US20110008902A1 - Method and device for studying transport of an agent across a bilayer membrane in bioanalytical sensor applications - Google Patents

Method and device for studying transport of an agent across a bilayer membrane in bioanalytical sensor applications Download PDF

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US20110008902A1
US20110008902A1 US12/863,499 US86349909A US2011008902A1 US 20110008902 A1 US20110008902 A1 US 20110008902A1 US 86349909 A US86349909 A US 86349909A US 2011008902 A1 US2011008902 A1 US 2011008902A1
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bilayer structure
membrane
sensor
liposomes
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Fredrik Hook
Magnus Branden
Seyed Tabaei
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Layeriab AB
Bio Rad Laboratories Inc
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Layeriab AB
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • 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
    • 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
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N2021/258—Surface plasmon spectroscopy, e.g. micro- or nanoparticles in suspension
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G01N21/211—Ellipsometry
    • 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
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
    • G01N2333/43565—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from bees
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a method and a device for the measurement of transport of an agent across a bilayer membrane.
  • SPR Surface plasmon resonance
  • liposomes in a solution.
  • a membrane protein of interest To the liposome membrane there are associated for instance a membrane protein of interest and the membrane protein mediated transport of an agent across the membrane is studied using methods involving for instance fluorescence and/or radioactivity measurements.
  • US 2004/0033624 disclose a membrane receptor reagent and assay device.
  • Liposomes are tethered to a surface with anchor groups.
  • the surface comprises reagent ligands that are tethered to the surface.
  • the ligands are able to bind reversibly to a receptor in the liposome membrane.
  • the membrane protein in the liposome is associated with a molecule with the capability to be excited by emitted energy from the surface and thereby produces a detectable signal.
  • the binding of the membrane protein in the liposome membrane to the reagent ligands on the surface tend to pull the liposomes towards the surface.
  • a test molecule with affinity to the membrane protein will bind competitively to the membrane protein and to some extent replace the reagent ligand on the surface. This will result in that membrane proteins come off the surface and are redistributed in the liposome membrane, while the liposome still is anchored to the surface. The membrane proteins will have a larger average distance from the surface and thereby they receive less energy from the surface, since the energy transfer from the surface is distance dependent. This can be detected.
  • the method in US 2004/0033624 requires some kind of label such as a fluorophore in order to function.
  • bilayer structure denotes a double layer structure of atoms or molecules and especially lipids.
  • the term encompasses bilayers of all geometries including but not limited to curved bilayers. Examples of bilayer structures include but are not limited to liposomes.
  • detection volume as used throughout the description and the claims denotes a volume in which the refractive index is measured.
  • ellipsometry sensor as used throughout the description and the claims denotes a sensor comprising an ellipsometer.
  • ionophore as used throughout the description and the claims denotes a lipid-soluble molecule with the capability to transport ions across a lipid bilayer.
  • lipid as used throughout the description and the claims denotes any fat-soluble molecules.
  • examples of lipids include but are not limited to; fats, oils, waxes, cholesterol, sterols, monoglycerides, diglycerides, and phospholipids.
  • liposome as used throughout the description and the claims denotes an essentially spherical vesicle comprising a lipid bilayer membrane. Liposomes may comprise a core of an aqueous solution.
  • the lipid membrane of the liposome may comprise components such as but not limited to proteins, glycolipids, steroids and other membrane-associated components.
  • membrane as used throughout the description and the claims comprises all types of membrane such as but not limited to a bilayer membrane.
  • a membrane may comprise molecules such as but not limited to proteins and lipids.
  • membrane protein as used throughout the description and the claims denotes a protein which is associated with a membrane.
  • sensor denotes a transducer which uses a type of energy, a signal of some sort, and converts it into a reading for the purpose of information transfer.
  • spacer denotes a molecule that is used to link together other molecules so that there is a space between the linked molecules.
  • a surface can in the present invention be used to support means on which structures can be tethered.
  • surface plasmon resonance sensor as used throughout the description and the claims denotes a sensor utilising the excitation of surface plasmons by light.
  • tether as used throughout the description and the claims denotes the attachment or entrapment of a material to a surface in a manner that confines, but not necessarily restricts the movement of the material.
  • the present invention provides a method for studying transport of an agent across a membrane comprising the steps
  • the bilayer structure is in contact with a solvent.
  • the solvent is water.
  • the bilayer structure comprises at least one membrane.
  • At least one membrane protein is associated with the bilayer structure. If it is desired to study transport across a membrane which is mediated by a membrane protein, the membrane protein is in one embodiment inserted in the bilayer membrane. Also transport of other molecules on the transport across a membrane can be studied. Examples include but are not limited to passive diffusion of molecules across membranes and transport which is facilitated by for example ionophores and permeation enhancers.
  • a permeation enhancer is a molecule which is added to the solution comprising the analyte and which influences the transport across the membrane.
  • At least one entity from the group consisting of a membrane protein, and an ionophore is associated with the bilayer structure.
  • the surface comprises a detection volume in which the refractive index is measured.
  • the bilayer structure tethered to the surface is entirely or partly within the detection volume.
  • the detection volume is a volume limited by the surface and a plane 250 nm from the surface and parallel to the surface.
  • the sensitivity of the detection volume decreases exponentially with the distance from the surface.
  • the detection volume is limited to a volume where the detection sensitivity is more than 1% of the maximum sensitivity at the surface.
  • An example of an embodiment where the sensitivity of the detection volume decreases exponentially with the distance from the surface includes but is not limited to an embodiment comprising a surface plasmon resonance sensor.
  • the bilayer structure is such that an analyte can not diffuse or move freely across the bilayer structure because of the bilayer. However, the analyte can still pass through the membrane.
  • the bilayer structure comprises a volume which is at least partly enclosed by the bilayer.
  • the analyte to be investigated is in one embodiment added to the solvent in contact with the bilayer structure. Depending on the properties of the analyte, it may be transferred very slowly or hardly at all across the bilayer membrane. For some analytes, the analyte may be transferred across the bilayer membrane at a higher rate. The transport of an analyte across the membrane depends on the properties of the analyte and on the molecules in the bilayer.
  • At least one molecule to be studied there is in one embodiment associated at least one molecule to be studied.
  • molecules include but are not limited to; ionophores, integral membrane spanning proteins, proteins binding to one side of the bilayer through a hydrophobic patch, mainly hydrophilic proteins attaching hydrophobically through a covalently bound alkyl chain, mainly hydrophilic proteins with a hydrophobic pocket that can attract a lipid chain, and hydrophilic proteins binding electrostatically.
  • the present invention provides a possibility to study transport across a bilayer membrane facilitated by a molecule.
  • the transport of the analyte across the bilayer membrane is measured by measuring the refractive index.
  • analytes include but are not limited to positive ions, negative ions, drug molecules, organic molecules, inorganic molecules, receptor ligands, sucrose, DNA, RNA, peptides, and proteins.
  • an analyte is added to the solvent in contact with the bilayer structure and is spread over a volume which is on one side of the bilayer in the bilayer structure.
  • the analyte dissolved in the solvent at a certain concentration has one refractive index.
  • concentration of the analyte may be different, which will give a different refractive index for that volume.
  • the senor utilises a technique that measures refractive index changes in close proximity to the surface. In one embodiment the sensor has the capability to measure changes in refractive index in the interval 0-250 nm from the surface.
  • the method comprises detecting a change in refractive index with a sensor selected from the group consisting of a surface plasmon resonance sensor, an ellipsometry sensor, and an optical waveguide laser spectroscopy sensor.
  • the bilayer structure comprises at least one liposome. In a further embodiment the bilayer structure comprises at least one layer of liposomes. In still a further embodiment the bilayer structure is a layer of tethered liposomes. In another embodiment the bilayer structure comprises at least two layers of liposomes. In yet another embodiment the bilayer structure is a multitude of liposomes tethered to the surface.
  • the liposomes are tethered to the surface with a spacer molecule. In one embodiment where there are several layers of liposomes there is at least one spacer that tethers the liposomes in one layer to the liposomes in an adjacent layer.
  • the method comprises measuring the refractive index in a volume enclosed by the bilayer structure.
  • the method comprises measuring the refractive index in a volume enclosed by liposomes tethered to the surface.
  • the method comprises measuring the refractive index in a volume enclosed by at least one liposome tethered to the surface.
  • the bilayer structure encloses a first volume of the detection volume and wherein the volume not enclosed by the bilayer structure but within the detection volume is a second volume and wherein the ratio between the first and second volumes is optimised for measurement of refractive index of the first volume.
  • the first volume should be as large as possible compared to the second volume.
  • a large first volume will give higher sensitivity.
  • suitable ratios between the first volume and the second volume include but are not limited to 0.001, 0.01, 0.1, 1, and 10. Also higher values of the ratio are encompassed by the present invention such as 20, 50, 100, 500, and 1000.
  • the presently invented method to study cell-membrane permeation advantageously admits direct measurements of the transfer rate of both uncharged and charged solutes across biological membranes.
  • the method is based on resolving the temporal change in refractive index upon a permeation-dependent change in the solute concentration inside liposomes confined to the evanescent field associated with, for example, a surface plasmon resonance active sensor surface.
  • a device comprising;
  • the ratio between the first volume and second volume is above about 0.001, preferably above about 0.01, more preferably above about 0.1, even more preferably above about 1, and most preferably above about 10.
  • a high ratio is desired because it leads to a better sensitivity for the refractive index measurement in the first volume.
  • One advantage of the present invention is that the sensitivity and accuracy can be increased by providing a high ratio.
  • the bilayer structure comprises at least one liposome tethered to the surface. In yet another embodiment the bilayer structure comprises a multitude of liposomes tethered to the surface.
  • the at least one liposome is tethered to the surface with at least one spacer.
  • spacers include but are not limited to a polymer, a nucleic acid, DNA, a His-tag, a biotinylated lipid attached to avidin covalently bound to the surface, and hydrophobically modified dextran. Also any combination of the above mentioned spacers can be used.
  • the at least one spacer is a polymer. In another embodiment the at least one spacer is a His-tag. In a further embodiment the there are both polymer spacers and His-tag spacers.
  • the distance between the bilayer structure and the surface is less than about 250 nm.
  • the sensor which measures the refractive index in the detection volume is selected from the group consisting of a surface plasmon resonance sensor, an ellipsometry sensor, and an optical waveguide laser spectroscopy sensor.
  • the senor utilizes the phenomenon surface plasmon resonance.
  • FIG. 1 shows the successive addition of Neutravidin/Biotin DNA (N/B), 0.1 M sucrose (S1), 2 mg/ml cholesterol DNA tagged POPC-liposomes (POPC), 0.1 M sucrose (S2), 40 ⁇ M melittin, 0.1 M sucrose (S3), 0.1 M sucrose (S4), 0.1 M sucrose (S5) to a PEG/PEG-biotin functionalized Biacore® sensor.
  • FIG. 2 shows the response (RU) from the five sucrose additions (S1-S5).
  • the insert shows a partial enlargement of FIG. 2 .
  • FIG. 3 shows the uptake of sucrose into the liposomes as a function of time, which is obtained by subtracting the change in RU upon addition of sucrose to closed liposomes (S2) from the change in RU upon addition of S3-S5.
  • FIGS. 4 a and 4 b show liposome permeability for glycerol, urea and hydroxyurea.
  • FIGS. 5 a and 5 b show the release and transport rate for glycerol over different temperatures.
  • FIGS. 6 a to 6 c show the behaviour of cholesterol comprising liposomes when subjected to cyclodexterin.
  • FIG. 7 illustrates membrane transport of iodide and chloride ions.
  • 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phophocholine was purchased from Avanti Polar Lipids®.
  • PEG-polymers (3 kDa), HS-PEG-NHCO-CH 2 CH 2 -OH (HS-PEG) HS-PEG-NHCO-CH 2 CH 2 -Biotin (HS-PEG-Biotin) were purchased from Rapp Polymere® GmbH.
  • the Sensor chips were purchased from GE Healthcare®.
  • the membrane protein analysis kit came from LayerLab®.
  • HEPES-2 is 10 mM HEPES, 150 mM NaCl, pH 7.4.
  • Liposomes were made by first evaporating the solvent, chloroform, from the POPC lipids using a flow of nitrogen gas. The dried lipids were then kept under the flow of nitrogen gas for at least one hour after which HEPES-2 was added to yield a 4 mg/ml concentration of POPC. The dissolved POPC lipids were vortexed for at least 30 min to yield multi-laminar liposomes. Uni-laminar liposomes were produced using a mini extruder and polycarbonate membranes with pore size of 50 nm from Avanti Polar Lipids® with a 50 nm pore size. The radiuses of the liposomes had a Gaussian size distribution with a mean radius of 36 nm. The liposomes were stored under nitrogen gas at 4° C. until use (within 2 days).
  • POPC liposomes (2 mg/ml, in HEPES-2) containing cholesterol-DNA tags (3 DNA tags per liposome) were injected at a flow rate of 5 ⁇ l/min.
  • the end of the DNA-part of the cholesterol-DNA tag is single-stranded and complementary in sequence to the Biotin-DNA at the surface enabling tethering of the liposomes to the sensor surface.
  • the bilayer structure enclosed a first volume of the detection volume and the volume not enclosed by the bilayer structure but within the detection volume is a second volume.
  • the ratio between the first and second volumes was in this particular case about 0.1.
  • FIG. 1 shows the successive addition of Neutravidin/Biotin DNA (N/B), 0.1 M sucrose (S1), 2 mg/ml cholesterol DNA tagged POPC-liposomes (POPC), 0.1 M sucrose (S2), 40 ⁇ M melittin, 0.1 M sucrose (S3), 0.1 M sucrose (S4), 0.1 M sucrose (S5) to a PEG/PEG-biotin functionalized surface.
  • FIG. 2 shows the response (RU) from the five sucrose additions (S1-S5).
  • sucrose changes the refractive index of the solution in the detection volume, i.e. the volume corresponding to the evanescent field above the sensor surface, which is manifested as a shift in RU.
  • the smaller increase in RU upon adding S2 compared to adding S1 corresponds to the reduced accessible volume of the evanescent field caused by the surface-bound liposomes.
  • Addition of melittin produces pores in the liposomes membrane and hence makes the interior volume of the liposomes accessible to the added sucrose molecules.
  • FIG. 2 shows the change in RU upon addition of sucrose as the number of pores/liposome is reduced (S3-S5).
  • FIG. 3 shows the uptake of sucrose into the liposomes as a function of time, which is obtained by subtracting the change in RU upon addition of sucrose to closed liposomes (S2) from the change in RU upon addition of S3-S5.
  • the amplitude of the uptake decrease from S3 to S5, which is explained by a decreasing number of liposomes having pores.
  • the rate also decreases from S3 to S5, which is explained by decreasing number of pores per liposome.
  • the amplitude of the response is in good agreement with the value that can be expected from filling the liposomes with 0.1 M of sucrose, which have a molecular weight (Mw) of 0.34 kDa.
  • Mw molecular weight
  • the Mw of the liposome is 35800 kDa, which gives 0.19 RU/kDa.
  • the efficiency of the SPR-based method and its compatibility with screening of multiple permeation events was evaluated by investigating the permeability of the non-electrolytes glycerol, urea and hydroxyurea, which are all biologically relevant molecules.
  • glycerol is transported across the membranes of the adipocytes, where fat molecules are stored, has been suggested to be an important factor in the development of obesity and type II diabetes [17].
  • Urea is a waste product in the metabolic process and is transported out from liver cells and released from the body via urine, while hydroxyurea is a drug that has been widely used in cancer chemotherapy [18] as well as in treatment of HIV-virus infections [19].
  • the applicability of the method is demonstrated for in situ alteration and simultaneous monitoring of liposome permeability by monitoring the increase in glycerol permeation upon a gradual cyclodextrin-induced reduction in the liposomal cholesterol content. Also shown is the compatibility of the method to probe transport of ions.
  • FIG. 4 a shows binding of 70 nm liposomes followed by repeated injection pulses of glycerol, urea, hydroxyurea at different concentrations (see inset).
  • FIG. 4 b shows a magnification of one of the rinsing steps, upon which the solute (glycerol) is released from the liposome interior. Also shown in FIG. 4 b (dashed curve) is an identical rinsing step without immobilized liposomes, illustrating that the time constant of the fluidic exchange is faster than 100 ms, which is sufficient to temporally resolve these transport measurements. The rate of glycerol release was examined in a temperature interval from 8 to 22° C.
  • FIGS. 4 and 5 demonstrate that the method can be used to accurately determine permeation coefficients of lipid membranes by following the release of solutes, rather than the uptake. Note that all these data were obtained from a single experiment using the same set of immobilized liposomes. The reproducibility between different experiments was better than 2%.
  • Example 7 70 nm liposomes containing 40% cholesterol were provided in accordance with Example 1. Binding of the liposomes followed by repeated injection cyclodextrin, which removes cholesterol from the lipid bilyaer of the liposomes is shown in FIG. 6 a .
  • the SPR response is proportional to the interfacial refractive index, which makes it possible to quantify the removal of cholesterol. After seven injections of cyclodextrin, the cholesterol content was reduced to 14%.
  • FIG. 6 a Each injection of cyclodextrin ( FIG. 6 a ) was followed by an injection pulse of glycerol, which enabled the time constant of glycerol transport versus cholesterol content to be quantified, ranging from around 7 s at 40% cholesterol to 1 s at 14% cholesterol, see FIG. 6 b . Note that a difference in transport rate was detectable for as little as 1% change in cholesterol content.
  • FIG. 6 c shows the variations in permeability (estimated from the rate of solute transfer and the vesicle area) and the amplitude of the response (which is proportional to the internal volume).
  • the permeability decreases linearly versus cholesterol content, while the internalized volume increases non-linearly.
  • the latter observation reflects a structural change of the membrane at a cholesterol content of around 25%.
  • the method according to the invention enables screening of multiple permeation events in a single measurement, and allows for in situ alterations in permeability to be quantified, which is exemplified by successive removal of cholesterol from the lipid bilayer.
  • the method improves the sensitivity and reduces the required amount of sample by orders of magnitude.
  • solute transport across the lipid bilayer membrane of liposomes immobilized in an electromagnetic evanescent field can be determined by following the changes in refractive index of the liposome-internalized volume upon solute release, rather than uptake ( FIG. 4 ). It is also demonstrated that the membrane properties can be varied in situ, and be correlated to changes in permeability ( FIG. 5 ). In FIG. 6 , we demonstrate that not only uncharged solutes, but also the transport of ions can be measured by following changes in the refractive index of the liposome-internalized volume upon solute release (or uptake).
  • the present invention admits the possibility of using this sensing principle for direct measurements of molecular transport of non-electrolyte solutes across membranes with a time resolution of 100 ms.
  • the method is unique in the sense that it enables direct, rather than indirect (c.f. the DLS and fluorescence quenching methods), measurements of solute transfer across lipid bilayers, which was so far possible only for charged molecules using patch-clamp [14] or chip-based alternatives [15].
  • it also offers all other advantages of surface-based methods, such as rapid sequential (or parallel) screening of multiple solutes as well as in situ perturbation of liposome permeability by addition of effector molecules.

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US9322830B2 (en) 2008-02-08 2016-04-26 Bio-Rad Laboratories, Inc. Method for studying transport of an agent across a bilayer membrane in bioanalytical sensor applications

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JP7179330B2 (ja) * 2019-01-28 2022-11-29 国立大学法人福井大学 脂質二重膜の水透過性の評価システム、脂質二重膜の水透過性の評価方法、および、脂質二重膜の水透過性を制御する薬剤のスクリーニング方法

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US20070116733A1 (en) * 2003-04-07 2007-05-24 Annette Graneli Surface immobilised multilayer structure of vesicles

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US20110008902A1 (en) 2008-02-08 2011-01-13 Layeriab Aktiebolag Method and device for studying transport of an agent across a bilayer membrane in bioanalytical sensor applications

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US20070116733A1 (en) * 2003-04-07 2007-05-24 Annette Graneli Surface immobilised multilayer structure of vesicles

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US9322830B2 (en) 2008-02-08 2016-04-26 Bio-Rad Laboratories, Inc. Method for studying transport of an agent across a bilayer membrane in bioanalytical sensor applications
US9784746B2 (en) 2008-02-08 2017-10-10 Bio-Rad Laboratories, Inc. Method for studying transport of an agent across a bilayer membrane in bioanalytical sensor applications

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EP2250502A4 (de) 2011-04-27
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US20160231326A1 (en) 2016-08-11
JP5302341B2 (ja) 2013-10-02
US20150031139A1 (en) 2015-01-29
JP2011512526A (ja) 2011-04-21
CN101939647B (zh) 2015-04-15
CN101939647A (zh) 2011-01-05
US9784746B2 (en) 2017-10-10
EP2250502B1 (de) 2014-05-07

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