EP1563305A1 - Verfahren zur immobilisierung membranassoziierter moleküle - Google Patents

Verfahren zur immobilisierung membranassoziierter moleküle

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Publication number
EP1563305A1
EP1563305A1 EP03810928A EP03810928A EP1563305A1 EP 1563305 A1 EP1563305 A1 EP 1563305A1 EP 03810928 A EP03810928 A EP 03810928A EP 03810928 A EP03810928 A EP 03810928A EP 1563305 A1 EP1563305 A1 EP 1563305A1
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EP
European Patent Office
Prior art keywords
membrane
gel
molecule
liposome
sol
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EP03810928A
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English (en)
French (fr)
Inventor
John D. Brennan
Michael A. Brook
Travis Besanger
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McMaster University
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McMaster University
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Publication date
Application filed by McMaster University filed Critical McMaster University
Publication of EP1563305A1 publication Critical patent/EP1563305A1/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention relates to methods for the immobilization of membrane- associated molecules, including membrane-associated biomolecules, to composites prepared by such methods and to the use of these composites, in particular for high- throughput drug screening, multianalyte biosensing or bioaffinity chromatography.
  • BACKGROUND TO THE INVENTION Immobilization of natural cellular receptors, which are mainly membrane associated proteins, is receiving substantial attention in the areas of research, clinical and environmental analysis, and in drug development. 1 ' 2 ' 3 ' 4 ' 5 ' 6 ' 7 ' 8 ' 9,10 ' 11 This is a result of increasing demand for robust and portable devices for medical, environmental and bioprocess monitoring.
  • bilayer lipid membranes BLMs
  • membrane-bound receptors A number of strategies have been reported for the immobilization of bilayer lipid membranes (BLMs) and membrane-bound receptors, including supporting of BLMs on the pores of filter paper, 18 covalent attachment of monolayer or bilayer lipid membranes to surfaces, 1 ' 4 ' 5 ' 19 tethering of phospholipid liposomes to a surface by deposition, 20 covalent attachment 21 or via avidin-biotin linkages, 22 and entrapment of BLMs
  • BLMs bilayer lipid membranes
  • membrane-bound receptors including supporting of BLMs on the pores of filter paper, 18 covalent attachment of monolayer or bilayer lipid membranes to surfaces, 1 ' 4 ' 5 ' 19 tethering of phospholipid liposomes to a surface by deposition, 20 covalent attachment 21 or via avidin-biotin linkages, 22 and entrapment of
  • An emerging method for the immobilization of biological species is their entrapment within inorganic matrixes formed by the sol-gel processing method.
  • 32 ' 33 This method involves formation of a colloidal sol solution owing to hydrolysis of a precursor such as tetraethyl orthosilicate (TEOS).
  • TEOS tetraethyl orthosilicate
  • a buffered solution containing the biomolecule of interest is then added to the sol to initiate rapid polycondensation of the silane.
  • a hydrated gel is produced that immobilizes the biological element without the need for a covalent tether.
  • TEOS tetraethylorthosilicate
  • TMOS tetramethyl orthosilicate
  • a new method for the immobilization of membrane-associated proteins or ionophore-liposome assemblies has been developed. This method is based on the immobilization of a reconstituted molecule-liposome assembly within a sol-gel- derived matrix that is prepared from protein- and membrane-compatible precursors, such as diglycerylsilane (DGS) and sodium silicate.
  • DGS diglycerylsilane
  • gA gramicidin A
  • AChR nicotinic acetylcholine receptor
  • Ca(II) ionophore ionomycin embedded within the membranes of 1,2-dioleoyl-s??- glycero-3-
  • gramicidin remained sensitive to the concentration of ions across the membrane and selective to passage of monovalent cations through the peptide channel, ionomycin retained the ability to transport Ca(II) across the membrane, while nAChR retained its ability to transport Ca(II) across the membrane in a ligand-gated fashion based on its interaction with agonists. Furthermore, following immobilization of gramicidin, the ability of divalent cations to block ion flux through the channel was also retained, while nAChR retained the ability to be inhibited by known antagonists, which block the ion channel, indicating that modulation of membrane-channel proteins is possible following entrapment in sol-gel derived silica.
  • the present invention relates to a method of immobilizing membrane-associated molecules in silica matrixes comprising combining a liposome- assembly comprising the membrane-associated molecule, with a protein- and membrane-compatible sol-gel precursor under conditions which allow a gel to form.
  • the present invention further relates to protein- and membrane-compatible sol-gels with a liposome-membrane associated molecule assembly immobilized therein. Further included within the scope of the present invention are methods for the detection of modulators of membrane-associated molecules comprising:
  • a liposome assembly comprising the membrane-associated molecule, said assembly being immobilized in a protein- and membrane-compatible sol- gel, to one or more test substances; and (b) detecting a change in one or more characteristics of the membrane-associated molecule.
  • the protein- and membrane-compatible sol-gel is prepared using a method as described herein.
  • a change in the one or more characteristics of the membrane-associated molecule in the presence of the one or more test substances compared to a control indicates that the one or more test substances are modulators of the membrane- associated molecule.
  • the methods of entrapment and for detecting modulators of membrane- associated molecules of the present invention provide a general method for analyzing these molecules and their inhibitors, agonists and/or antagonists.
  • the ability to immobilize membrane-associated molecules will allow development of bioaffinity chromatography or microarray technologies that will be useful for high throughput screening of potential inhibitors or effectors.
  • the present invention further relates to an improved method for preparing a sol gel immobilized liposome assembly comprising a membrane associated molecule, wherein the membrane-associated molecule is an ion-channel molecule, comprising:
  • the present invention also relates to an improved method for the detection of membrane potentials in a sol-gel immobilized liposome assembly comprising a membrane-associated molecule, wherein the membrane-associated molecule is an ion- channel molecule, comprising:
  • the present invention also includes kits, biosensors, microarrays, chromatographic and bioaffmiry columns comprising the silica matrixes comprising a liposome-protein assembly prepared as described herein.
  • Yet another aspect of the present invention provides a method of conducting a target discovery business comprising:
  • step (b) (optionally) conducting therapeutic profiling of the test substances identified in step (a) for efficacy and toxicity in animals;
  • step (c) licensing, to a third party, the rights for further drug development and/or sales or test substances identified in step (a), or analogs thereof.
  • Figure 1 shows tryptophan emission spectra of gramicidin A before and after reconstitution into phospholipid vesicles comprised of DOPC, both in solution and after entrapment into DGS derived silica.
  • Figure 2 is a schematic of the response of safranine O to the development of a membrane potential caused by an influx of potassium ions under various conditions.
  • FIG. 3 shows graphs indicating the change in steady-state fluorescence intensity (panel A) and anisotropy (panel B) of safranine O as membrane potential is developed across DOPC liposomes containing 0.39 mol % gramicidin A. Response follows influx of potassium ions after addition of liposomes to a solution of KI.
  • Figure 4 contains graphs showing the potential-induced decrease in fluorescence intensity as a result of the influx of potassium ions into unilamellar DOPC liposomes containing various levels of gramicidin A (a) in solution and (b) following entrapment in DGS derived silicate. Units are normalized as the ratio of intensity observed at time zero.
  • Figure 5 contains graphs showing the effect of different potassium iodide concentrations on the potential induced fluorescence response of safranine O for liposomes containing 0.39 mol % gramicidin A (a) in solution and (b) after entrapment in DGS derived silica.
  • Inset for (a) depicts typical time trials for the various potassium iodide concentrations. Data are normalized as the ratios of final and initial fluorescence intensities.
  • Figure 6 is a graph showing the inhibition of potassium ion flux through DOPC liposomes containing 0.39 mol % gramicidin A as a result of adding various concentrations of calcium ions to liposomes in the presence of 3 M KI.
  • Figure 7 is a graph showing the aging of samples containing DOPC liposomes with 0.39 mol % gramicidin A after entrapment in DGS derived silica in the presence of 25 % glycerol in distilled deionized water (•), in distilled deionized water ( ⁇ ) or without any external buffer or solution ( ⁇ ). Data are normalized as a percentage of response observed on day 1.
  • Figure 8 shows the response obtained upon addition of 3 H-epibetadine to Torpedo californica nAChR entrapped in sodium silicate derived silica (Panel A) and the response obtained for blank liposomes entrapped in sodium silicate derived materials (Panel B).
  • Figure 9 shows the specific binding of 3 H-epibetadine to human nAChR when entrapped in DGS derived materials relative to the binding obtained in the absence of entrapped nAChR.
  • Figure 10 shows the results of a competitive binding assay wherein varying concentrations of a non-radioactive antagonist (d-tubocurarine, Panel A) or agonist (nicotine, Panel B) is introduced along with 2.5 nM 3 H-epibetadine to IMR-32 nAChR entrapped in DGS derived materials.
  • the IC 50 and Ki values are in good agreement with those obtained from solution based experiments.
  • Figure 11 shows the concept of the fluo-3 based assay for measuring the Ca(II) ion flux across nAChR doped liposomes.
  • the channel In the absence of an agonist the channel remains closed and no ion flux is observed.
  • the nAChR ion channel opens and Ca(II) can pass into the membrane, resulting in a large increase in emission intensity from intraliposomal Fluo-3.
  • Figure 12 shows the changes in emission intensity of intraliposomal fluo-3 with time (Panel A) and the normalized concentration-dependent decrease in fluo-3 emission intensity (Panel B) due to blockage of the passage of Ca(II) ions upon addition of the antagonist d-tubocurarine to n-AChR doped liposomes entrapped in DGS derived glasses that were previously incubated with an excess of the agonist nicotine.
  • the decease in emission intensity correlates to a decrease in ion flux owing to closing of the nAChR channel upon binding the antagonist.
  • Figure 13 shows the changes in emission intensity of intraliposomal fluo-3 with time (Panel A) and the normalized concentration-dependent decrease in fluo-3 emission intensity (Panel B) due to enhanced passage of Ca(II) ions upon addition of the agonist cytisine to n-AChR doped liposomes entrapped in DGS derived glasses that were previously incubated with an excess of the antagonist d-tubocurarine.
  • the increase in emission intensity correlates to an increase in ion flux owing to opening of the nAChR channel upon binding the agonist.
  • Figure 14 is a graph showing the fluorescence intensity response of the calcium selective indicator dye Fluo-3 to the influx of calcium into DOPC liposomes in buffered solution following the addition of a calcium selective ionophore ionomycin to the membrane.
  • Figure 15 is a graph showing the response of fluo-3 to the addition of calcium ions for DOPC liposomes both with and without ionomycin present within the membrane following entrapment in sodium silicate derived silica.
  • Figure 16 are pictures showing a microarray of sol-gel entrapped liposomes containing ionomycin, and shows that our entrapment and signalling methods are amenable to the microarray format. Panel A shows the array before addition of calcium, panel B show the array after addition of calcium.
  • Phospholipid liposomes with reconstituted ionomycin, gramicidin A or nAChR ion channels were readily incorporated into sol-gel derived silicates without loss of ion channel activity when the sol gel was prepared from protein- and membrane-compatible, silica precursors such as organic polyol-derived silanes and sodium silicate.
  • silica precursors such as organic polyol-derived silanes and sodium silicate.
  • Steady-state fluorescence measurements of the tryptophan residues of gramicidin A indicated that it remained in its native conformation within the phospholipid membrane following entrapment. It was also found that ion channel activity was retained for reconstituted gramicidin A and that this activity was still sensitive to various electrochemical gradients caused by potassium ion concentration.
  • ion flux could be inhibited by the presence of divalent cations; moreover, the ion flux activity through gramicidin channels was retained for several weeks.
  • ion channel activity could be produced using either an ionophore (ionomycin) to produce Ca(II) flux across the membrane, or by using the ligand-gated ion channel (LGIC) nAChR, which produced agonist or antagonist dependent transmembrane fluxes of Ca(II).
  • LGIC ligand-gated ion channel
  • the present invention relates to a method of immobilizing membrane-associated molecules in silica matrixes comprising combining a liposome assembly comprising the membrane-associated molecule with a protein- and membrane-compatible sol-gel precursor under conditions which allow a gel to form.
  • Membrane-associated molecules which may be immobilized using the method of the invention include, for example, non-natural ionophores, ion channel proteins, ion-channel receptors, G-protein coupled receptors or membrane associated enzymes.
  • proteins include gramicidin, bacteriorhodopsin and the nicotinic acetylcholine receptor.
  • LGIC such as GABA A , Glycine, GLUCl, 5-HT 3 and nicotinic acetylcholine receptors
  • ATP gated channel superfamily of LGIC receptors as well as the glutamate cationic receptor superfamily of LGIC receptors
  • G-protein coupled receptors such as the dopamine receptor, histamine receptor and androgenic receptors
  • membrane transport proteins and membrane associated enzymes such as ⁇ - glutamyltranspeptidase or lipase.
  • non-natural ionophores include, for example, various ionophore antibiotics such as ionomycin, monensin, Ionomycin, laidlomycin, nigericin, grisorixin, dianemycin, lenoremycin, salinomycin, narasin, antibiotic X206, alborixin, septamycin, antibiotic A204, maduramicin and semduramicin, compound 47224, lasalocid (also including factors A, B, C, D and E), mutalomycin, isolasalocid A, lysocellin, tetronasin, echeromycin, antibiotic X-
  • ionophore antibiotics such as ionomycin, monensin, Ionomycin, laidlomycin, nigericin, grisorixin, dianemycin, lenoremycin, salinomycin, narasin, antibiotic X206, alborixin, septamycin, antibiotic A204,
  • the term "immobilized" means that the liposome assembly is physically, electrostatically or otherwise confined within the nanometer-scale pores of the biomolecule-compatible matrix. In an embodiment of the invention, the assembly does not associate with the matrix, and thus is free to rotate within the solvent-filled pores. In a further embodiment of the invention, the assembly is optionally further immobilized through electrostatic, hydrogen-bonding, bioaffinity, covalent interactions or combinations thereof, between the lipid bilayer and the matrix. In a specific embodiment, the immobilization is by physical immobilization within nanoscale pores.
  • liposome assembly comprising the membrane-associated molecule
  • membrane-associated molecule is either extrinsically or intrinsically associated with the lipid components in the liposome though hydrophobic, electrostatic, hydrogen-bonding, bioaffinity, covalent interactions or combinations thereof.
  • the membrane-associated molecule may be associated with the headgroups or acyl chains of the liposome or with both.
  • biomolecule-compatible and “membrane compatible” it is meant that the silica matrix either stabilizes proteins, membranes and/or other biomolecules against denaturation or does not facilitate denaturation.
  • biomolecule as used herein means any of a wide variety of proteins, enzymes, organic and inorganic chemicals, other sensitive biopolymers including DNA and RNA, and complex systems including whole or fragments of plant, animal and microbial cells that may be entrapped in the matrix.
  • the biomolecule-compatible and membrane-compatible matrix is a sol-gel.
  • the sol-gel is prepared using biomolecule- and membrane-compatible techniques, i.e. the preparation involves biomolecule- and membrane-compatible precursors and reaction conditions that are biomolecule- and membrane-compatible.
  • the biomolecule- compatible sol gel is prepared from a sodium silicate precursor solution.
  • the sol gel is prepared from organic polyol silane precursors. Examples of the preparation of biomolecule-compatible sol gels from organic polyol silane precursors are described in inventor Brennan's co-pending patent applications entitled "Polyol-Modified Silanes as Precursors for Silica", PCT patent application S.N.
  • the organic polyol silane precursor is prepared by reacting an alkoxysilane, for example tetraethoxysilane (TEOS) or tetramethoxysilane (TMOS), with an organic polyol.
  • TEOS tetraethoxysilane
  • TMOS tetramethoxysilane
  • the organic polyol is selected from sugar alcohols, sugar acids, saccharides, oligosaccharides and polysaccharides. Simple saccharides are also known as carbohydrates or sugars. Carbohydrates may be defined as polyhydroxy aldehydes or ketones or substances that hydroylze to yield such compounds.
  • the organic polyol may be a monosaccharide, the simplest of the sugars, or a carbohydrate.
  • the monosaccharide may be any aldo- or keto-triose, pentose, hexose or heptose, in either the open-chained or cyclic form.
  • Examples of monosaccharides that may be used in the present invention include one or more of allose, altrose, glucose, mannose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, threose, erythrose, glyceraldehydes, sorbose, fructose, dextrose, levulose and sorbitol.
  • the organic polyol may also be a disaccharide, for example, one or more of, sucrose, maltose, cellobiose and lactose.
  • Polyols also include polysaccharides, for example one or more of dextran, (500-50,000 MW), amylose and pectin.
  • the organic polyol is selected from one or more of glycerol, sorbitol, maltose, trehelose, glucose, sucrose, amylose, pectin, lactose, fructose, dextrose and dextran and the like.
  • the organic polyol is selected from glycerol, sorbitol, maltose and dextran.
  • Some representative examples of the resulting polyol silane precursors suitable for use in the methods of the invention include one or more of diglycerylsilane (DGS), monosorbitylsilane (MSS), monomaltosylsilane (MMS), dimaltosylsilane (DMS) and a dextran-based silane (DS).
  • DGS diglycerylsilane
  • MSS monosorbitylsilane
  • MMS monomaltosylsilane
  • DMS dimaltosylsilane
  • DS dextran-based silane
  • the polyol silane precursor is selected from one or more of DGS and MSS.
  • the biomolecule-compatible matrix precursor is selected from one or more of functionalized or non-functionalized alkoxysilanes, polyolsilanes or sugarsilanes; functionalized or non-functionalized bis- silanes of the structure (RO) 3 Si-R'-Si(OR) 3 , where R may be ethoxy, methoxy or other alkoxy, polyol or sugar groups and R' is a functional group containing at least one carbon (examples may include hydrocarbons, polyethers, amino acids or any other non-hydrolyzable group that can form a covalent bond to silicon); functionalized or non-functionalized chlorosilanes; and sugar, polymer, polyol or amino acid substituted silicates.
  • R may be ethoxy, methoxy or other alkoxy, polyol or sugar groups
  • R' is a functional group containing at least one carbon
  • examples may include hydrocarbons, polyethers, amino acids or any other non-hydrolyzable group that can form
  • the biomolecule compatible matrix further comprises an effective amount of one or more additives.
  • the additives are present in an amount to enhance the mechanical, chemical and/or thermal stability of the matrix and/or assembly components.
  • the mechanical, chemical and/or thermal stability is imparted by a combination of precursors and/or additives, and by choice of aging and drying methods. Such techniques are known to those skilled in the art.
  • the additives are selected from one or more of humectants and other protein stabilizing agents (for e.g. osmolytes).
  • Such additives include, for example, one or more of organic polyols, hydrophilic, hydrophobic, neutral or charged organic polymers, block or random co-polymers, polyelectrolytes, sugars (natural or synthetic), and amino acids (natural and synthetic).
  • the one or more additives are selected from one or more of glycerol, sorbitol, sarcosine and polyethylene glycol (PEG).
  • the additive is glycerol.
  • biocompatible matrix is a silica based glass prepared from, for example, a silicon alkoxide, alkylated metal alkoxide or otherwise functionalized metal alkoxide or a corresponding metal chloride, silazane, polyglycerylsilicate, diglycerylsilane or other silicate precursor, optionally in combination with additives selected from one or more of any available organic polymer, polyelectrolyte, sugar (natural or synthetic) or amino acids (natural and non natural).
  • the preparation of sodium silicate solutions for use as a sol-gel precursor is known in the art.
  • sodium silicate as a sol-gel precursor may be problematic if either sodium or potassium ions are to be transported through the membrane of the liposome due to interference from the sodium ions present in the precursor solution. In these circumstances, it is preferred to use the organic polyol silane precursors described above. In the case of ligand-gated ion channels, the sodium may not enter the internal compartment of the liposome in the absence of ligand, accordingly the residual sodium could be washed away before use allowing sodium silicate to be a suitable precursor for the transport of sodium or potassium ions through these types of membrane associated molecules.
  • the liposome-molecule assembly can be prepared using methods known to those skilled in the art. Typically a solution of the membrane-associated molecule, either with or without its intrinsic lipids (if any) present, is combined with a solution of a suitable lipid. Any lipid which forms liposomes may be used, for example, phospholipids, such as l,2-dioleoyl-s -glycero-3-phosphocholine (DOPC).
  • DOPC l,2-dioleoyl-s -glycero-3-phosphocholine
  • Suitable lipid components may include, but are not limited to: phospholipids, sphingolipids, glycolipids, synthetic and non-natural lipids, fluorescently labelled lipids, polymer- linked and polymerizable lipids (i.e., diacetylenic lipids), photoreactive lipids, fatty acids, fatty amines and hydrophobic moieties such as cholesterols, sterols etc. These may be used alone or in combination, and the resulting liposomes may contain mixtures of single or double chain surfactants, with chain lengths in the range of 4-30 carbons, with between 0 and 10 sites of unsaturation per chain.
  • lipid bilayer Upon formation of the lipid mixture, all organic solvents are removed (if necessary) and the resulting lipid films may be rehydrated in suitable buffer solutions followed by conversion to lipid vesicles (by sonication and/or extrusion, or any other suitable method) with the membrane associated molecule embedded within the lipid bilayer.
  • the liposome-molecule assembly may be combined with a protein- or membrane-compatible, sol-gel precursor solution under conditions which allow a gel to form.
  • gel it is meant a solution or “sol” that has lost flow.
  • the sols lose flow due to the hydrolysis and polycondensation of the precursor.
  • the hydrolysis and condensation of the polyol silane and sodium silicate precursors may suitably be carried out in aqueous solution.
  • a solution for example a homogeneous solution, of precursor, in acidified water is used, or in the case of DGS a solution of the precursor in water or buffer at neutral pH. Sonication may be used in order to obtain a homogeneous solution.
  • homogeneous it is meant having an essentially uniform composition or structure.
  • Conditions which allow the formation of a gel comprise adjusting the pH of the aqueous solution of precursor so that formation of a gel occurs.
  • the pH may be in the range of about 4 - 11.
  • the pH may be adjusted, for example, by the addition of suitable buffer solutions or resins.
  • the solutions lose flow, they can be formed, cast, moulded, shaped, spun, pin-printed as microarrays or drawn into desired shapes. Examples of such shapes include, but are not limited to, films, fibres, monoliths, pellets, granules, tablets, rods or bulk.
  • the solutions may also be placed into multi-well plates for high-throughput screening applications, or printed as microarrays for multianalyte sensing or screening. Accordingly, in an embodiment of the present invention, the method of immobilizing membrane-associated molecules in silica matrixes comprises:
  • the gel may be aged over a period of time under select conditions to lock the conformation of the gel, its pores, matrixes and interconnecting channels into fixed positions and permit long term storage.
  • the gels are aged in buffer or in a solution comprising an effective amount of a humectant, for example glycerol (suitably about 5-50% (v/v) of glycerol in water or buffer solution, preferably 25% (v/v) of glycerol in water or buffer solution).
  • a humectant for example glycerol (suitably about 5-50% (v/v) of glycerol in water or buffer solution, preferably 25% (v/v) of glycerol in water or buffer solution).
  • the protein- and membrane-compatible, sol- gel precursor solution and the liposome assembly are combined in the presence of an indicator molecule.
  • the liposome assembly further comprises an indicator molecule located on the interior of the liposome.
  • indicator refers to any compound that may be used to detect a change in the membrane-associated molecule's conformation or activity, including trans- membrane ion fluxes.
  • indicator molecules include compounds which have at least one detectable characteristic which is sensitive to changes in, for example, pH, membrane potential, ionic strength, divalent ion concentration or the hydrophilicity/hydrophobicity of its environment.
  • Specific examples of such an indicator molecules are the lipophilic cationic dye safranine O, the fluorescence of which is sensitive to changes in membrane potential, and the fluorescent dye fluo-3 , which is sensitive to the concentration of free Ca(II) in solution.
  • the protein- and membrane-compatible, sol- gel precursor solution and the liposome assembly are combined in the presence of one or more ligands (natural or unnatural) for the protein (for example a receptor) in question, that may optionally be labelled, for example, fluorescently labelled, for detection of activity of the protein.
  • ligands naturally or unnatural
  • label refers to any detectable moiety. A label may be used to distinguish a particular ligand from others that are unlabelled, or labelled differently, or the label may be used to enhance detection.
  • the present invention further relates to protein- and membrane-compatible sol-gels with a liposome/membrane-associated molecule assembly immobilized therein and prepared using the method as described hereinabove. (ii) Uses
  • the immobilization of membrane-associated molecules is important in several technologies including the development of biosensors, protein microarrays and bioaffinity columns.
  • the sol-gels prepared using the method described in the previous section can be used for any of these applications.
  • the gels may be used to screen for agonists, antagonists and modulators of any membrane associated molecule, such as non-natural ionophores, ion-channel receptors, G-protein coupled receptors or membrane-associated enzymes; microarraying of protein- membrane complexes for high-throughput screening of modulators of membrane- bound receptors; or immobilization of membrane-bound receptors into sol-gel derived monolithic columns for drug screening by frontal-affinity chromatography with mass spectrometric detection.
  • methods for the detection of modulators of a membrane-associated molecule comprising:
  • the protein- and membrane-compatible sol-gel is prepared using a method described herein.
  • a change in the one or more characteristics of the membrane-associated molecule in the presence of the one or more test substances compared to a control indicates that the one or more test substances are modulators of the membrane-associated molecule.
  • control is meant repeating the same method, under the same conditions but in the absence of the one or more test substances.
  • the one or more test substances can be any compound which one wishes to test including, but not limited to, proteins (including antibodies), peptides, nucleic acids (including RNA, DNA, antisense oligonucleotide, peptide nucleic acids, RNA or DNA aptamers, ribozymes or deoxyribozymes), fragments of proteins, peptides, and nucleic acids carbohydrates, organic compounds, inorganic compounds, natural products, library extracts, bodily fluids and other samples that one wishes to test for modulators of the membrane-bound protein.
  • the one or more test substance may be in liquid or gaseous form. Typically a solution of known concentration of the one or more test substances is employed.
  • the method for the detection of modulators of a membrane-associated molecule further involves a liposome assembly comprising a membrane-associated molecule in combination with other entities that facilitate the detection of modulation of the membrane-associated molecule by the one or more test substances.
  • the other entities are selected from one or more of indicator molecules and ligands (natural or unnatural) for the receptor protein being investigated.
  • the ligands may be labelled or unlabelled.
  • the method of detecting modulators of membrane-associated molecules may be "miniaturized" in an assay system through any acceptable method of miniaturization, including but not limited to multi-well plates, such as 24, 48, 96 or
  • the assay may be reduced in size to be conducted on a microfluidic-chip support, advantageously involving smaller amounts of reagents and other materials. Any miniaturization of the process which is conducive to high-throughput screening is within the scope of the invention.
  • the "one or more characteristics of the membrane-associated molecule” that may be used to detect modulators of the membrane-associated molecule include, but are not limited to, molecule-mediated transmembrane ion fluxes and conformational/environmental changes in the protein, membrane or a probe molecule that is associated with the protein or membrane, or entrapped within the liposome, or by binding of fluorescent or radioactive ligands by the entrapped protein.
  • the membrane-associated molecule is an ion channel protein or ionophore and the characteristic of the membrane-associated protein or ionophore that is detected is the flux of ions through the protein or ionophore from the exterior of the liposome to the interior.
  • a flux or movement of ions results in the formation of an electrochemical potential across the liposome membrane and/or in the presence of a specific ion within the liposome.
  • Certain fluorescent indicator molecules for example the lipophilic cationic dye safranine O, respond to the development of membrane potential by partitioning to certain locations in the assembly resulting in either an increase or decrease in fluorescence intensity and anisotropy.
  • the sol-gel entrapped liposomes comprising membrane associated molecules are formed into microarrays.
  • Microarrays may be formed by pin-printing the solution comprising the liposome assembly and the sol-gel precursors onto a suitable surface in array format before the solution gels. The solutions are then allowed to gel and dry on the surface.
  • sol-gel microarrays Suitable methods for forming sol-gel microarrays are known in the art (see, for example, inventor Brennan's co-pending PCT and U.S. regular applications entitled “Multicomponent Protein Microarrays", filed on November 3, 2003).
  • the present invention provides the first example of the use of transmembrane ion flux as a signalling method for microarrays.
  • Fluorescence is only one of many means of detecting change in one or more characteristics of the membrane-associated molecule. Because of the light- transmission capabilities of the matrixes of the present invention, UV, IR and visible light optical spectroscopy, as well as luminescence, adsorption, emission, excitation and reflection techniques are all suitable for detecting changes in the characteristics of the entrapped membrane associated molecule.
  • kits, biosensors, microarrays, chromatographic and bioaffinity columns comprising the silica matrixes comprising a lipo some-protein assembly prepared as described herein.
  • the kits of the present application comprise, in different combinations, the matrixes, reagents for use with the matrixes, signal detection and processing instruments, databases and analysis and database management software above.
  • the kits may be used, for example, to determine the effect of one or more test compounds on a membrane-associated molecule and to screen known and newly designed drugs.
  • Yet another aspect of the present invention provides a method of conducting a target discovery business comprising:
  • step (b) providing one or more assay systems for identifying test substances by their ability to effect one or more membrane-associated molecules based systems, said assay systems using a method of the invention; (b) (optionally) conducting therapeutic profiling of the test substances identified in step (a) for efficacy and toxicity in animals; and (c) licensing, to a third party, the rights for further drug development and/or sales or test substances identified in step (a), or analogs thereof.
  • the fluorescence indicator used to detect the development of a potential across the lipid membrane or the presence of a specific ion inside the liposome due to ion flux was located on the inside of the liposome assembly only.
  • the present invention further relates to a method for preparing a sol gel immobilized liposome assembly comprising a membrane associated molecule, wherein the membrane-associated molecule is an ion-channel molecule, comprising:
  • the present invention also relates to an improved method for the detection of membrane potentials in a sol-gel immobilized liposome assembly comprising a membrane-associated molecule, wherein the membrane-associated molecule is an ion- channel molecule, comprising:
  • the indicator molecule can be any compound that interacts with the surface of the sol gel, for example, the lipophilic cationic dye, safranine O.
  • the indicator molecule acts by detecting the ion directly upon entry into the interior of an entrapped liposome, for example the calcium dependent fluorophore, fluo-3.
  • the indicator molecule is removed from the solution external to the protein-liposome assembly using dialysis or gel filtration chromatography.
  • the silica precursor is biomolecule- and membrane-compatible.
  • the liposome assembly further comprises a ligand (natural or unnatural, labelled or unlabelled) for the membrane associated molecule (for example a receptor).
  • EXAMPLES Materials l,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), Egg phosphatidylcholine (EggPC), Egg Phosphatidylethanolamine (EggPE) and sphingomylin were purchased from Avanti Polar-Lipids, Inc (Alabaster, AL). Gramicidin A, High Purity (95%) was purchased from Calbiochem (San Diego, CA).
  • the human nicotinic acetylcholine receptor was purchased from Perkin Elmer Life Sciences (Boston, MA) while Torpedo californica nAChR was purified from the electric organ of the organism according to established protocols.
  • 53 Diglyceryl silane (DGS) was provided by Dr. Michael Brook of McMaster University and was prepared by a method that is described elsewhere.
  • 54 The fluorescent dye Fluo-3 was purchased from Molecular Probes (Eugene, OR).
  • the fluorescent dye safranine O, sodium silicate solution, Sephadex G25, sucrose, ethylenendiaminetetraacetic acid (EDTA), SM-2 Biobeads, Pottasium phosphate, ethylenediaminetetraacetic acid (EDTA), ethyleneglycolamine- tetraacetic acid (EGTA), potassium chloride, sodium aside, ionomycin, iodoacetamide, (-)-nicotine, cytisine, d-tubocurarine, phenylmethanesulfonylfluoride
  • Tubing with a molecular weight cut-off of 3500 Da was purchased from Spectrum
  • DOPC stock solutions were purchased in chloroform at a concentration of 20 mg.mL "1 . DOPC stock solution was dispensed in disposable glass vials and the organic solvent was removed by evaporation under a dry nitrogen gas stream to remove the bulk of the organic solvent, followed by evaporation under vacuum for two hours.
  • the resulting dried lipid films were then rehydrated in a buffer consisting of 25 mM EGTA, 25 mM EDTA, 10% Sucrose (w/w), 10 mM KCl, 0.529 mM Fluo- 3, pH 7.6 to a final lipid concentration of 2mg/mL.
  • the hydrated liposomes were then extruded through 600 nm pores, with an Avanti - MINIEXTRUDER at room temperature to create a mono-disperse suspension of unilamellar liposomes 600 nm in diameter.
  • the external Fluo-3 was removed by filtration through a column packed with Sephadex G25 to yield liposomes with dye only on the interior of the liposome.
  • the Gramicidin A stock was prepared in a solution of chloroform, trifluoroethanol and dimethylsulf oxide (19:5:1 volume ratio) to a final concentration of 4.79 x 10 "4 M.
  • Gramicidin A stock solutions were mixed with the lipid stock solutions (20 mg.mL "1 DOPC in chloroform) in disposable glass vials to provide final ratios of gA:lipid of either 0.39 mol% or 0.94 mol%.
  • the organic solvent was removed by evaporation under a dry nitrogen gas stream to remove the bulk of the organic solvent, followed by evaporation under vacuum for two hours.
  • the resulting dried gA:lipid films were then rehydrated in an appropriate buffered solution followed by high frequency sonication for one hour with a VWR Scientific Aquasonic Model
  • Lipid films consisting of EggPC, EggPE, Sphingomylin and cholesterol in mol ratios of 55:27:9:9 percent, respectively, were prepared as described earlier, and rehydrated to a final lipid concentration of 3.0 mg/mL and Fluo-3 concentration of 65 ⁇ M.
  • the liposome stock was then mixed with a small amount of DMM bringing the solution to a final concentration of 0.2 mM.
  • IMR-32 nAChR stock was added to the detergent lipid mixture and allowed to incubate at 4°C for 90 min. Following this 15 mg of SM2-Biobeads were added to the mixture and incubated for 1 hr.
  • Diglyceryl silane (DGS) derived sol-gels were prepared by adding 0.212 g of solid DGS and 5 ⁇ L of 0.1 N HC1 to 650 ⁇ L of distilled deionized water followed by sonication at 0 °C for 1.5 hours until all of the silane precursor had been dissolved and the solution had become homogeneous and transparent. Samples used for the collection of Trp spectra were prepared by placing 70 ⁇ L of the solution in the well of a microwell plate. For ion channel studies, the dialysed liposomes were mixed in a
  • Sodium silicate or DGS derived sol precursors were prepared by methods described previously. 58
  • the sol solution was mixed 1 :1 (v/v) with the buffered liposome or reconstituted nAChR solution, in the bottoms of 96 well microtiter plates to a final volume of 100 ⁇ L.
  • the nAChR containing stock described above was uses for entrapment without further dilution.
  • the stock sample as provided directly from Perkin Elmer was diluted four fold in 25mM HEPES, lOOmM KCl, 5 mM EGTA, pH 7.4 and mixed 1:1 (v/v) in DGS derived sol. The samples were aged for 1 hr at 4°C.
  • the stock sample was diluted four fold in 150 mM HEPES, 100 mM KCl, 5 mM EDTA, pH 7.4, and mixed 1:1 (v/v) with sodium silicate derived sol.
  • nAChR stock (3.72 nmol/mg protein Torpedo californica nAChR or 168 fmol/mg IMR-32 nAChR) or 20 mg/mL Asolectin liposomes were mixed with an equal volume of DGS or sodium silicate sol in the well of a microwell plate, where formation of a solid gel commenced.
  • the monoliths were allowed to cure for 1 hour, following which 10 ⁇ L of either buffer or 10 mM nicotine was added and allowed to incubate at 4 °C for 2.5 hrs. 160 ⁇ L of 3 H-epibatidine in buffer was added to the monoliths to a final concentration of 1.0 - 3.0 nM, and incubated for 18 hr at 4 °C. After incubation, 155 ⁇ L of 3 H-epibatidine solution was drawn off the top of the monolith and dissolved in 20 mL of Liquiscint scintillation fluid. The radioactive decay from 3 H-epibatidine was then counted for 5 min to determine the ratio of free ligand existing in solution.
  • Nicotine was added to determine the amount of specific binding to the receptor itself, and the Asolectin liposome samples were used to evaluate the amount of non-specific binding to the matrix. Using the information from these samples the amount of receptor bound ligand could be determined.
  • 10 ⁇ L various concentrations of either (-)-nicotine or d-tubocurarine were added to the tops on the DGS monoliths containing IMR-32 nAChR or Asolectin liposomes and allowed to incubate for 2.5 hrs. 160 ⁇ L of 3.0 nM 3 H-epibatidine was then added and the samples were incubated for 18 hrs. Free ligand was determined as described above. Ion-channel activity of IMR-32 nAChR
  • IMR-32 nAChR was used for the Fluo-3 based assays due to its increased calcium permeability as compared to the nAChR derived from Torpedo californica.
  • IMR-32 nAChR containing liposomes with an intraliposomal solution of Fluo-3 were entrapped as described above in 1 : 1 (v/v) in DGS derived silica.
  • the buffered sol was then dispensed in the bottoms of standard 96-well microwell plates and allowed to cure for 1 hr at 4°C.
  • Antagonism of the nAChR ion channel was measured by addition of 25 ⁇ L of the nAChR antagonist d-tubocurarine to the top of the nAChR-containing monolith in the 96-well plate.
  • Ligand-gated ion-flux was monitored through time dependent changes in fluorescence intensity upon the addition of 50 ⁇ L of 3 M CaCl 2 using a TECAN-Safire microwell platereading fluorescence system. Fluo-3 emission was monitored and 526 nm with an excitation wavelength of 488 nm, emission and excitation bandpasses of 5 and 7.5 nm, and a detector gain of 130 V, over a period of 45 min.
  • agonism of the nAChR ion-channel was monitored using the same assay except the channel was first antagonized by incubating the samples in 0.012 M d-tubocurarine, which was then followed by the addition of various concentrations of (-)-cytisine along with 3 M CaCl 2 .
  • the time dependent responses were then normalized as a function of their initial fluorescence intensity before the addition of calcium as seen in Figure 12.
  • the normalized changes in fluorescence intensity for the various concentrations of agonist or antagonist were then scaled as a percentage between their maximum and minimum response and an apparent dissociation constant could be determined by fitting the response to the "Hill" equation.
  • Sodium silicate derived sol precursors were prepared by methods described previously. 59 The sol solution was mixed 1:1 (v/v) with the buffered solution of ionomycin doped DOPC liposomes in the bottoms of 96 well microtiter plates to a final volume of 80 ⁇ L.
  • a Virtek Chipwriter Pro (Virtek Engineering Sciences Inc., Toronto, ON) robotic pinspotter equipped with a SMP 3 Stealth microspotting pin (Telechem Inc., Sunnyvale, CA) was used to print the ionmycin:liposome samples onto glass microscope slides from 96-well plates. Printing temperature was ambient with a humidity of approximately 50-70%. Completion of an array of 25 spots (5 x 5) took about 1 minute to perform, including pin wash cycles when using a printhead speed of 16 mm/s.
  • Trp residues within proteins has been widely used to probe the conformation and dynamics of proteins within sol-gel derived silica.
  • 60,61,62 In the case of gramicidin A, each homodimeric subunit of the ion channel contains four tryptophan residues, which NMR and crystallographic data have shown to be buried within the lipid bilayer.
  • the tryptophan residues of gramicidin have been shown to have distinctly different fluorescence emission spectra when located in the bilayer relative to being in solution. 49
  • the fluorescence emission properties of gA can therefore be used to indicate if gramicidin has survived the entrapment process and remained in the bilayer.
  • Figure 1 shows the emission spectra of gramicidin A before and after reconstitution into phospholipid vesicles comprised of DOPC, both in solution and after entrapment into DGS derived silicate.
  • the results clearly show that the emission maximum of gramicidin embedded in DOPC liposomes stays constant at 340 nm in solution and in DGS derived silicate; whereas gramicidin in the absence of liposomes is red-shifted, with a peak emission intensity at 350 nm both in solution and when entrapped.
  • the use of the diglyceryl silane precursor which liberates glycerol as a byproduct of hydrolysis, was able to retain the emission properties of reconstituted gA upon entrapment, and as discussed below, also provided an environment that was conducive to maintaining the ion- channel activity of entrapped gA.
  • the lipophilic cationic dye safranine O was used to follow the development of an electrochemical potential of K + across the phospholipid membrane. As shown in Figure 2, the changes in emission properties depend on whether the probe is located inside or outside of the membrane. As shown in Figure 2a, upon addition of KCl or KI to a membrane with the probe in the external solution, the influx of potassium ions through gA into the interior of the liposomes, combined with the exclusion of chloride ions, creates an electrochemical gradient across the membrane that is net positive on the interior and net negative on the exterior. Safranine O responds to development of such a membrane potential by partitioning into the hydrophobic lipid core due to the electrostatic attraction of the dye to the net-negative side of the membrane.
  • a further alteration of the assay was to use potassium iodide in place of potassium chloride to generate the ion gradients.
  • Iodide is a well-known quencher that is membrane impermeable, thus iodide abolishes any contribution to the fluorescence intensity from residual safranine O that is on the exterior of the liposome, enhancing the overall response from the probe that is located inside the membrane.
  • Figure 3 shows the changes in both fluorescence intensity (Panel A) and anisotropy (Panel B) that were obtained for reconstituted gA within DGS derived silicate upon addition of low and high levels of KI. Both the intensity and anisotropy decrease upon addition of KI, with the magnitude of the decrease becoming larger at the higher level of KI, as expected. These responses are consistent with the repulsion of the dye from the hydrophobic membrane owing to the influx of K + into the membrane, and provide evidence that ion channel activity can be monitored for reconstituted ion channels even after entrapment into sol-gel derived silica, proving that both the membrane and the ion channel are able to withstand the entrapment conditions.
  • ion flux was monitored for reconstituted gA both in solution and after entrapment to allow a direct comparison of the fluorescence responses.
  • liposomes that contained gA and an internal solution of safranine O were added to solutions of KI, and the changes in emission intensity were immediately measured. This method avoided dilution of the sample, as would occur if KI were added to a liposome solution, making it possible to accurately determine the initial intensity of the solution before the ion flux began.
  • the KI was added to the top of the monolith within the microwell plate to initiate a response. In this case, the liposomes were not diluted and thus the determination of the initial intensity was straightforward.
  • Figure 4 shows the response of safranine O to development of membrane potential for liposomes that contained varying levels of gA, both in solution and following entrapment. Even in the absence of gA, there is a significant fluorescence response that is due to the passive transport of K + directly through the lipid membrane. However, it is apparent that incorporation of gramicidin A into the phospholipid membrane results in development of a much larger potential at much faster rates over the time-course of the experiment, and that the response is increased in rate and magnitude as the level of gA increases.
  • Figure 5 shows the response of DOPC liposomes containing 0.93 mol % gramicidin to a range of K + concentrations.
  • the rate at which the emission intensity changes and the extent of the overall fluorescence response both increased as higher salt concentrations were introduced.
  • Example 3 Inhibitors of gA Ion Channel Activity
  • DGS derived silicate was examined by adding various levels of CaCl 2 to the entrapped samples along with 3.0 M KI. As shown in Figure 6, the presence of calcium ions produces a significant and concentration-dependent decrease in the potential induced fluorescence response to ion flux, consistent with inhibition of the ion-channel activity.
  • the inhibitory effect requires the presence of several hundred millimolar of Ca 2+ , which in expected given that Ca 2+ must compete with molar levels of K + for access to the ion channel.
  • a benefit of the "inverted" safranine O assay is that it avoids the potential for the direct interaction of Ca 2+ with the fluorescent probe.
  • Akerman et al have demonstrated the addition of divalent cations can directly inhibit the ability of safranine to embed into the membrane. However, entrapping the dye within the liposome leads to the exclusion of Ca 2+ from the vicinity of the probe.
  • FIG. 8 shows the response obtained upon addition of the radioligand 3 H-epibetadine to Torpedo californica nAChR entrapped in sodium silicate derived silica (Panel A) and the response obtained for blank liposomes entrapped in sodium silicate derived materials (Panel B).
  • the total specific binding of the entrapped receptor (ca. 1000 cps) is approximately 25% the specific binding activity obtained for free AChR (ca. 4000 cps), indicating that a significant fraction of the entrapped nAChR is either denatured or inaccessible to analyte.
  • the amount of specific binding is more than sufficient to conclusively prove that a fraction of the receptor remains active after entrapment.
  • Figure 9 shows the specific binding of 3 H-epibetadine to IMR-32 nAChR when entrapped in DGS derived materials relative to the binding obtained in the absence of entrapped nAChR.
  • the amount of specific binding is ca 500 cps, which is about half the amount observed for nAChR in sodium silicate glasses.
  • No activity was observed from Torpedo californica nAChR in DGS derived glasses, suggesting that sodium silicate based materials may be superior for entrapment of nAChR.
  • Figure 10 shows the results of a competitive binding assay wherein varying concentrations of a non-radioactive antagonist (d-tubocurarine, Panel A) or agonist
  • IC 50 and K ⁇ values for both d-tubocurarine and nicotine are in good agreement with those obtained from solution based experiments, and are in relatively good agreement with literature values, showing that the entrapment process does not dramatically alter the dissociation constants for the entrapped nAChR.
  • the key drawback of the radioligand binding assay is that a similar response (i.e., decrease in radioactivity) is observed upon binding of either agonists or antagonists, and thus no discrimination of the functional response of the nAChR to such ligands can be done.
  • an assay based on enhancement and diminution of ion channelling was developed to provide more detailed information on the mode of action of the ligand, as described below.
  • Example 6 Modulation of nAChR ion channelling by an antagonist
  • Figure 11 shows the concept of the fluo-3 based assay for measuring the Ca(II) ion flux across nAChR doped liposomes, which is based on the enhancement in the emission intensity of fluo-3 upon binding of Ca(II).
  • the channel In the absence of an agonist the channel remains closed and no ion flux is observed.
  • the nAChR ion channel opens and Ca(II) can pass into the membrane, resulting in a large increase in emission intensity from intraliposomal Fluo-3.
  • Figure 12 shows the changes in emission intensity of intraliposomal fluo-3 with time (Panel A) and the normalized concentration-dependent decrease in fluo-3 emission intensity (Panel B) due to blockage of the passage of Ca(II) ions upon addition of varying levels of the antagonist d-tubocurarine to n-AChR doped liposomes entrapped in DGS derived glasses that were previously incubated with an excess of the agonist nicotine to cause channel opening.
  • the decease in emission intensity correlates to a decrease in ion flux owing to closing of the nAChR channel upon binding the antagonist.
  • the results show that in the absence of antagonist, the presence of nicotine produced the expected rapid increase in fluorescence intensity upon addition of Ca(II). However, in the presence of the antagonist d-tubocurarine, the response is reduced owing to the blockage of a portion of the AChR ion channels.
  • AChR channel can be modulated by antagonists, showing that the AChR:liposome assembly entrapped in sol-gel glass is suitable for drug-screening studies.
  • Example 7 Modulation of Entrapped AChR Ion Gating using an Agonist
  • Figure 13 shows the changes in emission intensity of intraliposomal fluo-3 with time (Panel A) and the normalized concentration-dependent decrease in fluo-3 emission intensity (Panel B) due to enhanced passage of Ca(II) ions upon addition of the agonist cytisine to nAChR doped liposomes entrapped in DGS derived glasses that were previously incubated with an excess of the antagonist d-tubocurarine.
  • the increase in final emission intensity upon addition of Ca(II) in the presence of higher levels of cytisine correlates to an increase in ion flux owing to opening of the nAChR channel upon binding the agonist.
  • Panel B shows that the increase in intensity occurs in a manner that depends on the concentration of cytisine added.
  • the increase in ion flux provides clear evidence that the cytisine acts as an agonist and thus opens the AChR ion channel, producing a Ca(II) flux across the membrane.
  • the decrease in signal upon addition of d-tubocurarine, described in Example 6 provides evidence that this ligand acts as an antagonist.
  • Figure 14 shows the fluorescence intensity response of the calcium selective indicator dye Fluo-3 to the influx of calcium into DOPC liposomes in buffered solution following the addition of a calcium selective ionophore ionomycin to the membrane.
  • Ca(II) is initially present only outside the liposome, while fluo-3 is present only inside the liposome.
  • the addition of ionomycin results in the incorporation of the ionophore into the membrane, and produces a channel through which Ca(II) can move into the interior of the liposome.
  • Figure 15 shows the response of fluo-3 to the addition of calcium ions for
  • DOPC liposomes both with and without ionomycin present within the membrane following entrapment in sodium silicate derived silica The data clearly show that the presence of ionomycin results in the formation of a pore within the lipid membrane, which in turn produced a flux of Ca(II) from the exterior to the interior of the liposome upon addition of Ca(II) to the entrapped liposome.
  • Example 9 Liposome Microarrays using Transmembrane Ion Flux Signalling
  • Pin-printed sol-gel derived microarrays were constructed from samples illustrated in example 5. The microarrays were constructed with both negative and positive controls present. Negative controls consisted of buffered sodium silicate glass or fluo-3 loaded DOPC liposomes without ionomycin, while the positive control was entrapped fluorescein-dextran.
  • the array contained fluo-3 loaded DOPC liposomes with ionomycin present within the membrane bilayer. It was clearly seen that upon addition of calcium ions to the exterior of the pin-printed array that only the samples containing the ionomycin ion channel underwent a change in fluorescence intensity, consistent with transmembrane ion flux and a corresponding increase in fluo-3 intensity (Figure 16).
  • This example shows that the microarray format can be used to deposit intact liposome-ionophore assemblies onto surfaces, and to probe a functional response (i.e., transmembrane ion flux). Based on the other examples presented above, it is clear that such a microarray formation and readout method can be directly transferred to ligand gated ion channels such as the nicotinic acetylcholine receptor.
  • Figeys D Adapting arrays and lab-on-a-chip technology for proteomics.
  • DGS Besanger, T.R.; Chen, Y; Deisingh, A.K.; Hodgson, R.; Jin, W.; Mayer, S.; Brook, M.A.; Brennan, J.D. Screening of Inhibitors using Enzymes Entrapped in Sol-Gel Derived Materials. Analytical Chemistry, 2003, 75, 2382- 2391.

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