WO1996038726A1 - Couches doubles de phospholipides immobilisees par covalence sur des surfaces solides - Google Patents

Couches doubles de phospholipides immobilisees par covalence sur des surfaces solides Download PDF

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Publication number
WO1996038726A1
WO1996038726A1 PCT/IB1996/000496 IB9600496W WO9638726A1 WO 1996038726 A1 WO1996038726 A1 WO 1996038726A1 IB 9600496 W IB9600496 W IB 9600496W WO 9638726 A1 WO9638726 A1 WO 9638726A1
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layer
lipid
functional groups
proximal
bilayer
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PCT/IB1996/000496
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English (en)
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Stephan Heyse
Michael SÄNGER
Hans Sigrist
Horst Vogel
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Ecole Polytechnique Federale De Lausanne (Epfl)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/002—Electrode membranes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/08—Materials for coatings
    • A61L29/085—Macromolecular materials
    • 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
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00734—Lipids

Definitions

  • the invention concerns devices carrying on the surface covalently immobilized lipid bilayers which bilayers optionally contain biological receptor molecules, processes for their preparation, and their use as biosensors or implantation devices.
  • the common structural element of cell membranes is a double layer (herein "bilayer") of lipid molecules held in place by intermolecular forces.
  • a lipid is generally defined as a molecule carrying at one end a hydrophobic hydrocarbon chain, and at the other end a hydrophilic polar group.
  • Membranes separate compartments, each membrane being associated with an inside and an outside. Many biologically important signal transduction processes occur at the level of cell membranes. Specialized membrane receptors selectively detect and bind ligand and thereby filter these extracellular signals, pass them on across membranes, amplify and integrate them.
  • a biosensor is a device which converts biological activity into a quantifiable signal.
  • the (bio) chemical information is transformed into a form of energy which is measured by the transducer.
  • the transducer part is a device capable of transmitting this energy. It transduces the chemical information from the sample to an analytical signal.
  • Transducer systems include electrochemical devices, piezo ⁇ electric crystals, surface acoustic devices, thermistors and optical devices.
  • a modified procedure was applied to solid surfaces which are totally or partially covered by a molecular layer of physisorbed fatty acids or phospholipids transfered by LB-techniques to hydrophilic surfaces (Kalb et al. 1992) or by SA of thioalkanes or thiolipids (Lang et al. 1994) to gold or silver surfaces resulting in a hydrophilic surface of the supporting material.
  • a second step the first layer is completed and simultaneously a second lipid layer is formed (Lang et al. 1994) .
  • lipid bilayers as membrane models for studying ligand-receptor interactions occurring at cell membrane surfaces have been introduced by McConnell (1986) .
  • Lipid bilayers on solid supports represent geometrically well defined systems: the hydrophilic polar groups of the lower
  • proximal lipid monolayer contact the support, whereas the polar groups of the upper (distal) monolayer face the surrounding aqueous phase. Hydrophobic forces hold together the two lipid leaflets.
  • the photoactivatable headgroups of the reactive lipids in the lower leaflet are in direct contact with the support.
  • the direct attachment of the first proximal monolayer to the support results in a very inflexible and rigid membrane which lacks the space and water required for the proper folding of the extra-membraneous parts of membrane proteins.
  • the described photoim obilization procedure (radical forma ⁇ tion) has not been applied to hydroxylated surfaces.
  • Gitler et al., EP 441 120 disclose biosensors with a lipid bilayer on a recording electrode.
  • King et al., WO 92/17788 provide electrode membrane combinations for use in biosensors.
  • Cornell et al., WO 89/01159 describe amphiphilic bilayer membranes with a plurality of ion channels incorporated claimed to be useful as biosensors. None of the prior art discloses the novel and improved devices envisioned in the present invention and the uses thereof.
  • bioreceptors into such bilayer in a mode that naturally occuring conditions of biological membranes are mimiced and substrate/receptor binding can be measured by electrical or optical methods.
  • the present invention concerns a solid device carrying a coating which coating comprises
  • Fig. 1 Schematic cross-sections of different waveguide devices useful as biosensors.
  • A Binding of thiolipids from mixed micelles to a maleimide- modified waveguide surface to form an imperfect third, proximal lipid layer.
  • Fig. 2 Representation of an example of the four layers of the coat of a coated device.
  • X represents a group CH2 (CH3)3 + , a chemically reactive group, a receptor molecule, e.g. biotin, a carbohydrate, or a polymer.
  • the graph shows the change of the apparent thicknesses during formation of the lipid layers versus time and finally the binding of a streptavidin layer to the lipid bilayer on a r ⁇ aleimide-modified waveguide as measured with the integrated Optics Scanner from ASI AG .
  • the proximal lipid layer consists of the thiolipid DOPSH. Fifteen to sixty minutes after its attachment a steady state is reached. The actual average thickness of the thiolipid layer bound to the surface is evaluated as the difference between the signal of the buffer before adding DOPSH micelles and the measured stable signal of the thiolipid layer after the second washing step (end of period A) . This value was arbitrarily defined as 0 A on the thickness axis. Accordingly, the measurement starts at negative values. The two polarizations of the laser light (TE- and TM-modes) yield different thickness values for the isotropically calculated thiolipid layer. This can only be explained by the assumption that the thiolipid binding changes the overall anisotropy of the system.
  • Fig. 4 The graph shows the change of the apparent thicknesses versus time of the fourth, proximal lipid layer containing (NANP) 3-lipopeptide during formation of said layer and after addition of anti- (NANP) n antibody on a waveguide surface as measured with the integrated Optics Scanner from ASI AG.
  • A After one washing step with octyl glucoside (7-15 min) , a second lipid layer is formed by applying a 50 mM octyl glucoside solution thereof.
  • a solid coated device according to the present invention is amenable for electrical or optical signal detection and propagation. It can be used with membrane incorporated receptor molecules as a biosensor, which can selectively bind drugs, hormons, proteins, viruses etc.
  • the biosensor devices amenable for electrical signal detection are such wherein the lipid bilayer with incorporated receptor molecules is electrically coupled to a transducer such that changes in electrical resistance and capacitance of an electrode upon which the bilayer is mounted can be monitored.
  • the biosensor devices amenable for optical signal detection are such wherein the lipid bilayer with incorporated receptor molecules is in intimate contact with a transducer such that the binding of liga ' nd molecules to the receptors can be opti-
  • SUBST1TUTESHEET(RULE26) -lo ⁇ cally monitored This is conveniently performed by measuring the changes in the effective refractive indices of guided modes using waveguiding techniques.
  • the devices of the present invention are further permanently or temporary implantable devices for humans and animals, such as, pace makers, artificial metal or polymer joins, cathe ⁇ ters, and the like.
  • the basic uncoated material of such devices consists, depending on its prospected use, of metal, e.g. titanium, metal oxides, alloy, glas, ceramic or a polymer material.
  • the surface of the basic uncoated device is composed for example and without limitation, of glass, diamond or diamond-like materials, silicium, silicium dioxide (Si0 2 ) , silicon nitride (e. g.
  • Si 3 N 4 tantaliumoxide (Ta2 ⁇ 5) , titanium dioxide (Ti0 2 ) , titanium nitride, titanium carbide, platinum, tungsten, aluminum, or indium/tin oxide and carries on the surface functional groups, such as carboxyl, amino, thiol or in particular hydroxyl groups, to which the coating can be covalently attached.
  • Such basic materials carrying such functional groups are known in the art or can be produced according to conventional methods.
  • a silicon nitride film with varying amounts of NH- groups can be produced from SiH 4 and NH 3 by plasma discharge deposition according to Gmelin (1995) or Efimov et al. (1992) .
  • Uncoated devices for biosensor use are commercially availabel sensor chips, e.g, planar optical waveguides having a Ti ⁇ 2 Si ⁇ 2 2:1 surface, obtainable e.g. from ASI AG, Ziirich, Switzerland, or optical fibers, obtainable e.g. from ATOF Ag, Luzern, Switzerland.
  • the devices of the present invention are further permanently or temporary implantable devices for humans and animals, such as pace makers, artificial metal or polymer joins, cathe ⁇ ters, and the like.
  • Uncoated devices for implantation use are suited for implantation into a human or animal, and consist of a metal, such as a biocompatible metal, e.g.
  • PTT polyethyleneterephtalate
  • PTFE polytetra-fluoroethylene
  • composition and formation of the different layers of the coating are described in the following steps.
  • Step 1 Formation of the first layer containing first functional groups
  • the first layer is covalently attached to the functional groups of the basic device in a manner known per.se. It is preferably created by reacting the surface of the basic device with a reactive organosilane carrying a first functional group on the other end. Many functionalized organosilan compounds are available. Silylation of various types of surfaces with alkoxysilanes carrying functional groups has been disclosed e.g. by Kallury et al. (1989). Preferred silane coupling agents for creating a first layer containing first functional groups are for example of the formula
  • Y is an optionally protected functional group, such as amino, protected amino, hydroxy, protected hydroxy, mercapto, protected mercapto, carboxyl or protected carboxyl.
  • Z is lower alkoxy having from 1 to 6 carbon atoms, such as in particular methoxy, or ethoxy, propoxy, butoxy, pentoxy or hexoxy, or halogen such as Cl, Br or J, and n is an integer from 1 to 8, in particular 3.
  • such agents are amino-lower alkyl-tri-lower alkoxysilanes, wherein lower alkyl has from 1 to 8 carbon atoms, and is e.g methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl, and lower alkoxy has from 1 to 3 carbon atoms, and is e.g. methoxy, ethox or propoxy.
  • Particular preferred silanization compounds are 3- aminopropyltriethoxysilane and 3-mercaptopropyltrime- thoxysilane, resulting in an "amino device surface" or a "thiol device surface", respectively.
  • Activation of the device surface, leading to an increased number of surface exposed hydroxyl functions, is preferably performed prior to silane coupling.
  • Siloxane films on the device surface are preferably formed by reacting the device in an organic solvent containing the silane coupling agent for several hours at elevated temperatures.
  • silane coupling is performed by exposing the device surface to the silane in the vapor-phase.
  • Step 2 Formation of second, linking layer containing second functional groups
  • the linking layer containing second functional groups is created by reacting the first functional groups, with a homobifunctional- or heterobifunctional crosslinking agent.
  • Homobifunctional crosslinking agents carry at both ends of the molecules identical functional groups suitable for reacting at first with the functional groups of the first layer and subsequently with the headgroup functions of the phospholipids of the third layer.
  • Preferred homobifunctional crosslinking agents are for example
  • A is an activating group, such as preferably N- succinimidyloxy, or alkyl- or arylcarbonyloxy, halogen, e.g. chloro, bromo or iodo, alkan- or arylsufoyloxy, e.g. methan- or benzolsulfonyloxy, or the two A together represent oxygen to form an inner anhydride
  • X is a group (CH2. n ' wherein n is an integer from 1 to about 12, preferably 2 to 6, in particular 6, or X is a group (CH2. m COO- (CH2) m -OCO- (CH2. m , wherein m is an integer from 2 to 4, preferably 2,
  • n is an integer from 1 to about 12, preferably 2 to 6, in particular 2, or
  • B is the 1-maleimido rest and n is an integer from 2 to 12 , preferably 4 to 8 , in particular 6.
  • said second functional groups of the linking layer are in particular a N-succinimidyloxy ester group, or another activated carboxylic acid ester group, a carboxylic acid group or a maleimido group.
  • Heterobifunctional crosslinking agents carry at each end of the molecule different functional groups: one of them is selectively reactive with the functional groups of the first layer whereas the other functional group is selectively reactive with the headgroup function of the phospholipids of the third, proximal phospholipid layer.
  • Preferred heterobifunctional crosslinking agents carry for example at one end a succinimidyloxy ester function, suitable for reacting with primary amines, and at the other end a 1- maleimido group, the double bond of which being suitable for addition reactions with thiols.
  • Preferred heterobifunctional crosslinking agents are of the formula
  • A is an activating group, such as preferably N- succinimidyloxy, or alkyl- or arylcarbonyloxy, halogen, e.g. chloro, bromo or iodo, alkan- or arylsufoyloxy, e.g. methan- or benzolsulfonyloxy
  • B is the 1-maleimido rest
  • m is an integer from 0 to 12, preferably 0 to 6, and
  • p is an integer from 1 to about 6, preferably 2 to 4, in particular 2, whereby m is selected depending on the degree of the desired hydrophilicity, and m and p together are selected on the distance desired between the surface of the uncoated device and the third, proximal lipid layer.
  • the linker group according to formula (V) wherein m is above > 0 is of hydrophilic nature.
  • the first layer together with the second, linking layer separate the third, proximal phospholipid layer from the surface of the uncoated device depending on the length of the carbon chains expressed by the integers n, m and p.
  • Said integers can be adapted to the size and structure of the receptor protein so that the distance of the proximal phospholipid layer to the surface of the device leaves sufficient space for the receptor protein.
  • Reaction of one of the functional groups of the crosslinkig agents with the functional groups of the first layer is achieved in a conventional manner, depending on the functional group of the first layer and the crosslinking agent.
  • the application of heterobifunctional crosslinking agents for tethering proteins to surfaces has been for instance described by Hong et al (1994) .
  • Activated esters and anhydrides of the formulas II to V react easily with a hydroxy or amino group in an organic solvent or organic solvent/buffer mix, if need be in the presence of a condensing catalyst agent. After the reaction with the functional groups of the first layer, the excess of crosslinking agent is removed by washing procedures.
  • the functional group of the first layer is a carboxyl group
  • This activated group may react with a crosslinker agent carrying instead of a group -COA a hydroxy or amino group.
  • the amino group of the' first layer is reacted with a heterobifunctional crosslinker of the formula IV, wherein A is succinimidyloxy, to give a linking layer which is covalently bound by an amide bond to the amino group of the first layer and which has a maleimido functional group at the outside.
  • Step 3 Formation of the third, proximal phospholipid layer
  • a phospholipid comprises a glycerol bridge which links two long fatty acids (either saturated or unsaturated) with a polar head containing a phospho group.
  • the fatty acids by convention occupy the 1st and 2nd position of the glycerol moiety while the phospho containing polar head group is in position 3.
  • the phospho group is bound by an ester linkage to a lower alkyl group carrying a functional group able to covalently reacting with the functional group of the second layer.
  • Such group is for example a group -CH2-R3 in formula VI.
  • Typical phospholipids forming the third layer and binding covalently to the second layer are for example of the formula
  • R!-CO and R 2 -C0 independently from each other are rests of a fatty acid and R 3 is a group CH2-SH, CH2- (0- CH2CH2) n -SH, wherein n is from about 1 to 6, preferably 2, 3 or 4, CH 2 NH 3 + or CH(C00 _ )NH 3 + .
  • R!-CO and R 2 -CO are rests of a natural or synthetic fatty acid having of from 12 to 20 carbon atoms, e. g. the rest of lauric, myristic, palmitic, stearic, arachidic, oleic, linolic, linolenic, or arachidonic acid. Such acids are found in natural bilayer membranes.
  • compounds of the formula VI are selected from the group of naturally occurring or synthetically accessible phosphatidyl-thioethanols, phosphatidyl-ethanolamines and phosphatidylserines, in particular e.g. DMPSH, DOPSH (see Abbreviations) or thiolipids of the class mentioned in WO 93/215280 and Lang et al. , 1994, which citations are hereby incorparated by reference.
  • DMPSH naturally occurring or synthetically accessible phosphatidyl-thioethanols
  • DOPSH DOPSH
  • thiolipids of the class mentioned in WO 93/215280 and Lang et al. , 1994 which citations are hereby incorparated by reference.
  • Prefered are the phospholipids which form at ambient temperature the third layer in the fluid state.
  • Reacting thiolipids with the maleimido groups of the second layer yields a proximal phospholipid layer covalently bound to the second linking layer through a thioether linkage.
  • Reacting phosphatidylethanolamines or phosphatidylserines with N-succinimidyl ester groups of the second layer results in a proximal phospholipid layer covalently bound to the second linking layer through amide linkage.
  • Reacting phosphatidylethanolamines or phosphatidylserines with carboxylic acid groups of the second layer in the presence of a coupling agent yields a proximal phospholipid layer covalently attached to the second linking layer through an amide linkage.
  • Water-soluble carbodiimides are preferably selected as coupling agents.
  • a phase transfer catalyst e. g. a detergent solution of above mentioned phospholipids in aqueous media.
  • the detergent OG (see Abbreviations) is preferably selected for this purpose. Due to its high critical micellar concentration (CMC) of 25 mM, OG can be easily removed from the resulting phospholipid layer by washing with aqueous media.
  • the proximal phospholipid layer is usually an imperfect layer as it covers only about 60 to 50% of the device surface.
  • the imperfect layer is usually filled up and completed by incorporation of a surplus of phospholipids used in the next step.
  • Step 4 Formation of the fourth, distal lipid layer
  • the proximal phospholipid layers serves as a hydrophobic template for the non-covalent deposition of the fourth, distal lipid layer.
  • Lipids of the distal lipid layer can be of natural or synthetic origin, but are preferably selected from the phosphatidylcholine group, preferably phosphatidylcholines with one or two unsaturated fatty acid chains, most preferably l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) .
  • POPC l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine
  • the lipids of the distal lipid layer are deposited in a conventional manner, e. g. by the vesicle or mixed micelle fusion method, on the third layer to give a lipid bilayer structure.
  • Vesicle fusion and mixed micelle fusion have been described by Lang et al. (1992), Lang et al. WO 93/215280, and Lang et al. (1994), respectively. These citations are hereby incorporated by reference.
  • Vesicle fusion comprises the formation of small unilamellar vesicles (liposomes) composed of the lipids which should form the distal lipid layer and applying them onto the proximal - 19- phospholipid layer. This procedure results in the deposition of a lipid layer onto the proximal phospholipid layer. Excess vesicles are removed by washing, e.g. with buffer B.
  • Mixed micelle fusion compries detergent dilution and the formation of an aqueous dispersion composed of the lipids envisioned to form the distal lipid layer and a detergent.
  • OG is preferably selected for this purpose.
  • the mixed micelle dispersion is desposited onto the proximal phospholipid layer and is then several times (> 10) diluted in a 1:1 manner with an aqueous buffer. This procedure results in the deposition of a lipid layer onto the proximal phospholipid layer. For both procedures, care has to be taken that the resulting lipid bilayer is always covered with a layer of water.
  • proximal phospholipid layer About 40 to 50% of the device surface may remain uncovered in the previous step by the proximal phospholipid layer. However, this imperfect proximal phospholipid layer is completed and filled up with lipids upon deposition of the distal lipid layer either by vesicle or mixed micelle fusion
  • the proximal lipid monolayer of the resulting lipid bilayer is composed of phospholipids covalently attached to the (second) linking layer, and non-covalently bound lipids of the same species that constitute the distal lipid layer.
  • Step 5 Incorporation of receptor molecules into the lipid bilayer:
  • Lipids carrying chemically reactive groups may be part of the distal lipid layer. Said functionalized lipids are incorporated into the distal lipid layer during lipid deposition. Hence, functionalized lipids are conveniently already constituents of the small unilamellar vesicles or mixed micelles applied for lipid deposition.
  • Lipids carrying functional groups include as headgroup functions for example maleimides, carboxylic acids, activated carboxylic acids, primary amines, and photoactivatable headgroups.
  • Water-soluble biomolecules including enzymes, antigens, antibodies, lectins, and oligonucleotides may be covalently immobilized on such functionalized distal lipid layers through formal chemical reactions including amide formation, formation of thioethers and insertion of photogenerated intermediates into chemical bonds of target molecules.
  • Lipids bearing receptor molecules may be a constituent of the distal lipid layer. Such lipids are capable of non-covalently binding their respective water-soluble ligands to the distal lipid layer.
  • Such systems include biotinylated lipids for binding of avidin or streptavidin, avidin or streptavidin derivatives, lipid-bound peptides or proteins, antigens for the binding of their respective antibodies, glycolipids for the binding of their respective lectins, and cell receptor ligands for the binding of their respective receptor proteins.
  • Membrane bound receptor molecules may be incorporated into the lipid bilayer.
  • Said receptor molecules include proteins which are bound to the lipid membrane via a lipid anchor, or membrane proteins which cross the lipid layer once or several times and thereby extend on either side of the lipid bilayer.
  • Transmembrane proteins are preferably incorporated into a lipid bilayer membrane which is decoupled from the device surface via a second linking layer carrying several hydrophilic oligooxyethylene spacer groups. This arrangement enables receptor proteins which extend beyond the membrane to adopt a configuration which is more closely conform to that found in nature and enables them to respond to the binding of a ligand in a correspondingly natural fashion.
  • transmembrane receptor molecules are preferably inserted into micelles thus forming mixed micelles prior to application for forming the distal lipid layer by detergent dilution.
  • a solid device comprises in particular such devices, wherein said (second) linking layer provides a distance from said surface to said lipid bilayer to allow for an aqueous layer between said surface and said lipid bilayer, wherein into the lipid bilayer are inserted biological receptor molecules, e.g. such which are selected from the group consisting of antigens, haptens, lectins, bioreceptors, such as neural receptor ligands, oligonucleotides and antibodies capable of biospecifically binding with their respective analyte.
  • biological receptor molecules e.g. such which are selected from the group consisting of antigens, haptens, lectins, bioreceptors, such as neural receptor ligands, oligonucleotides and antibodies capable of biospecifically binding with their respective analyte.
  • the invention pertains in particular to a solid coated device, wherein the device is composed of waveguiding materials, such as mixtures of Si0 2 and Ti0 2 , tantalium oxide (Ta 2 0 5 ) , hafniumoxide, zirconiumoxide, or gallium arsenide.
  • waveguiding materials such as mixtures of Si0 2 and Ti0 2 , tantalium oxide (Ta 2 0 5 ) , hafniumoxide, zirconiumoxide, or gallium arsenide.
  • the invention pertains in particular to a solid device, wherein the device surface is composed of an electrically conductive material, such as a metal or metal oxide carrying OH groups.
  • the invention pertains in particular to a solid coated device, wherein the first layer is bound via a covalent silicium-oxygen bond to the surface of the device.
  • the invention pertains in particular to a solid coated device, wherein the second, linking layer is bound via a covalent carbon-carbon bond, or an ether, ester or amide bond to the first layer.
  • the invention pertains in particular to a solid device, wherein the third, proximal phospholipid layer is bound via a covalent thioether or amide bond to said second, linking layer.
  • the fourth, distal lipid layer is formed in particular from mixtures of bilayer forming lipids, glycolipids, and lipids with polymeric polar head groups, said lipids, after addition in the form of mixed micelles or liposomes to said third, proximate phospholipid layer, filling up and forming together with said proximal phospholipid layer a bilayer, which may in particular contain reconstituted or surface-linked biologi ⁇ cally active agents selected from the group consisting of antigens, haptens, antibodies, carbohydrates, extracellular proteins, trophic factors and bioreceptors including cell receptor ligands, capable of biospecifically binding with cell surface constituents.
  • the invention covers a process for the preparation of said solid coated device comprising the steps of (1) covalently attaching a first layer carrying a first group of functional groups to the uncoated device,
  • step 5 wherein said fourth, distal lipid layer is formed by contacting said third, proximal phospholipid layer with mixed micelles or small unilamellar vesicles.
  • the invention concerns the use of a present solid coated device as a biosensor.
  • the devices of this invention find particular application when used as biosensors with membrane incorporated receptor molecules, which can selectively bind drugs, proteins, viruses etc.
  • Water-soluble receptor molecules may be covalently linked to the lipid membrane, the lipid membrane acting in this particular arrangement predominantly as an interfacing layer, which suppresses unspecific analyte binding (Lang et al., WO 93/215280).
  • receptor molecules are incorporated into cell membranes via a lipid-anchor or are intrinsic membrane proteins, whose polypeptide chains cross the lipid layer of natural membranes once or several times.
  • Examples of the first type are glycosyl-phosphatidylinositol anchored proteins like acetylcolinesterase and alkaline phosphatase.
  • Members of the second type are channel forming proteins like nicotinic acetylcholine, GABA, glycine and 5-HT 3 (serotonin) receptor, and G-coupled receptor proteins like the muscarinic acetylcholine receptor and the beta-adrenergic receptor.
  • the devices of this invention will find particular application in the reconstitution of membrane-associated receptor molecules into covalently attached supported lipid bilayers and their exploitation as sensing elements in biosensors.
  • the devices of this invention find use in a variety of sensing devices, especially those wherein the lipid bilayer with incorporated receptor molecules is in intimate contact with a transducer such that changes in electrical resistance and capacitance of an electrode upon which the bilayer is mounted can be monitored (Lang et al., WO 93/215280).
  • the devices of this invention find further use in sensing devices wherein the lipid layer with incorporated receptor molecules is in intimate contact with a transducer such that the binding of ligand molecules to the receptors can be optically monitored. This is conveniently performed by measuring the changes in the effective refractive indices of guided modes using waveguiding techniques.
  • the invention further concerns the use of a present device as implantation device for the human or animal body.
  • covalently linked phospholipid bilayers on permanent (e.g. pace maker) or temporary implant devices (e.g. catheters) suppresses undesired protein adsorption, protein deposition or cell adherence.
  • the invention thus allows to covalently bind phospholipids on implant surfaces and to introduce phospholipid constituents into the second lipid layer which are effective in protein or cell repulsion.
  • a second beneficial application of the invention concerns lubrication between load bearing implant components. Friction forces in artificial metal-polymer joins which are currently in use, lead to material abrasion.
  • abrased material accumulates at the implant/tissue interphase and adverses healing.
  • Binding of molecular glycolipid films onto metal-based artificial join surfaces allows to use metal- metal joins for load bearing implants.
  • Covalently immobilized lipid bilayer films on either metal surface favorably counteract abrasion through lubrication and retention of molecular solvent layers.
  • APTES 3-aminopropyl-triethoxysilane Merck, purified by distillation under vacuum biotin-DPPE N- ( (6- (biotinoyl)amino) -hexanoyl)-1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine, Molecular Probes, Eugene, Oregon (USA) buffer A 25 mM sodium phosphate buffer, pH 8.0 buffer Ab 66 mM sodium phosphate buffer, pH 7.0, with
  • NANP peptide sequence Asn-Ala-Asn-Pro OG N-octyl-beta-D-glucopyranoside (Sigma or Bachem AG, Bubendorf / Switzerland) POPC l-palmitoyl-2-oleoyl-sn-glycero-3-phospho- choline, Avanti Polar Lipids, Alabaster/- Alabama (USA)
  • Example 1 Preparation of an optical biosensor containing a DOPS/POPC bilayer
  • Step 1 Covalent attachment of the first layer: Silanization of waveguide sensor chips with APTES
  • the above described waveguide sensor chips (ASI AG; 4.8 cm x 1.6 cm) are cleaned by incubating them for 5 min in a hot (90°C) 1:1:5 mixture of NH4OH/H2O2/H2O, followed by rinsing three times with double-distilled water.
  • the chips are then treated for 5 min with a hot (90°C) 1:1:5 mixture of HCI/H2O2/H2O and again washed extensively with double- distilled water, then rinsed three times with acetone before being vacuum dried for 12 hours at ambient temperature.
  • Silanization is performed by incubating a clean and dry single chip in 30 ml of dry toluene containing 0.5 ml (2.15 mmol) of APTES. After refluxing for about 3-4 hours the solvent is removed at the end of the reaction, and the chip is washed with chloroform (five times), acetone (twice), and methanol (five times) . The silanized chip is dried under a stream of nitrogen and kept in acetonitrile at 4 C until use. The number of 3-aminopropylsilanyl groups per sensor chip surface area is determined with ninhydrine as described by Sarin et al., 1981, and is about 1.6 nmol NH2/cm 2 (100% covered) .
  • Step 2 Covalent attachment of the second, linking layer: 3- aleimidopropionylation of the amino group of the first layer 3-Aminopropyl-silanized sensor chips are removed from acetonitrile, dried under a stream of nitrogen and stored in buffer A for at least 12 hours. The sensor chips are then placed within two tight fitting metal plates, the grating area on the waveguide surface being accessible by a circular
  • Step 3 Covalent attachment of the third, proximal lipid layer: Addition of the thiolipid DOPS to the maleimido double bond DOPSH/OG mixed micelle solutions are prepared by dissolving a dried thiolipid DOPSH film (0.5 mg) in 500 ⁇ l of a 50 mM solution of OG in buffer B. The presence of thiols in the dispersion is assayed by the development of yellow color after 1:1 mixing with Ellman reagent (10 mM DTNB in water, Riddles et al., 1983) .
  • the waveguide chip is assembled with the open O-ring cuvette and mounted on the turntable of the Integrated Optics Scanner (IOS-1) .
  • the waveguide/cuvette assembly is rinsed twice with buffer B, then the baseline of the waveguide is recorded.
  • the buffer is replaced by the mixed micelle solution and the thiolipid binding is monitored with the IOS-1.
  • the waveguide surface is rinsed with buffer B.
  • a surplus of the only physisorbed thiolipid DOPS is removed by two to three washings with each 200 ⁇ l 50 mM OG.
  • the thickness of the third layer is given in Table 1, No. 1.
  • Step 4 Non-covalent attachment of the fourth, distal lipid layer: Formation of the DOPS/POPC bilayer
  • step 4 The products of step 4 are treated a) with a vesicle dispersion (Lang et al., 1994), or b) with a mixed micelle solution of POPC (Lang et al. , 1994) to form a lipid bilayer by vesicle spreading.
  • a vesicle dispersion Liang et al., 1994
  • a mixed micelle solution of POPC Liang et al. , 1994
  • vesicle dispersion is produced by drying down a chloroform solution of 1 mg of POPC under nitrogen, adding 50 ⁇ l of buffer B to the lipid film and sonicating the aqueous mixture 3-4 times for 3 minutes in a bath-type sonicator
  • the thickness of the POPC layer as averaged over 9 experiments is shown in Table 1, No. 3.
  • the average thickness of the entire DOPS/POPC bilayer determined from 4 experiments (like the one shown in Fig. 3) is given in Table 1, No. 9.
  • the mixed micelle solution is produced by dissolving 1 g of POPC in chloroform, drying the solution under nitrogen down to obtain a film of POPC which is dissolved in 1 ml of 50 mM of OG in buffer B to give a concentration of 1 mg/ml of POPC. 200 ⁇ l of this solution is placed on the thiolipid- modified waveguide of step 3. After 5 minutes, dilution is started by adding 200 ⁇ l buffer B and removing 200 ⁇ l of the sample. This procedure is repeated 10 times, allowing the sample to equilibrate between the dilution cycles for at least 1.5 minutes.
  • Example 2 Preparation of an optical biosensor containing a DMPS/POPC bilayer
  • Step 3 the thiolipid DMPS is added to the maleimido double bonds.
  • Step 4 a DMPS/POPC bilayer is produced from the product of Step 3 and POPC.
  • the average thickness of 3 experiments of the POPC layer is shown in Table 1, No. 5 (from micelles) .
  • Example 3 Preparation of an optical biosensor containing a DOPS/POPC/2% biotin bilayer (see Fig. 3) In analogy to Example 1, Step 4, a), a DOPS/POPC/2% biotin bilayer is produced from the product of Step 3 and a mixture of POPC and biotin-DPPE.
  • Example 4 Preparation of an optical biosensor containing a DMPS/POPC/2% lipopeptide bilayer (see Fig. 4)
  • the lipopeptide used is that shown under Sources and Abbreviations.
  • Step 4 b) a DMPS/POPC/2% lipopeptide bilayer is produced from the product of Example 2, Step 3 and POPC/2% lipopeptide.
  • dilution of mixed micelle solutions is used exclusively for producing the second layer on top of the thiolipid layer.
  • Mixtures of POPC and lipopeptide dissolved in chloroform: methanol 1:1 (v:v) are dried and subsequently dissolved in 50 mM of OG solution in buffer B (final concentration 1 mg lipid/ l solution) .
  • 200 ⁇ l of this solution is placed on the thiolipid-modified waveguide of Example 3.
  • dilution is started by adding 200 ⁇ l of buffer B, mixing, and removing 200 ⁇ l of the sample. This procedure is repeated 10 times, allowing the sample to equilibrate between the dilution cycles for at least 1.5 minutes.
  • Example 5 Preparation of an optical biosensor containing DOPS/POPC/lipopeptide bilayer
  • a DOPS/POPC/lipopeptide bilayer is produced from the product of Example 2, Step 3, and mixed micelles of POPC and different lipopeptide ratios varying from 0.5-4%.
  • the average thickness of the POPC/lipopeptide layer of 9 experiments is shown in Table 1, No. 8 (from micelles) .
  • Non-covalent binding of Streptavidin to biotin-containig bilayers of Example 3 is measured by injecting a solution of streptavidin in buffer B into the cuvette volume, to give final concentrations of 0.4 - 1.7 ⁇ M streptavidin. Binding is allowed to take place for 5-45 minutes, then the unbound strepavidin is removed from the reaction solution by dilution (10 times 1:1 with buffer B) .
  • Antibody (monoclonal anti- (NANP) n -antibody Sp3E9) binding to POPC bilayers of Example 4 containing 0-4 mol % of lipopeptide is initiated by injecting a solution of 0.3 mg/ml Anti- (NANP)n antibody in Ab-buffer to give final antibody concentrations of 100-200 nM. The binding is allowed to continue for 15-40 minutes, then unbound antibodies are removed by washing with Ab-buffer.
  • Specifically bound antibodies are displaced from the membrane surface by adding 66 ⁇ l of a 0.6 mg/ml (NANP) 6 solution in Ab-buffer (final (NANP) 6 concentration 75 ⁇ M) .
  • Example 8 Preparation of an implantation device containing a DOPS/POPC bilayer
  • Titanium implant surfaces e.g. the headpiece or the corresponding pan of an hip joint, carrying a vapor deposited surface layer of titanium nitride are treated with 10 % nitric acid in distilled water during 20 minutes at 80 °C. This treatment generates hydroxyl functions on the surface of the titanium substrate. The surface is rinsed four times with bidistilled water.
  • Step 1 Covalent attachment of the first layer: Silanization of the implantaion device with APTES
  • Silanization of the activated surfaces is performed by incubating the surface in 150 ml of dry toluene containing 2.5 ml (10.75 mmol) of APTES. The solvent is removed at the end of the reaction, and the implant surface is washed with chloroform (five times) , acetone, (twice) , and methanol (five times) . Headpiece and recipient pan surfaces are dried with nitrogen and stored in acetonitrile at 4 °. Silanized device surfaces are washed twice with each 150 ml buffer A.
  • Step 2 Covalent attachment of the second layer: 3- Maleimidopropionylation of the amino group of the first layer:
  • the silanized device surface of Step 1 is treated with 200 ml of 25 mM heterobifunctional crosslinker SMP in buffer A/DMF 4:1 (v/v). After incubation for 30 min at ambient temperature, excess reagent is removed by washing once with DMF and ten times with buffer B.
  • Step 3 Covalent attachment of the third layer: Addition of a thiolipid to the maleimido double bond Covalent thiolipid binding to the 3-maleimidopropyl group of the linker modified surfaces of Step 2 is carried out in situ using teflon coated counter part mimics to displace the solvent and thus reduce the total volume required for thiolipid binding.
  • Teflon coated pan mimics are used for headpiece modification.
  • the Teflon coated counterparts are perforated for solvent (reagent) inlet. Inlets density: one perforation per cm 2 , perforations are connected to a feeder tubing and a solvent delivery system. The setup allows homogeneous surface perfusion.
  • Thiolipid e.g.
  • DOPS or DMPS, and OG containing mixed micelle solutions are prepared by dissolving a dried thiolipid film (5 mg) in 5 ml 50 mM OG in buffer B. The reaction is carried out for 4 hours at ambient temperature and physically adsorbed lipid is removed by washing with 50 mM OG (twice) and buffer B (5 times) .
  • Step 4 Non-covalent attachment of the fourth layer: Formation of lipid/lipid bilayer
  • the second lipid layer is formed by incubation of the surface product of Step 3 with heterologous (soybean, egg yolk) , synthetic or autologous (extracted from red cells or fat tissue) lipids. Formation of the second lipid layer on top of the thiolipid layer is attained by dissolving the chosen lipid or lipid mixtures (e.g. egg PC) in chloroform: methanol 1:1 (v:v) .
  • the lipid is dried and dissolved in 50 mM of OG in buffer B. Mounted in the counter part mimic, the lipid/OG solution is brought in contact with the thiolipid modified layer of Step 3 by the solvent delivery system. After 15 min, detergent dilution is initiated by dispensing buffer B at a rate of 0.4 ml/min via the feeder tubings during 50 min. Bilayer covered joint surfaces are washed with and stored in saline until use.
  • Lipid bilayers were produced by first binding thiolipid from mixed micelle solutions to waveguides and subsequently assembling phospholipids or mixtures of phospholipids either with biotinylated lipids or with lipopeptides, by the method indicated in brackets.
  • LP stands for (NANP) 3-lipopeptide.

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Abstract

L'invention porte sur un système solide présentant un revêtement fixé par covalence comprenant: une première couche contenant de premiers groupes fonctionnels, auxquels est liée par covalence une deuxième couche de liaison portant de seconds groupes fonctionnels; une troisième couche proximale de phospholipides est liée par covalence à la deuxième couche, une quatrième couche distale de lipides étant liée de manière non covalente à la troisième couche, ceci de manière telle que les couches proximale et distale de lipides forment ensemble une couche double dans laquelle peuvent être insérées des molécules réceptrices. L'invention porte également sur leur procédé de préparation et leur utilisation comme biocapteurs ou dispositifs d'implantation.
PCT/IB1996/000496 1995-05-30 1996-05-24 Couches doubles de phospholipides immobilisees par covalence sur des surfaces solides WO1996038726A1 (fr)

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EP0847766A2 (fr) * 1996-12-10 1998-06-17 SORIN BIOMEDICA S.p.A. Dispositif pour implantation et trousse contenant ce dispositif
WO2001032230A2 (fr) * 1999-11-04 2001-05-10 Kazunori Kataoka Micelle polymere comme monocouche ou surface a couches stratifiees
US6329209B1 (en) 1998-07-14 2001-12-11 Zyomyx, Incorporated Arrays of protein-capture agents and methods of use thereof
DE10048822A1 (de) * 2000-09-29 2002-04-18 Nimbus Biotechnologie Gmbh Verfahren zur Immobilisierung von Lipidschichten
WO2002072873A1 (fr) * 2001-03-14 2002-09-19 Biacore Ab Procede de preparation de membranes a film lipidique sur support et utilisation de celles-ci
WO2002074983A1 (fr) * 2001-03-15 2002-09-26 Iongate Biosciences Gmbh Ensemble de detection et dispositif et procede pour controler des substances actives et/ou des sites actifs d'un point de vue pharmacologique a l'aide d'un amperemetre et/ou d'un potentiometre
EP1283270A1 (fr) * 2001-08-07 2003-02-12 Warner-Lambert Company Nouvelle membrane supportée, préparation et utilisations
US6576478B1 (en) 1998-07-14 2003-06-10 Zyomyx, Inc. Microdevices for high-throughput screening of biomolecules
WO2003052420A2 (fr) * 2001-10-03 2003-06-26 Purdue Research Foundatio Dispositif et procedes utilisant une membrane biofonctionnalisee asymetrique
US6682942B1 (en) 1998-07-14 2004-01-27 Zyomyx, Inc. Microdevices for screening biomolecules
US6745156B2 (en) 1997-04-24 2004-06-01 Bright Ideas, L.L.C. Petroleum exploration and prediction apparatus and method
US6872522B1 (en) 1996-06-25 2005-03-29 Michael Mecklenburg Broad specificity affinity arrays: a qualitative approach to complex sample discrimination
US6897073B2 (en) 1998-07-14 2005-05-24 Zyomyx, Inc. Non-specific binding resistant protein arrays and methods for making the same
US6904367B2 (en) 2002-10-04 2005-06-07 Daniel R. Cook Petroleum exploration and prediction apparatus and method
US6952649B2 (en) 2002-10-04 2005-10-04 Cook Daniel R Petroleum exploration and prediction apparatus and method
US7037517B2 (en) 1999-11-04 2006-05-02 Johnson & Johnson Vision Care, Inc. Polymer micelle as monolayer or layer-laminated surface
US7135343B2 (en) 2002-06-17 2006-11-14 Agilent Technologies, Inc. Biomolecule resistant and their methods of use in assays
WO2006127167A2 (fr) * 2005-04-12 2006-11-30 Sru Biosystems, Inc. Biodetecteur de membrane proteolipidique et de membrane lipidique
WO2008021290A2 (fr) 2006-08-09 2008-02-21 Homestead Clinical Corporation Protéines spécifiques d'organes et procédés d'utilisation
WO2008077248A1 (fr) * 2006-12-22 2008-07-03 Miv Therapeutics Inc. Revêtements pour des dispositifs médicaux implantables pour une administration de liposomes
US8111401B2 (en) 1999-11-05 2012-02-07 Robert Magnusson Guided-mode resonance sensors employing angular, spectral, modal, and polarization diversity for high-precision sensing in compact formats
EP2444464A1 (fr) * 2010-10-21 2012-04-25 Centre National de la Recherche Scientifique (CNRS) Nouveau (bio)matériau neutre
US8293542B2 (en) 2000-10-30 2012-10-23 X-Body, Inc. Real time binding analysis of antigens on a biosensor surface
US9134307B2 (en) 2007-07-11 2015-09-15 X-Body, Inc. Method for determining ion channel modulating properties of a test reagent
US9206410B2 (en) 2009-03-03 2015-12-08 Grifols Therapeutics Inc. Compositions, methods and kits for preparing plasminogen and plasmin prepared therefrom
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US10359573B2 (en) 1999-11-05 2019-07-23 Board Of Regents, The University Of Texas System Resonant waveguide-granting devices and methods for using same
US10865409B2 (en) 2011-09-07 2020-12-15 X-Chem, Inc. Methods for tagging DNA-encoded libraries
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US6745156B2 (en) 1997-04-24 2004-06-01 Bright Ideas, L.L.C. Petroleum exploration and prediction apparatus and method
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US6329209B1 (en) 1998-07-14 2001-12-11 Zyomyx, Incorporated Arrays of protein-capture agents and methods of use thereof
US6897073B2 (en) 1998-07-14 2005-05-24 Zyomyx, Inc. Non-specific binding resistant protein arrays and methods for making the same
US6475808B1 (en) 1998-07-14 2002-11-05 Zyomyx, Incorporated Arrays of proteins and methods of use thereof
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US7037517B2 (en) 1999-11-04 2006-05-02 Johnson & Johnson Vision Care, Inc. Polymer micelle as monolayer or layer-laminated surface
US7674478B2 (en) 1999-11-04 2010-03-09 Johnson & Johnson Vision Care, Inc. Polymer micelle as monolayer or layer-laminated surface
WO2001032230A3 (fr) * 1999-11-04 2001-12-20 Kazunori Kataoka Micelle polymere comme monocouche ou surface a couches stratifiees
WO2001032230A2 (fr) * 1999-11-04 2001-05-10 Kazunori Kataoka Micelle polymere comme monocouche ou surface a couches stratifiees
US8111401B2 (en) 1999-11-05 2012-02-07 Robert Magnusson Guided-mode resonance sensors employing angular, spectral, modal, and polarization diversity for high-precision sensing in compact formats
US10359573B2 (en) 1999-11-05 2019-07-23 Board Of Regents, The University Of Texas System Resonant waveguide-granting devices and methods for using same
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US8293542B2 (en) 2000-10-30 2012-10-23 X-Body, Inc. Real time binding analysis of antigens on a biosensor surface
WO2002072873A1 (fr) * 2001-03-14 2002-09-19 Biacore Ab Procede de preparation de membranes a film lipidique sur support et utilisation de celles-ci
WO2002074983A1 (fr) * 2001-03-15 2002-09-26 Iongate Biosciences Gmbh Ensemble de detection et dispositif et procede pour controler des substances actives et/ou des sites actifs d'un point de vue pharmacologique a l'aide d'un amperemetre et/ou d'un potentiometre
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US7135343B2 (en) 2002-06-17 2006-11-14 Agilent Technologies, Inc. Biomolecule resistant and their methods of use in assays
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WO2012052539A1 (fr) * 2010-10-21 2012-04-26 Centre National De La Recherche Scientifique (Cnrs) Nouveau (bio)matériau neutre
EP2444464A1 (fr) * 2010-10-21 2012-04-25 Centre National de la Recherche Scientifique (CNRS) Nouveau (bio)matériau neutre
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WO2016109423A1 (fr) * 2014-12-30 2016-07-07 X-Chem, Inc. Procédés de marquage de banques codées par de l'adn
CN114206485A (zh) * 2019-08-01 2022-03-18 米麦斯生物有限公司 一种活性磷脂膜及其相关制备方法
CN114206485B (zh) * 2019-08-01 2024-03-19 米麦斯生物有限公司 一种活性磷脂膜及其相关制备方法
WO2022067358A1 (fr) * 2020-09-25 2022-03-31 Stellenbosch University Revêtement de matériaux avec des composés biotensioactifs
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