  • the extra signal in the center of the experimental spectrum of lipid adsorbed in AAO pores appears to be mostly from a residual 2 H resonance of AAO hydroxyl groups.
  • the small deviation in signal intensity between measured and calculated spectra in the frequency range of ⁇ 5 kHz could indicate existence of a few percent of lipid with lower headgroup order.
  • Figure 5 shows 300 MHz ⁇ NMR MAS spectra at a rotor spinning frequency of 5 kHz and a temperature of 30°C.
  • Trace A POPC MLVs
  • Trace B POPC in AAO pores
  • Trace C POPC in AAO pores exposed to 5 mM Pr 3+ .
  • Inset Expansion of ⁇ -choline signal in POPC in AAO samples. The POPC resonance assignments are given in the lower spectrum.
  • Figure 6 shows 31 P MAS NMR spectra at a rotor spinning frequency of 5 kHz and a temperature of 30°C.
  • Trace A POPC MLVs
  • Trace B POPC single bilayers adsorbed on AAO pores
  • Trace C POPC adsorbed onto AAO pores and exposed to Pr 3+ .
  • the integral intensity of the superimposed ⁇ POPC +PEG methylene resonance at 3.57 ppm relative to the ⁇ POPC resonance at 3.15 ppm is 8.6:9.
  • Figure 8 show the normalized methylene intensity plotted versus temperature for DMPC supported on AAO and DMPC MLVs. The lines are included as a guide to the eye.
  • Figure 9 shows 500 MHz ⁇ PFG-MAS NMR diffusion measurements on crushed POPC/AAO with trapped PEG8000 at a spinning frequency of 10 kHz, temperature of 30.0 °C, and a diffusion time of 200 ms.
  • Trace A Water resonance and Trace B: choline resonances of spectra acquired at 16 different gradient strengths from 0.01 - 0.37 T/m.
  • Trace C The signal intensity decay of the choline resonance as a function of k, fit to Equation 1.
  • Figure 10A shows a model for lipid adsorption consistent with the NMR data. A single bilayer forms a good seal with the AAO surface by the interaction of a small percentage of the lipids.
  • the lipids adsorb as wavy tubules with an average length of 0.4 ⁇ m. These tubules posses undulation with a radius of curvature of 100 - 400 nm. Trapped between the tubules and the AAO surface are pockets of water with an average thickness of 3 nm.
  • Figure 10B shows an illustration of shape of the lipid bilayer tubules inside the AAO pore.
  • Figure 11 shows the solid state 2 H NMR spectrum of POPC -d 3 ⁇ lipid tubules in anopore filters containing bovine rhodopsin at a lipid/protein molar ratio of 100/1.
  • the anopore disks were aligned with their normal parallel to the B 0 field of the NMR instrument. The spectrum indicates that bilayers adhere to the surface of the cylindrical pores as lipid tubules.
  • Figures 12A-H show 2 H NMR spectra of 18:0(d35)-22:6 PC membranes containing bovine rhodopsin at protein/lipid molar ratios from zero to 1/100. On the left the experimental spectra and on the right the simulated spectra are shown. The simulation yields the chain order parameters as a function of rhodopsin concentration, the mosaic spread of bilayer orientations, and the resonance linewidth. Results confirm formation of lipid tubules containing reconstituted bovine rhodopsin.
  • Figure 13 shows chain order parameter profile of the sn-1 hydrocarbon chain in 18:0(d35)-22:6 PC as a function of rhodopsin concentration reported as a function of molar protein/lipid ratio. In the presence of rhodopsin a small reduction of sn-1 chain order parameters for carbon atoms 2-8 is observed.
  • Figure 14 shows ! H MAS NMR spectrum of a cytoplasmic membrane preparation of E. coli BL21 cells recorded at a MAS spinning frequency of 5 kHz at ambient temperature. Trace A: membrane pellet, Trace B. membranes deposited inside the pores of an anopore filter.
  • Figure 15 shows a comparison of the conventional competitive filter- binding assay (Whatman GF/B filters) with ligand-binding performed on Anopore membranes. Results indicate that depositioning of the cannabinoid receptor in single tubular lipid bilayers at the inner surface of pores did not alter the ligand binding properties of the receptor. Furthermore, nonspecific binding of ligands was reduced significantly, allowing much more accurate ligand binding measurements.
  • Figure 16 shows the use of Anopore filters in a ligand binding study. Shown are scintillation count rates for binding of CP55,940 to the cannabinoid receptor CB2. Depositioning of membranes as tubular lipid bilayers in Anopore filters reduced nonspectific binding to less than 15% of total radioactivity, therefore greatly increasing the accuracy and reproducibility of ligand binding assays. Each data point represents the average of three filters.
  • Figure 17 shows the reconstitution of the CB2 fusion protein into a SOPC lipid bilayer.
  • Panel A Excitation: 532 nm (green laser); Emission: 580 nm.
  • PMT 320; Sensitivity: normal;
  • Panel B Excitation: 532 nm (green laser); Emission: 610 nm.
  • PMT 320; Sensitivity: normal.
  • Alexa 532 Exc conflicton:532 nm; Emission: 561 nm; T Red: Excitation: 583 nm; emission: 602 nm.
  • Figure 18 shows the deposition of SOPC into Anodisk filters. Presented is the lipid fluorescence count as a function of SOPC concentration in the reconstitution mixture. Reconstitution from lipid dispersions in octylglucoside is significantly more efficient than reconstitution from the triple detergent mix that is used for protein solubilization.
  • Figure 19 shows the deposition of CB2/SOPC into Anodisk filters.
  • cannabinoid receptor CB2 fluorescence count as a function of CB2 concentration in the dispersion.
  • CB2 binding per filter saturated at a concentration of 10 micrograms.
  • Figure 20 shows the deposition of SOPC/CB2 into Anodisk filters.
  • lipid fluorescence count as a function of cannabinoid receptor CB2 concentration in the reconstituted membranes. In the concentration range from 0 - 10 ⁇ g of CB2 a reduction of the amount of deposited lipid with increasing protein content was observed. The count rate remained constant at a protein content of 10 ⁇ g or higher.
  • Figure 21 illustrates the depositioning of CB2 into Anodisk filters using a Western blot with anti-MBP antibody.
  • 1 CB2-POPC mixloaded onto Anopore Filter; 2, 2a: flowthrough solution/wash with Tris buffer; 3: CB2 eluted from the Anopore filter with 2% SDS
  • This invention relates to compositions and methods for forming membranes containing membrane proteins that may be deposited into solid supports for use in biosensors.
  • the present invention particularly concerns the development of a procedure capable of forming a high surface area, supported single-lipid bilayer in which the membrane is separated from the support by a closed and stable aqueous cushion.
  • the present invention particularly concerns the development of a procedure for depositing single lipid membranes onto a solid support so as to provide, for example, a total exposed surface area of 500 cm 2 in a filter with a diameter of only 13 mm and thickness of 60 ⁇ m, orienting approximately 2.4 x 10 "7 moles of lipid.
  • 2 H NMR spectra on chain deuterated phosphatidylcholine it was established that lipids adsorb as wavy, tubular bilayers to the inner pore surface.
  • H magic angle spinning NMR it is found that the sample preparation procedure resulted in formation of a single lipid bilayer inside every pore.
  • lipid tubule length may vary from a fraction of a micrometer to several micrometers depending on membrane composition and preparation procedures.
  • the membranes are separated from the solid surface by a thick water layer with an average thickness of 30 A such that lipids are completely unperturbed by the solid support.
  • This makes the membranes suitable for incorporation of biological components, such as sensitive integral and peripheral membrane proteins, e.g. G- Protein Coupled Membrane Receptors (GPCR), receptor tyrosine kinases, ion channels, etc.
  • GPCR G- Protein Coupled Membrane Receptors
  • Adhesion between membrane and solid support is, nevertheless, quite strong. Membranes are not removed from the surface by passing water through the pores at high rate. Furthermore, the invention evidences that the supporting water layer is well sealed by the membrane from the bulk water phase. This permits entrapment of solutes, like soluble components of signal transduction pathways between the solid support and the bilayers. It is demonstrated that water soluble polymers with a molecular weight of 8,000 can be trapped in this water layer. Also, the substrate used for sample preparation, anodized aluminum oxide, is compatible with magnetic resonance spectroscopy, optical spectroscopy, radiotracer studies, neutron- and x-ray experiments, calorimetry, etc., permitting easy detection of receptor binding events as well as structural studies.
  • the large accessible membrane surface raises sensitivity of assays based on our technology by up to three orders of magnitude per unit of chip area.
  • An additional advantage is that substrates are applied by slowly flowing solutions through pores. The flow rate of liquid through the pores is easily controlled, permitting substrate binding studies to be conducted under very reproducible conditions. Because of the unique features of those solid-supported membranes, including lack of perturbation from the substrate, large surface area, strong adhesion to the support, stability to water flow, and the ease of sample preparation, the invention provides considerable promise for preparation of biosensors.
  • the invention concerns a composition
  • a composition comprising a single bilayer lipid membrane supported on a solid support, wherein the membrane comprises a lipid tubule having a wavy tubular geometry and rims, wherein the tubule has rims that are in contact with the solid support, and a center region between the rims that is spaced apart from the support by an aqueous cushion, the aqueous cushion being located between the membrane and the support.
  • the invention further concerns the embodiment of such a composition wherein the solid support is a porous aluminum oxide support.
  • the invention further concerns the embodiment of such compositions wherein the lipid comprises a phospholipid.
  • the invention further concerns the embodiment of such compositions wherein the membrane comprises an incorporated biological molecule.
  • the invention further concerns the embodiment of such compositions wherein the incorporated biological molecule is an integral membrane protein or a peripheral membrane protein.
  • the invention further concerns the embodiment of such compositions wherein the integral membrane protein or the peripheral membrane protein is a receptor molecule, an enzyme, or an antigen.
  • the invention further concerns the embodiment of such compositions wherein the incorporated biological molecule is a receptor molecule capable of binding an agonist or an antagonist of a signal transduction pathway.
  • the invention further concerns the embodiment of such compositions wherein the receptor molecule is a G-protein coupled membrane receptor.
  • the invention further concerns the embodiment of such compositions wherein the solid support is porous, and wherein an agonist or antagonist of the receptor, a G- protein, or an analog of such molecules is provided to the composition by flowing a solution through the pores of the porous support so as to permit the receptor agonist or antagonist to bind to the receptor from inside the lipid tubule from the side of the lipid monolayer opposite from the AAO surface.
  • the invention further concerns the embodiment of such compositions wherein the agonist or antagonist of the receptor, the G-protein, or the analog of such molecules receptor agonist or antagonist is contained in the aqueous cushion.
  • the invention further concerns a biosensor device comprising a solid support, a biological component, and a physical transducer; wherein: the biological component comprises a single bilayer lipid membrane supported on the solid support, wherein the membrane comprises a lipid tubule having a wavy tubular geometry and rims, wherein the tubule has rims that are in contact with the solid support, and a center region between the rims that is spaced apart from the support by an aqueous cushion, the aqueous cushion being located between the membrane and the support; the membrane comprises an incorporated biological molecule selected from the group consisting of an enzyme, a receptor molecule, and an antigen; and the physical transducer serves to generate a signal in response to the binding of a target molecule to the incorporated biological molecule.
  • the biological component comprises a single bilayer lipid membrane supported on the solid support, wherein the membrane comprises a lipid tubule having a wavy tubular geometry and rims, wherein the tubule has rims that
  • the invention further concerns the embodiment of such a biosensor device wherein the solid support is a porous aluminum oxide support.
  • the invention further concerns the embodiment of such biosensor devices wherein the lipid comprises a phospholipid.
  • the invention further concerns the embodiment of such biosensor devices wherein the incorporated biological molecule is a receptor molecule.
  • the invention further concerns the embodiment of such biosensor devices wherein the receptor molecule is a G-protein coupled membrane receptor.
  • the invention further concerns the embodiment of such biosensor devices wherein the solid support is porous, and wherein an agonist or antagonist of the receptor, a G-protein, or an analog of such molecules is provided to the composition by flowing a solution through the pores of the porous support so as to permit the receptor agonist or antagonist to bind to the receptor from inside the lipid tubule from the side of the lipid monolayer opposite from the AAO surface.
  • the invention further concerns the embodiment of such biosensor devices wherein the aqueous cushion contains a biological molecule that interacts with the receptor molecule.
  • the invention further concerns the embodiment of such biosensor devices wherein the aqueous cushion contains a G-Protein, and the incorporated biological molecule is a G-protein coupled membrane receptor.
  • the invention further concerns the embodiment of such biosensor devices wherein the physical transducer comprises an optical sensor, an electrochemical sensor, a potentiometric sensor, a conductometric sensor, or a piezoelectrical sensor; wherein the sensor generates a detectable physical signal in response to the binding of a target biological molecule to the biosensor's incorporated biological molecule.
  • the physical transducer comprises an optical sensor, an electrochemical sensor, a potentiometric sensor, a conductometric sensor, or a piezoelectrical sensor; wherein the sensor generates a detectable physical signal in response to the binding of a target biological molecule to the biosensor's incorporated biological molecule.
  • the invention further concerns the embodiment of such biosensor devices wherein the physical transducer is an optical sensor selected from the group consisting of a colorimetric optical sensor operating in the visible or non-visible spectral range.
  • the optical sensor is an optical sensor that detects fluorescent light.
  • the invention further concerns a method of detecting a pharmacological agent that binds to a biological molecule, wherein the biological molecule is selected from the group consisting of an enzyme, a receptor molecule, an antigen, and an antibody, wherein the method comprises the steps: (A) incubating the pharmacological agent in the presence of a biosensor device, the biosensor device comprising a solid support, a biological component, and a physical transducer; wherein the biological component comprises a single bilayer lipid membrane supported on the solid support, wherein: the membrane comprises a lipid tubule having a wavy tubular geometry and rims, wherein the tubule has rims that are in contact with the solid support, and a center region between the rims that is spaced apart from the support by an aqueous cushion, the aqueous cushion being located between the membrane and the support; the membrane comprises the biological molecule incorporated therein; and the physical transducer serves to generate a signal in response to the binding of a target
  • the present invention relates to compositions and methods for forming membranes that may be deposited into solid supports e.g., for use in biosensors.
  • the invention relates to compositions and methods for forming a high surface area, supported single-lipid bilayer matrix in which the membrane is separated from the support by a closed and stable aqueous cushion.
  • Such compositions comprise a lipid bilayer, a solid support, and where used for a biosensor, a biological molecule (such as a GPCR).
  • lipids may be employed in accordance with the methods of the present invention.
  • such lipids will be glycerol based lipids (e.g., phosphocholine, phosphatidyl-DL-glycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, etc.) and their derivatives and salts.
  • lipids will have fatty acid side chains of 10-20 carbon atoms, more preferably of 12-16 carbon atoms.
  • the membrane bilayer compositions of the biosensor devices of the present invention will comprise l-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC).
  • POPC l-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine
  • Any of a variety of solid supports may be employed in the biosensor devices of the present invention, however, the most preferred solid support is a porous aluminum oxide support. In particular, the porous aluminum oxide AnoporeTM support (Structure Probe, Inc., West Chester, PA, USA) is preferred. Although any of a variety of solid supports may be employed in accordance with the principles of the present invention, the most preferred solid support is a porous aluminum oxide support. In particular, the porous aluminum oxide AnoporeTM support (Whatman, Inc.) is preferred.
  • the AnoporeTM support is fabricated from a unique form of aluminum oxide with a highly controlled, uniform capillary pore structure that is tightly controlled at 0.2 ⁇ m.
  • the starting purity of the aluminum metal use in the first step for the manufactured of the membranes is quite high.
  • the support provides the advantages of high flow rates, efficient particle retention, rigid, uniform surface, transparency (when wet), ability to retain virtually no background stain, low levels of extractable materials, promotes sieving of particles at the surface, is temperature resilient (stable to 400° C) and electron beam radiation resistant.
  • the proteins of the biological component of the biosensor can be prepared either by reconstituting a lipid bilayer using, for example, purified membrane protein(s), or by forming bilayers from the cellular membranes of spheroplasts.
  • the present invention provides a facile process for reconstituting single lipid bilayers that contain functional membrane proteins into AnoporeTM filters.
  • a desired membrane protein is preferably recombinantly expressed as a fusion protein containing a C-terminal "tail" (e.g., a Hisio tail) (Grisshammer, R. et al. (1997) "QUANTITATIVE
  • membrane proteins and lipids are solubilized in detergent micelles preferably using either a mixture of 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate (Chaps), cholesteryl hemisuccinate (CHS), and dodecyl-,B-D-maltoside (LM )] (see, Tucker, J. et al.
  • Liposomes are formed from the mixed detergent-lipid-protein components by lowering the detergent concentration below the critical micelle concentration (cmc) (see, U.S. Patent No. 6,143,321; Urbaneja, M.A. et al. (1990) "DETERGENT SOLUBILIZATION OF PHOSPHOLIPID VESICLE. EFFECT OF ELECTRIC CHARGE,” Biochem J. 270(2):305-308; Marsh, D. et al. (1986) "PREDICTION OF THE CRITICAL MICELLE CONCENTRATIONS OF MONO- AND DI-ACYL PHOSPHOLIPIDS,” Chem Phys Lipids 42(4):271-277; Lasch, J. et al.
  • cmc critical micelle concentration
  • liposomes with lipid bilayers are formed spontaneously when the added buffer causes the detergent concentration to fall below the critical micelle concentration (about 20 mM) (see e.g. Mitchell, D.C. et al. (2001) "OPTIMIZATION OF RECEPTOR-G PROTEIN COUPLING BY BILAYER LIPID COMPOSITION I - KINETICS OF RHODOPSIN- TRANSDUCIN BINDING," J. Biol. Chem. 276(46):42801-42806; Litman, B. J. (1982) "PURIFICATION OF RHODOPSIN BY CONCANAVALIN A AFFINITY
  • the liposomes formed after dilution form bilayers that cover the cylindrical surface on pores in the Anopore® support.
  • the residual detergent is easily flushed out the lipid bilayers by passing detergent- free buffer through the support.
  • the present invention greatly shortens and simplifies the procedure for detergent removal.
  • bilayers can be prepared by extruding spheroplasts through stacked membranes (especially stacked AnoporeTM porous aluminum oxide membranes).
  • the spheroplasts will be bacterial spheroplasts that have been genetically engineered to express membrane proteins. Spheroplasts from genetically engineered E. coli are particularly preferred.
  • Multilayer components may be removed by controlled flushing with buffer.
  • the supported membranes of the present invention will be at least partially spaced apart from the support by a water (or aqueous buffer) layer or cushion.
  • Water-soluble biopolymers may be advantageously entrapped in this water layer or cushion.
  • the bilayer membranes of the present invention have the advantages that the incorporated protein is functional, and the effective membrane area is up to three orders of magnitude larger than the surface area of a flat chip.
  • the formed membranes are stable at moderate flow rates through pores; the flow rate can be controlled by, for example, an infusion pump.
  • the bilayer membranes produced in accordance with the methods of the present invention are wavy tubules as shown in Figures 10A and 10B. The upper and lower ends of tubules are in contact with the solid support, and seal off a layer of trapped water.
  • Membrane protein that is incorporated into such bilayers can be in an orientation that "faces" the support, or in an orientation that "faces" away from the support. Where desired, buffer, charge, pH, and membrane curvature parameters may be altered in order to cause more membrane proteins to adopt a particular orientation relative to the support.
  • the supported membranes of the present invention can be employed in biosensor devices.
  • biosensor device is intended to denote an analytical device that is capable of generating a signal in response to the presence of, and preferably, the binding of, a target molecule.
  • the biosensor devices of the present invention possess a biological component, which comprises an immobilized, or substantially immobilized, single bilayer membrane, and one or more membrane proteins incorporated therein; and a physical transducer, which serves to initiate the generated signal of the device.
  • the biosensor devices of the present invention may be augmented to additionally contain signal enhancers and signal multipliers. Likewise, co-factors, accessory proteins, and other relevant biomolecules may be incorporated into the biosensor.
  • the biological component of the biosensors of the present invention will typically comprise one, two, three, four or more membrane-bound proteins.
  • a single species of such protein may be incorporated into the membranes of the present invention, or, in alternative embodiments, two, three, four or more different species of receptor proteins.
  • Such additional species may have related functions, such as, for example, all being cytokine receptors, etc., or may have diverse functions, or may have subsets of receptors with related functions.
  • Different proteins (or sets of proteins) can be incorporated into membrane located at different regions of the support, so as to produce a solid support having a pattern of pores each containing different types of sensor protein.
  • membrane associated proteins can be incorporated in the membranes of the present invention.
  • biological components may be enzymes, or other proteins, whose expression is characteristic of disease (e.g., bone specific alkaline phosphatase, aldose reductase, myoglobin, troponin I, etc.). More preferably, however, they will be receptor molecules.
  • Suitable receptor molecules include cell-surface receptors (including protein-tyrosine kinase receptors (e.g., EGFR, PDGFR, MCSFR, SCFR, insulin-R, VEGFR, Trk, Met, Ron, Axl, Eph), ion channel receptors; or receptors for TNF and related factors (e.g., Trk, Met, Ron, Axl, Eph, Fas, TNFRI, TNFRII, CD40, CD30, CD27, 4-1BB, LNGFR, OX40), serine-threonine kinase receptors (e.g., TGF ⁇ R), transmembrane 7 or G protein-coupled receptors (e.g., receptors for CCR1, CCR2 ⁇ , ⁇ , CCR3, CCR4, CCR5, CXCR1, CXCR2, CXCR3, CXCR4, BLR1, BLR2, V28, and class I and class II cytokines), CD4 + receptors, class I (hematopoi
  • pylori or M. tuberculosi, hepatitis virus, rubella, CMV or immunodeficiency virus (HIV, FIV), prostate specific antigen, etc.); or membrane- associated antibodies to such antigens, or autoimmune immunoglobulins, thyroglobulin, anti-thyroglobulin, IgE, IgG, or IgM immunoglobulins, tumor markers (e.g., prostate specific antigen, AFP CEA, etc.). It is, however particularly preferred that such biological molecule be a
  • GPCR so as to form a biosensor capable of detecting ligands that affect GPCR activity.
  • the G protein peptides simulating elements of G-protein function will be included in the reconstituted membrane preparations so as to be present in the aqueous cushion separating the bilayer from the support.
  • the external side of the bilayer will exhibit the attributes of the external side of a biological membrane.
  • the G protein will not be included in the reconstituted membrane preparation, but will be provided in the buffer being flushed through the support.
  • the external side of the bilayer will exhibit the attributes of the internal side of a biological membrane.
  • the biological component(s) can be recombinantly produced (i.e., produced in a heterologous host cell), genetically produced (i.e., produced in a homologous host cell), or synthetically produced (i.e., synthesized using in vitro chemical synthetic procedures).
  • Such biological component(s) can be incorporated into the bilayer membranes of the present invention by any of a variety of means, such as by reconstitution or by extrusion of spheroplasts membranes, as discussed above.
  • the physical transducer component of the biosensors of the present invention is preferably configured to exploit the open pore nature of the preferred solid support (i.e., Anopore®), so as to permit detection of signal by flowing a fluid through the pores of the support.
  • the physical transducer component of the biosensors of the present invention preferably comprises an optical sensor, such as a colorimetric (i.e., a sensor that modulates the production of a visible spectrum optical signal in response to target molecule binding), a non- visible spectrum optical sensor (i.e., a sensor that modulates the production of a non-visible spectrum optical (e.g., infra-red, UV, fluorescent, etc.) signal in response to target molecule binding).
  • a colorimetric i.e., a sensor that modulates the production of a visible spectrum optical signal in response to target molecule binding
  • a non-visible spectrum optical sensor i.e., a sensor that modulates the production of a non-visible spectrum optical (e.g., in
  • the use of the translucent AnoporeTM support facilitates the use of colorimetric and non-visible spectrum optical sensors.
  • An optical sensor that detects fluorescent light is particularly preferred.
  • Receptor activation may be sensed by retention/release of radioactive substances such as tritium labeled receptor agonists or antagonists.
  • the physical transducer component of the biosensor of the present invention may be configured as an electrochemical sensor (e.g., an amperometric sensor (i.e., a sensor that modulates a current in response to target molecule binding), a potentiometric sensor (i.e., a sensor that modulates a pH change in response to target molecule binding), a conductometric sensor (i.e., a sensor that modulates resistance in response to target molecule binding), or a piezoelectrical sensor (i.e., a sensor that modulates a piezoelectric response to target molecule binding).
  • an electrochemical sensor e.g., an amperometric sensor (i.e., a sensor that modulates a current in response to target molecule binding), a potentiometric sensor (i.e., a sensor that modulates a pH change in response to target molecule binding), a conductometric sensor (i.e., a sensor that modulates resistance in response to target molecule binding), or a pie
  • the signal generated by the physical transducer may be quantitative (i.e., varying in intensity, duration, etc., with the concentration of the target molecule in a sample), qualitative (i.e., producing a signal in response to a threshold concentration of the target molecule), or partially qualitative and partially quantitative.
  • the biosensor devices of the present invention may be employed in ligand binding studies to detect ligand concentrations in a sample.
  • ⁇ -, 2 H, 13 C, and 31 P NMR with and without "magic angle” spinning can be used to verify that the facile sample preparation method of the present invention results in the attachment of single lipid bilayers to the solid support.
  • MAS NMR spectra are simpler, better resolved, and have larger signal amplitudes, as only the isotropic chemical shift and / couplings contribute to the resonance frequency.
  • Chemical shift anisotropy and dipole-dipole couplings are averaged by fast tumbling of the molecules in solution. The physical reason for this averaging is that the orientation of the molecule with respect to the static field changes on a timescale that is faster than the inverse anisotropy of the interactions. Such an irregular fast tumbling of the sample inside the spectrometer cannot be realized technically. However, as was shown by Lowe, I.J. (1959) ("FREE
  • the technique is called “magic angle spinning” (MAS) (see, STRUCTURE DETERMINATION II (NMR, EPR), G. Jeschke (WS 2003/04 9) "High-Resolution Solid-State NMR: Magic Angle Sample Spinning and Cross Polarization," web site: mpip-mainz.mpg.de/ ⁇ jeschke/lect9.pdf).
  • the preferred solid-supported membranes of the present invention are separated from the solid surface by a thick water cushion over most of their area. Therefore it can be expected that proteins in reconstituted membranes, prepared according to our procedure, will function without any interference from the solid support. Furthermore, the present sample preparation procedure will raise sensitivity of assays by up to three orders of magnitude per unit of biochip area. It is therefore envisioned that the invention has utility in the production of biosensors for pharmaceutical applications, environmental screening, detection of warfare agents, etc.
  • the solid supported membranes can be used for rapid screening of binding affinity of drugs to membrane receptors, e.g. G-protein coupled membrane receptors (GPCR).
  • GPCR G-protein coupled membrane receptors
  • the solid substrate is compatible with optical studies, radiotracer binding studies, NMR, EPR, small angle neutron scattering, x-ray diffraction, calorimetry, and other methods, permitting the development of binding assays as well as structural studies on proteins and bound ligands.
  • l-Palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC), 1- palmitoyl(d 3 ⁇ )-2-oleoyl-sn-glycero-3 phosphocholine (POPC-d 3 ⁇ ), l-palmitoyl-2- oleoyl-sn-glycero-3 phosphocholine-d (POPC-d 4 ), and 1,2-dimyristoyl-sn- glycero-3 -phosphocholine (DMPC) were purchased from Avanti Polar Lipids (Alabaster, AL).
  • D 2 O (99.8% D) and deuterium-depleted H 2 O (2-3 ppm D) were purchased from Cambridge Isotope Labs (Andover, MA).
  • Anopore aluminum oxide filters (13 mm diameter) with nominal pore sizes of 0.02, 0.1, and 0.02 ⁇ m were purchased from SPI Supplies (West Chester, PA).
  • Praseodymium (III) nitrate hexahydrate (99.9%) was purchased from Aldrich (Milwaukee, WI).
  • Polyethylene glycol 8000 (PEG8000) with an average molecular weight of 8455 g/mol was purchased from Spectrum.
  • Piperazine-N,N'-bis(ethanesulfonic acid) sesquisodium salt (Calbiochem; San Diego, CA) was used to prepare 10 mM PIPES buffer with 100 mM NaCl (Sigma; St. Louis, MO) in deuterium-depleted water for use in 2 H NMR measurements on POPC-d 4 .
  • lipids were loaded using one of two different extruders.
  • the majority of the experiments utilized a mini-extruder (Avanti; Alabaster, AL) equipped with 1-mL syringes.
  • One to five 13 -mm diameter anopore filters were placed into the extruder and flushed with 1 mL of water 11 times before bringing them into contact with the lipid.
  • the water was removed and 5 mg lipid dispersed in 1 mL of water, buffer, or aqueous PEG solution was passed through the filters 15 times.
  • the lipid dispersion was removed and one or two 1 mL aliquots of water, buffer, or aqueous PEG solution was passed through the filters at an rates of 3 - 12 mL/min, either by hand or using an infusion pump, to remove any remaining liposomes and to remove additional bilayers that are loosely attached to the first bilayer at the AAO surface.
  • the filters were then flushed with 1 mL of an isotonic solution (100 mOsmole) of NaCl in D 2 O at a rate of 0.04 mL/min using an infusion pump to remove any PEG not trapped beneath the bilayers.
  • the extrusion was performed at room temperature, but for DMPC, the lipid and extruder were maintained at 35 °C during loading using a dry heat incubator (Fisher Scientific; Pittsburgh, PA).
  • H spectra are acquired at a resonance frequency of 300.14 MHz with a 3.6 ⁇ s 90°pulse and a 4 s delay between scans.
  • the spectral width is 5 kHz and the number of acquisitions is 32.
  • P spectra are acquired at a resonance frequency of 121.4 MHz with a 2.5 ⁇ s 90°pulse and a repetition rate of one acquisition per second.
  • the spectral width is 50 kHz, and the number of acquisitions varied from 25,000 to 90,000.
  • the data are transferred to a personal computer and processed as described by Holte, L.L. et al. ((1995) " 2 H NUCLEAR MAGNETIC RESONANCE ORDER PARAMETER PROFILES SUGGEST A CHANGE OF MOLECULAR SHAPE FOR PHOSPHATIDYLCHOLINES CONTAINING A POLYUNSATURATED ACYL CHAIN," Biophys. J. 68:2396-2403) and Huster, D. et al. ((1998) "INFLUENCE OF
  • Diffusion measurements are conducted at sixteen different values of gradient strength varying from 0.01 - 0.37 T/m with a stimulated echo sequence using sine-shaped bipolar gradient pulses (Cotts, R.M. et al. (1989) J. Magn.Reson. 83:252-266) of 5 ms duration. A longitudinal eddy current delay of 5 ms is used. Diffusion times are varied from 20 - 200 ms. At every gradient strength, 64 scans are acquired with a recycle delay of 4 s.
  • Mathcad is used to fit the diffusion data to the equation that relates signal intensity to the diffusion constant for powder samples with filter pores oriented at random to the orientation of the magnetic field gradient (Gaede, H.C. et al. (2003) "LATERAL DIFFUSION RATES OF LIPID, WATER, AND A HYDROPHOBIC DRUG IN A MULTILAMELLAR LIPOSOME,” Biophys J 85:1734-1740),
  • Equation (3) a formula for the calculation of the FID for a 2 H NMR experiment on a lipid bilayer is obtained. Resonances corresponding to all possible values of ⁇ and ⁇ are superimposed by integration:
  • y realn ⁇ J Jli cos(2 ⁇ v q • s( ⁇ , ⁇ ) • S(mo ⁇ )i • n-DW)-exp(-n-DW/T 2 ⁇ )p( ⁇ )d ⁇ d ⁇ (4) i 0 0 0
  • the molecular order parameters S(mol)i were determined by dePakeing of spectra obtained by 2 H NMR experiments on randomly oriented bilayers (multilamellar liposomes) (Sternin, E. et al. (1983) J. Magn.Reson. 55:274-282; Mccabe, MA. et al. (1995) J. Magn. Reson. Ser.B 106:80-82).
  • the angular distribution function p( ⁇ ) and the line broadening, T 2 ⁇ > were adjusted to match experimental signal intensities.
  • the value of ⁇ measured in units of degrees, represents the width of the Gaussian distribution function.
  • p( ⁇ ) sin ⁇ .
  • the simulation included an angular distribution function to describe the spread in bilayer orientation.
  • the bilayer normals have a circular distribution relative to the external magnetic field which was the starting point for the simulation.
  • a mosaic spread of cylinder axis orientation was considered.
  • T 2 ⁇ a second adjustable parameter
  • the spectra in both the MLVs and the single bilayers at AAO are a superposition of two quadrupolar splittings with values of 6.3 and 5.2 kHz, corresponding to the quadrupolar splittings of POPC choline ⁇ and ⁇ resonances, respectively (Koenig, B.W. et al. (1996) Langmuir 12:1343-1350).
  • the extra signal in the center of the experimental spectrum of lipid adsorbed to AAO appears to be mostly from a residual 2 H resonance of AAO hydroxyl groups.
  • there was an additional small but systematic deviation in signal intensity between measured and calculated spectra in the frequency range of +5 kHz that could indicate existence of a few percent of lipid with lower, but not well defined headgroup order.
  • Lipids 6:343-350 This commonly used assay was employed here to determine whether the membranes formed were well-sealed and if they formed single or multiple bilayers. Addition of the shift reagent Pr 3+ at a concentration of 5 mM after adsorption of bilayers to AAO pores, either by dropwise addition or slow extrusion at rates of 0.02 mL/minute, shifted a fraction of the ⁇ choline resonance downfield by 0.12 ppm (Bystrov, V.F. et al. (1971) Chem.Phys. Lipids 6:343-350).
  • the seal was also perturbed by higher flow rates of water.
  • Pr 3+ shift reagent was applied by extrusion of a Pr 3+ solution through the AAO pores at rates of 12 mL/minute, then more than 50% of the ⁇ -choline resonances were shifted. Often two or more ⁇ choline peaks were detected, indicating that different regions of the monolayers were exposed to different concentrations of the shift reagent. However, extrusion at rates of 0.02 mL/minute resulted again in 1:1 peak ratios.
  • Fluid-gel phase transitions of lipids To probe the influence of the AAO support on the phase transition temperature of supported lipids, ⁇ MAS NMR spectra of DMPC supported on AAO pores were acquired as a function of temperature. At a MAS frequency of 5 kHz the ! H resonances of lipid hydrocarbon chains are well resolved in the fluid phase but broadened beyond detection in the gel state. To follow the main phase transition of DMPC, the normalized intensity of the 100 Hz line-broadened methylene resonance at 1.3 ppm was plotted versus temperature in Figure 8 for both DMPC in multilamellar liposomes and for single DMPC bilayers adsorbed on AAO pores.
  • the water resonance is a superposition of signals of at least two water pools. At our experimental conditions two thirds of water resides outside pores while one third is inside.
  • the signal decay of the water resonance as a function of gradients strength is complex due to the random orientation of filter pieces as well as water- filled pores in the spinning rotor, water interaction with lipid and AAO surfaces, chemical exchange of protons with AAO hydroxyl groups, and permeation of water through lipid bilayers. From the signal decay at low gradient strength it was estimated that within very generous error limits most of the water moves at the rate of free water, D ⁇ 2 TO "9 m 2 /s .
  • the width of the angular distribution of 20° must result predominantly from a distribution in bilayer orientations rather than the AAO surface, though it is likely to contain some contributions from the pores, such as deviations from parallelism, roughness along the long axis, and distortion from cylindrical shape.
  • the lipid bilayers must maintain curvature in excess to curvature from the cylindrical symmetry, suggesting that bilayers do not adhere flatly to the inner surface of the pores.
  • the T 2 value for the proton ⁇ resonance of AAO-adsorbed POPC of 8.8 ms may be compared to 97 ms, the value for the ⁇ -resonance of DMPC MLVs at 30°C (Huster, D. et al. (1999) J. Phys. Chem. B 103:243-251).
  • the reduced ! H NMR T 2 for POPC on AAO suggests an additional reorientation of the lipids with correlation times in the millisecond range.
  • a POPC lateral diffusion constant of 9.5 X 10 "12 m 2 /s see Fillipov, A. et al. (2002) Biophys. J.
  • the order parameters of the lipid choline resonances of the outer monolayers at the AAO surface are identical to order parameters in the inner monolayer. Therefore the headgroup order and motions of the majority of lipids in the outer monolayer adjacent to the AAO surface are not influenced by the association, in good agreement with existence of a thick water layer between AAO and lipid underneath most of the bilayer. The data are consistent with only a small percentage of the lipids acting as points of attachment to the AAO surface.
  • Lipid order parameters and phase transitions The deuterium spectra of the perdeuterated palmitic acid chain in POPC-d 3 ⁇ were used to assess order and dynamics of lipids in adsorbed single bilayers.
  • the molecular chain order parameters S(mol)i are indistinguishable from order parameters in multilamellar liposomes, which is remarkable considering the high sensitivity of order parameters to changes in lipid area per molecule as demonstrated in experiments conducted as a function of temperature or hydration.
  • E m J/mol equivalent to a lowering of the main lipid phase transition temperature AT - — ⁇ T j of about 0.9
  • Multilamellar liposomes become cylindrical after being forced into the pores.
  • the cylinders are stable until their length reaches a critical value that depends on the surface bending energy.
  • Cylinders with variable diameter have lowest energy if they belong to the family of surfaces with constant total curvature J, called Delaunay surfaces (Delauney, C.
  • lipid cylinders have a tendency to form constrictions and to break up at a certain length, forming short, capped tubules or spherical vesicles.
  • Rhodopsin purification and reconstitution into POPC membranes Rod outer segment discs from bovine retinas were solubilized in the detergent octylglucoside (OG), and the rhodopsin purified by affinity chromatography according to the procedure of Litman et al. (Litman BJ. (1982) "PURIFICATION OF RHODOPSIN BY CONCANAVALIN A AFFINITY CHROMATOGRAPHY,” Methods Enzymology, 81, 150- 153) using a Pharmacia concanavaline A columnm (Pharmacia Biotech, Piscataway, NJ).
  • Rhodopsin concentration after purification was determined by measuring light adsorption at 500 nm using a diode array UV/Vis spectrophotometer Agilent 8453 (Agilent Waldbronn, Germany). Care was taken to minimize exposure of samples to light during the experiment.
  • the lipid dispersion was added to the purified rhodopsin dispersed in OG to yield a rhodopsin/lipid molar ratio in the range from 1/100 to 1/1000.
  • the OG concentration was kept at a concentration in the range from 40-100 mM OG and the OG/lipid molar ratio was 10/1.
  • Small liposomes with membrane incorporated rhodopsin were formed according to the dilution/reconstitution method of Jackson and Litman (Jackson M.L. and Litman, BJ. (1985) "RHODOPSIN-EGG PHOSPHATIDYLCHOLINE RECONSTITUTION BY AN OCTYL GLUCOSIDE DILUTION PROCEDURE," Biochim.
  • a mini-extruder (Avanti Polar Lipids, Alabaster, AL) or stainless steel thermobarrel extruder (Lipex Biomembranes, Inc; Vancouver, BC Canada) were loaded with up to five 13 mm diameter Whatman Anopore filters (SPI Supplies, West Chester, PA) with a nominal pore diameter of 0.2 ⁇ m.
  • Anopore filters were packed between two filter supports.
  • a total of 5 ml of rhodopsin reconstituted into POPC-d 3 ⁇ liposomes were sent through the filters in 1 ml increments at a rate of 0.06 ml/s using a gas-tight Hamilton syringe.
  • the rhodopsin from the 4 th milliliter was partially adsorbed (rhodopsin concentration 0.19 mg/ml), and the rhodopsin from the 5 th milliliter passed the filters essentially without binding within anopore (rhodopsin concentration 0.35 mg/ml, compared to 0.37 mg/ml in the incoming solution).
  • rhodopsin concentration 0.35 mg/ml 0.35 mg/ml, compared to 0.37 mg/ml in the incoming solution.
  • a total of 1.3 mg of rhodopsin did bind within the 5 anopore disks, corresponding to 0.26 mg of rhodopsin in 0.52 mg of POPC-d 3 ⁇ bilayers per disk. Again, extrusion was performed in complete darkness to prevent the bleaching of rhodopsin.
  • the Anopore membranes were placed on a stainless steel grid and sealed against the upper barrel by an O-ring. In this approach, water was flushed through the filters several times before the lipid dispersion was passed through ten times using compressed argon at pressures in the range from 2 - 8 bar.
  • the anopore filter disks with rhodopsin in lipid membranes were loaded into a flat glass cell that was filled with PIPES/NaCl buffer prepared in deuterium depleted water (Cambridge Isotopes, Cambridge MA) and sealed with a silicon stopper. The cell was inserted in darkness into a Doty flat cell probe for a DMX500 solid-state NMR.
  • the carrier frequency of the spectrometer was adjusted to be exactly at the center of the symmetric spectra.
  • FID free induction decay
  • the location of the maximum of the quadrupolar echo was determined with a resolution of l/10 th of a dwell time unit, and the time base of the spectra was corrected such that the FID began exactly at the echo maximum using a spline interpolation function to calculate new digital data points.
  • the FID was multiplied with an exponential decaying window function corresponding to a line broadening of 100 Hz. After the Fourier transformation of the FID the spectra shown in Figure 11 and Figures 12A-H were obtained.
  • the spectra are characteristic for lipid bilayers oriented preferentially with their bilayer normal perpendicular to the magnetic field. This orientation of the bilayer normal is consistent with membranes adhering to the inner surface of pores as lipid tubules. With increasing amounts of protein in the samples we observed an increase of the linewidth of resonances and some increase of mosaic spread. The mosaic spread of preferred bilayer orientations was reduced after freezing the sample in a deep freezer before investigating it at ambient temperature. Exact values of sn-1 chain order parameters and of mosaic spread were determined by simulating the experimental spectra using a program written for Mathcad 200 li Professional (MathSoft Engineering & Education, Inc., Cambridge, MA).
  • Order parameter analysis revealed a small reduction of hydrocarbon chain order in the order parameter plateau region (carbon atoms 2-8) due to the presence of the protein (see Figure 13).
  • Mosaic spread could be reasonably well modeled as Gaussian distribution with a half width of 8 degrees (pure 18:0(d35)-22:6 PC bilayers) to about 20 degrees (18:0(d35)-22:6 PC bilayers containing rhodopsin at a lip id/protein molar ratio of 100/1.
  • the presence of the protein in the reconstituted membranes is also detectable as a substantial reduction of spin-spin relaxation times (broadening of resonance lines) and small reductions of spin-lattice relaxation times of rapidly moving lipid segments.
  • the anopore filters containing reconstituted rhodpsin had a bright pink color that turned to yellow within one minute after exposure to light. This transition is consistent with conversion of dark-adapoted rhodopsin to a meta- I/meta-II rhodopsin equilibrium, demonstrating that rhodopsin was successfully adsorbed and functional.
  • Example 3 Adsorption of E. coli Protoplast Membranes with Incorporated Human Peripheral Cannabinoid Receptor (CB2) into AAO Pores Human peripheral cannabinoid receptor (CB2) was expressed in E. coli BL21 cells as a fusion protein, containing E. coli maltose-binding protein (MBP) attached at the N-terminal end of CB2, and thioredoxin followed by a stretch often histidine residues attached at the C-terminal end of CB2. Cell density was of the order of 10 9 cells per ml. Every cell expressed about 1,000 copies of CB2.
  • MBP E. coli maltose-binding protein
  • E-coli cytoplasmic membrane preparation By Western Blot analysis using specific antibodies we established that CB2 is preferentially located in the cytoplasmic membranes of E. coli. Formation of spheroplasts and fractionation to obtain cytoplasmic membranes were conducted according to the protocols by R.L. Weiss (Weiss, R.L. (1976) "PROTOPLAST FORMATION IN ESCHERICHIA COLI," J. Bacteriol. 128:668-670) and Thai and Kaplan (Tai, S.-P. et al.
  • E. coli cells were collected by centrifugation, washed twice with 0.1 M Tris-HCl pH 8.0 buffer and re-suspended in 0.1 M Tris-HCl buffer pH 8.0 containing 20% (w/v) sucrose, such that the resulting cell suspension had an optical density of 10 at 600 nm.
  • a cocktail of protease inhibitors F. Hoffmann-La Roche Ltd, Basel, Switzerland was added to prevent enzymatic digestion of CB2.
  • the temperature was adjusted to 37° C, and a solution of lysozyme (2 mg/ml) was added slowly, under constant stirring, until a final lysozyme concentration of 0.1 mg/ml was reached.
  • the cell suspension was incubated at 37° C for additional 15 minutes.
  • a solution of 0.1 M EDTA pH 7.0 was added slowly under continuous stirring, until a final EDTA concentration of 10 mM was reached. Incubation continued for another 10 minutes.
  • the spheroplasts were centrifuged at 12,000 g for 20 minutes, the pellet collected and washed once with 0.1 M Tris-HCl buffer, pH 8.0, containing 20% sucrose. Spheroplasts were centrifuged again at 12,000 for 20 minutes. The pellet of spheroplasts was re-suspended in ice-cold water, resulting in osmotic lysis of sphoroplasts.
  • MAS NMR experiments one milliliter of the cytoplasmic membrane preparation was suspended in Tris buffer prepared in 99.9% D 2 O (Cambridge Isotopes, Cambridge MA) and pelleted at 150,000 g for thirty minutes. The pellet with a volume of approximately 15 ⁇ L was transferred by centrifugation to a 4 mm outer diameter MAS rotor outfitted with a Kel-F insert to generate a spherical sample volume of 11 ⁇ L (Bruker Biospin Inc., Billerica MA).
  • the sample was spun at a MAS frequency of 5 kHz using a Bruker H-X resonance MAS probe for a solid state DMX300 NMR spectrometer (Bruker Biospin, Billerica MA) equipped with a Bruker widebore 300 MHz magnet.
  • Proton resonance spectra were acquired at ambient temperature using a ⁇ /2 pulse length of 4 ⁇ s and Cyclops phase cycling. About 1,000 free induction decays (FID) with a relaxation delay of 4 s were acquired. Before Fourier transformation the FID was multiplied with an exponential window function equivalent to a signal broadening of 1Hz.
  • Radioligand [ 3 H]CP55,940 bound to the CB2 receptor deposited into the Anopore filters could be competed off by increased concentrations of unlabeled ligand CP 55,940.
  • Ligand-binding parameters determined for the CB2 receptor deposited into Anopore filters were identical to the ligand-binding characteristics of CB2 receptor measured by a conventional filter-binding assay ( Figure 15).
  • the conventional competitive filter-binding assay was performed by incubating a suspension of E. coli membranes with radioligand [3H]CP55,940 and variable concentrations of the unlabeled competing ligand. Upon incubation, the reaction mixture was rapidly filtered through Whatman GF/B paper filters, and retained activity on the filters was counted with a scintillation counter.
  • This assay typically results in significant nonspecific binding of the hydrophobic ligand (CP55,940) in the multilamellar deposits on the filter surface which requires to work with the active receptor at much higher concentrations and reduces accuracy and reproducibility of the binding parameters K and B max . In the convential assay nonspecific binding constitutes 40-50% of the total radioactivity count.
  • Example 5 Reconstitution of Purified Recombinant Cannabinoid Receptor, Adsorption into AAO Pores, and Detection of Membrane Adsorption by Fluorescence Spectroscopy Human peripheral cannabinoid receptor (CB2) was expressed in E. coli BL21 cells as a fusion protein, containing E. coli maltose-binding protein (MBP) attached at the N-terminal end of CB2, and thioredoxin followed by a stretch often histidine residues attached at the C-terminal end of CB2.
  • MBP E. coli maltose-binding protein
  • Recombinant protein was solubilized from the bacterial membranes in a mixture of 0.5% CHAPS, 0.1 % cholesteryl hemisuccinate and 1% of dodecylmaltoside, and purified to approximately 90% purity by affinity chromatography on Ni-agarose (Qiagen) and ion-exchange chromatography on HiTrap-Q Sepharose (GE-Amersham Biosciences).
  • fusion protein was covalently labeled with AlexaFluoro 532 carboxylic acid, succinimidyl ester (Molecular Probes) according to the protocol recommended by the manufacturer.
  • Non-reacted fluorescent dye was (partially) removed by sequential gel-filtration (PD-10 desalting columns, Amersham), centrifugation in the Centricon-30 filter device (TVIillipore) and dialysis (Slide-A-Lyzer, Pierce). About 200 ⁇ g of labeled protein was obtained.
  • lipid 150 ⁇ g of l-stearoyl-2-oleoyl-sn-glycero-3 -phosphocholine (SOPC, Avanti) were dissolved in methanol and mixed with a methanol solution of 1.5 ⁇ g of Texas Red l,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red DHPE, Molecular Probes). The methanol was removed under the stream of nitrogen, and lipids were dispersed in a solution of 3% octylglucoside (OG) in 50 mM Tris-HCl buffer pH 7.5.
  • OG octylglucoside
  • Filters containing a mixture of fluorescence labeled CB2 and SOPC produced bright fluorescence at 580 nm (excitation at 532 nm), characteristic for AlexaFluoro 532 fluorophore. Intensity of fluorescence of sample 3 containing labeled CB2 as well as labeled lipid was lower than the fluorescence of sample 1 which contained labeled CB2 and non-labeled lipid, the effect that can possibly be attributed to quenching of fluorescence of the AlexaFluoro532 fluorophore by the Texas Red.
  • Quantification of the amounts of lipid and CB2 protein deposited onto Anopore filters was performed as follows.
  • SOPC was mixed with the Dil-Cl 8 DilC i s ( 1 , 1 ' -dioctadecyl-3 , 3 ,3 ' ,3 ' -tetramethy lindodicarbocyanine, 4- chlorobenzenesulfonate salt) fluorescently labeled lipid at a ratio of 1000: 1 (w/w, SOPC to Dil-Cl 8) in either 3% OG or mixture of triple detergents (0.5% CHAPS, 0.1% CHS, 0.1% DM).
  • SOPC/ Dil C18 mix was rapidly diluted (20-fold) into 50 mM Tris-HCl buffer pH 7.5, and solution was slowly filtered through the Anopore filter.
  • the fluorescence of the lipid retained on the filter was measured by scanning of the wet filter on Typhoon 8600 fluorescence scanner at following settings: excitation: 633 nm, emission: 670 nm, PMT: 450, sensitivity: normal.
  • FIG. 21 demonstrates that CB2 receptor can be eluted from the Anopore filter in the presence of strong ionic detergent SDS, and that no degradation of the receptor occurred during deposition/ elution procedures.

Abstract

La présente invention concerne des réactifs et des procédés pour former des membranes contenant des protéines membranaires périphériques et intégrées, à l'intérieur d'un support solide poreux à utiliser dans des capteurs biologiques. L'invention concerne en particulier la mise au point d'une procédure pour déposer des membranes monocouches à l'intérieur des pores de façon à obtenir une aire de pore exposée totale dont l'ordre de grandeur est supérieur à l'aire correspondante du filtre.
PCT/US2005/000069 2004-01-06 2005-01-04 Membranes a support solide disposees a l'interieur de substrats poreux, et leur utilisation dans des capteurs biologiques WO2005069004A2 (fr)

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EP2337842A1 (fr) * 2008-09-15 2011-06-29 Searete LLC Nanostructure tubulaire ciblée sur une membrane cellulaire
EP2337842A4 (fr) * 2008-09-15 2013-09-11 Searete Llc Nanostructure tubulaire ciblée sur une membrane cellulaire
US9187330B2 (en) 2008-09-15 2015-11-17 The Invention Science Fund I, Llc Tubular nanostructure targeted to cell membrane
US9617157B2 (en) 2008-09-15 2017-04-11 Deep Science, Llc Tubular nanostructure targeted to cell membrane
US10683365B2 (en) 2008-09-15 2020-06-16 Deep Science, Llc Tubular nanostructure targeted to cell membrane

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