EP1180242A1 - Vesicule contenant des polymeres et procedes de mise en evidence par detecteur, fondes dessus - Google Patents

Vesicule contenant des polymeres et procedes de mise en evidence par detecteur, fondes dessus

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Publication number
EP1180242A1
EP1180242A1 EP00938651A EP00938651A EP1180242A1 EP 1180242 A1 EP1180242 A1 EP 1180242A1 EP 00938651 A EP00938651 A EP 00938651A EP 00938651 A EP00938651 A EP 00938651A EP 1180242 A1 EP1180242 A1 EP 1180242A1
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EP
European Patent Office
Prior art keywords
detection method
biological
vesicle
sensor platform
bioanalytical
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EP00938651A
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German (de)
English (en)
Inventor
Dietmar KRÖGER
Horst Vogel
Michael Pawlak
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Bayer AG
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Zeptosens AG
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Publication of EP1180242A1 publication Critical patent/EP1180242A1/fr
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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
    • G01N33/54366—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

Definitions

  • the invention relates to a functionalized, polymer-reinforced (sterically stabilized) vesicle, which comprises a biological or biochemical recognition element for the recognition and binding of a ligand, a method for the production and its use.
  • a reagent for bioanalytical applications both in solution and on a solid surface, which ensures the functionality and native conformation of a biological molecule serving as a recognition element, for example membrane receptors, for the detection of a ligand specifically binding to it and at the same time minimized non-specific interactions with this recognition element.
  • the present invention relates to a biological or biochemical reagent comprising
  • Another object of the invention is a functionalized, polymer-reinforced (sterically stabilized) vesicle, which additionally contains labels as signal-generating components in a bioanalytical method, which can be selected from the group of ESR or NMR spin labels, mass labels, electrochemical labels , Luminescence labels or fluorescence labels.
  • the at least one label as a signal-generating component can be located inside the vesicle or can be bound to the outer shell of the vesicle directly or via a spacer or via the polymer.
  • a large number of similar labels, in particular of similar luminescent or fluorescent labels, can also be associated with the vesicle.
  • luminescent or fluorescent labels can be conventional luminescent or fluorescent labels or so-called luminescent or fluorescent nanoparticles based on semiconductors (WCW Chan and S. Nie, "Quantum dot bioconjugates for ultrasensitive nonisotopic detection", Science 281 (1998) 2016 - 2018) act.
  • the invention further relates to methods for producing said functionalized vesicle and its use in bioanalytical detection methods. These can be selected from the group consisting of the detection of optical signal changes, electrochemical detection, impedance spectroscopy, electron spin resonance, nuclear magnetic resonance, quartz crystal measurements or a combination of these methods.
  • Another object of the invention is a method for producing a biological or biochemical reagent according to the invention, characterized in that
  • C) brings together at least one biological or biochemical or synthetic recognition element to be bound or adsorbed to the vesicle or to be bound or adsorbed to a polymer molecule in order to recognize and bind a ligand in sequential mixing and evaporation steps.
  • the invention further relates to a bioanalytical detection method using a biological or biochemical reagent according to the invention.
  • the analyte can be detected by means of an optical signal change.
  • the optical signal change is determined using an optical sensor serving as a sensor platform. This sensor platform is preferably selected from the group consisting of optical waveguide sensors and surface plasmon sensors.
  • the optical signal change to be determined is based on the change in the effective refractive index in the near field of the sensor surface. It is also preferred if the optical to be determined Signal change based on a luminescence or fluorescence generated in the near field of the sensor.
  • a planar or fibrous waveguide can be used as the sensor platform. It is preferred if a planar thin-film waveguide is used, which comprises a first optically transparent layer (a) on a second optically transparent layer (b).
  • the invention further relates to a bioanalytical detection method using a biological or biochemical reagent according to the invention, characterized in that the excitation light is coupled into the thin-film waveguide serving as a sensor platform via one or more gratings (c).
  • optically transparent layer (b ') with a lower refractive index than that of layer (a) and a thickness between the optically transparent layers (a) and (b) and in contact with layer (a) from 5 nm to 1000 ⁇ m, preferably from 10 nm to 1000 nm.
  • the function of the intermediate layer is to reduce the surface roughness under layer (a) or to reduce the penetration of the evanescent field of light guided in layer (a) into the one or more layers below or to improve the adhesion of layer (a) the one or more underlying layers or the reduction of thermally induced voltages within the optical sensor platform or the chemical isolation of the optically transparent layer (a) from underlying layers by sealing micropores in layer (a) against underlying layers.
  • the biological or biochemical recognition elements there are a number of methods for applying the biological or biochemical recognition elements to the optically transparent layer (a). For example, this can be done by physical adsorption or by electrostatic interaction. The orientation of the recognition elements is then generally statistical. In addition, there is a risk that if the composition of the sample containing the analyte or the reagents used in the detection method differ, some of the immobilized recognition elements will be washed away. It can therefore be advantageous if biological or biochemical detection elements (e) an adhesion-promoting layer (f) is applied to the optically transparent layer (a).
  • This adhesive layer should also be optically transparent. In particular, the adhesive layer should not protrude beyond the depth of penetration of the evanescent field from the wave-guiding layer (a) into the medium above. Therefore, the adhesion promoting layer (f) should have a thickness of less than 200 nm, preferably less than 20 nm.
  • Another object of the invention is a bioanalytical detection method with a sensor platform, which is characterized in that the thin-film waveguide serving as the sensor platform comprises several measuring ranges for the simultaneous or sequential determination of one or more analytes from one or more samples.
  • the lattice structure (c) is a diffractive lattice with a uniform period.
  • the resonance angle for coupling the excitation light via the grating structure (c) to the measuring areas is then uniform in the entire area of the grating structure.
  • the corresponding resonance angles for the coupling can differ significantly, which can make the use of additional adjustment elements in an optical system necessary to accommodate the sensor platform or can lead to spatially very unfavorable coupling angles .
  • the grating structure (c) is a multi-diffractive grating.
  • the material of the second optically transparent layer (b) can consist of glass, quartz or a transparent thermoplastic from the group formed by polycarbonate, polyimide or polymethyl methacrylate.
  • the refractive index of the waveguiding, optically transparent layer (a) is significantly larger than the refractive index of the adjacent layers. It is particularly advantageous if the refractive index of the first optically transparent layer (a) is greater than 2.
  • the first optically transparent layer (a) can consist of Ti0 2 , ZnO, Nb 2 ⁇ 5 , Ta 2 ⁇ 5 , Hf0 2 , or Zr0 2 . It is particularly preferred if the first transparent optical layer (a) consists of Ti0 2 or Ta 2 Os.
  • the thickness of the wave-guiding optically transparent layer (a) is the second relevant parameter for generating the strongest possible evanescent field at its interfaces with neighboring layers with a lower refractive index.
  • the strength of the evanescent field increases with decreasing thickness of the waveguiding layer (a) as long as the layer thickness is sufficient to guide at least one mode of the excitation wavelength.
  • the minimum “cut-off" layer thickness for guiding a mode depends on the wavelength of this mode. It is larger for longer-wave light than for short-wave light. However, as the "cut-off" layer thickness is approached, undesired propagation losses also increase sharply to what further limits the choice of preferred layer thickness.
  • layer thicknesses of the optically transparent layer (a) which only allow the guidance of 1 to 3 modes of a predetermined excitation wavelength
  • layer thicknesses which lead to monomodal waveguides for this excitation wavelength are very particularly preferred.
  • the discrete mode character of the guided light only refers to the transverse modes.
  • the resonance angle for the coupling of the excitation light in accordance with the above-mentioned resonance condition depends on the diffraction order to be coupled in, the excitation wavelength and the grating period.
  • the first diffraction order is advantageous.
  • the grating depth is decisive for the level of the coupling efficiency. In principle, the coupling efficiency increases with increasing grid depth.
  • the coupling efficiency also increases at the same time, so that it is used to excite luminescence in a measuring area (d) arranged on or adjacent to the lattice structure (c), depending on the geometry of the Measuring ranges and the irradiated excitation light bundle, gives an optimum. Because of these boundary conditions, it is advantageous if the grating (c) has a period of 200 nm - 1000 nm and the modulation depth of the grating (c) is 3 nm to 100 nm, preferably 10 nm to 30 nm.
  • the ratio of the modulation depth to the thickness of the first optically transparent layer (a) is equal to or less than 0.2.
  • a thin metal layer preferably made of gold or silver, optionally on an additional layer, is located between the optically transparent layer (a) and the immobilized biological or biochemical detection elements dielectric layer with a lower refractive index than the layer (a), for example made of silica or magnesium fluoride, is applied, the thickness of the metal layer and the possible further intermediate layer being selected such that a surface plasmon in the Excitation wavelength and / or the luminescence wavelength can be excited.
  • Another object of the present invention is an optical system for determining one or more luminescences, with at least one excitation light source of a sensor platform according to at least one of the named
  • the excitation light emitted by the at least one light source is coherent and is irradiated onto the one or more measurement areas at the resonance angle for coupling into the optically transparent layer (a).
  • At least one spatially resolving detector is used for the detection.
  • At least one detector from the group formed by CCD cameras, CCD chips, photodiode arrays, avalanche diode arrays, multichannel plates and multichannel photomultipliers can be used as the at least one spatially resolving detector.
  • optical components from the group that are used by lenses or lens systems can be used between the one or more excitation light sources and the sensor platform according to one of the aforementioned embodiments and / or between the said sensor platform and the one or more detectors for shaping the transmitted light bundles, planar or curved mirrors for deflection and, if necessary, additionally for shaping the shape of light bundles, prisms for deflection and, if necessary, for spectral division of light bundles, dichroic mirrors for spectrally selective deflection of parts of light bundles, neutral filters for regulating the transmitted light intensity, optical filters or monochromators for spectrally selective transmission of parts of light bundles or polarization-selective elements for the selection of discrete ones Polarization directions of the excitation or luminescent light are formed.
  • the excitation light is irradiated in pulses with a duration of between 1 fsec and 10 minutes.
  • the emission light from the measurement areas is measured in a temporally resolved manner.
  • the excitation light can be irradiated and the emission light to be detected sequentially from one or more measurement areas for individual or more measurement areas.
  • the sequential excitation and detection can be carried out using movable optical components which are formed from the group of mirrors, deflection prisms and dichroic mirrors.
  • the sensor platform is moved between steps of sequential excitation and detection.
  • the one or more excitation light sources and the components used for detection can be spatially fixed.
  • Another object of the invention is a complete analytical system for luminescence detection of one or more analytes in at least one sample on one or more measurement areas on a sensor platform, comprising an optical layer waveguide, with a sensor platform according to one of the aforementioned
  • Embodiments as well Feeding means to bring the one or more samples into contact with the measuring areas on the sensor platform.
  • samples and any additional reagents can be supplied in parallel or crossed microchannels, under the influence of pressure differences or electrical or electromagnetic potentials.
  • the one or more luminescence or fluorescence labels used for the detection of the analyte on the analyte or in a competitive assay on an analogue of the analyte or in a multi-stage assay is bound to one of the binding partners of the analyte or the biological or biochemical or synthetic recognition elements used.
  • the bioanalytical detection method according to the invention is used for the simultaneous or sequential, quantitative or qualitative determination of one or more analytes from the group of antibodies or antigens, receptors or ligands, chelators or "histidine tag components", oligonucleotides, DNA or RNA strands, DNA - or RNA analogs, enzymes, enzyme factors or inhibitors, lectins and carbohydrates.
  • the samples to be examined can be naturally occurring body fluids such as blood, serum, plasma, lymph or urine or egg yolk.
  • a sample to be examined can also be an optically cloudy liquid, surface water, a soil or plant extract, a bio- or synthesis process broth.
  • the samples to be examined can also be taken from tissue parts of living or deceased organisms.
  • lipid vesicles with their double-layer membranes have mainly been used in two ways.
  • lipid vesicles are used in research as model systems for biological membranes.
  • the vesicles then serve as carrier systems for water-insoluble membrane proteins and receptors.
  • lipid vesicles are used commercially in the pharmaceutical business as transport systems of therapeutically active reagents for the targeted therapeutic treatment of diseases.
  • the term "vesicle” is used exclusively, regardless of the different use of both terms in the literature.
  • vesicle-forming lipids contain amphiphatic lipids with hydrophobic and polar groups.
  • the hydrophobic components are referred to as “lipid chains” and the hydrophilic or polar components as "polar head groups”.
  • lipid molecules can spontaneously form double-layer structures, such as lipid vesicles.
  • the above-mentioned lipid molecules can also be easily inserted into already formed double layers or vesicles in such a way that the lipid chains are integrated into the hydrophobic interior of the lipid double layer and the polar head groups into the region formed by the head groups of the existing vesicles.
  • the vesicle-forming lipids preferably have two hydrocarbon chains with a typical length of 14 to 22 carbon atoms with different unsaturated character, for example with alkyl chains.
  • Such lipid molecules can be isolated, for example, from biological membranes of different organisms and are referred to as "naturally occurring lipids".
  • Typical examples are glycero- phospholipids such as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid and sphinogomyelin.
  • lipids include the classes of glycolipids, sterols such as cholesterol, cardiolipin, plasmalogen and lipids which are found in archaebacteria, for example those with hydrocarbon chains coupled to ether groups, branched hydrocarbon chains or hydrocarbon chains with aliphatic ring structures or so-called bipolar lipids in which a lipid molecule is involved extends over a lipid double layer (for a detailed description see, for example, RB Gennis: "Biomembranes", Advanced Texts in Chemistry (publisher: CR Cantor), Springer, Heidelberg 1989).
  • Other examples include artificial lipids, such as those commercially available, for example, from Avanti Polar Lipids, Alabaster, USA.
  • lipids that do not belong to naturally occurring lipid classes. They can contain activatable or polymerizable groups, both in the lipid chains and in the polar head groups. Further examples are positively charged lipids such as dodecylammonium bromide (DODAB) or boloamphiphiles as well as lipids which contain fluorinated or halogenated lipid-hydrocarbon chains.
  • DODAB dodecylammonium bromide
  • boloamphiphiles as well as lipids which contain fluorinated or halogenated lipid-hydrocarbon chains.
  • Synthetic lipids are ideally suited for the formation of vesicles, either as pure compounds , as mixtures of different synthetic lipids or in mixtures which comprise synthetic and naturally occurring lipids.
  • Vesicles are typically prepared according to one of the following protocols or in a combination thereof (as described, for example, by F. Szoka and D. Papahadjopoulos, Rev. Biophys. Bioeng. 9 (1980) 467-508).
  • n Multilamellar vesicles are typically made by rehydrating dried lipid films or powders under various conditions, for example adding water or an aqueous buffer solution to a lipid film that has been previously deposited in a suitable vessel from a lipid solution in an organic solvent by its evaporation .
  • MLVs of different sizes are formed.
  • Small unilamellar vesicles are produced by sonication of MLVs or by extrusion (extruding) of MLVs through filters. In the latter case, the diameter of the vesicles is typically of the same order of magnitude as the pore size of the filters used.
  • Another very useful manufacturing method is the detergent dilution method. Lipids or MLVs are dissolved in the presence of detergents, which are subsequently removed by means of dilution, dialysis, chromatography, adsorption, ultrafiltration or centrifugation, so that SUVs ultimately form.
  • LUV's Large unilamellar vesicles
  • LUV's are typically made by removing a detergent or by injection methods, whereby lipids dissolved in organic solvents are injected into water or an aqueous buffer solution, or by reverse phase evaporation methods. In the latter case, LUVs are formed by emptying droplets of organic solvents in which the lipids have been dissolved and which are dispersed in the aqueous phase.
  • Very large vesicles are produced, for example, by vibration-free swelling of uniformly dried lipid films in aqueous solution.
  • vesicles with a diameter of 20-1000 nm are preferred, particularly preferably 50-400 nm, very particularly preferably 50-200 nm.
  • the polymers mentioned under B) serve both to stabilize the vesicle and to reduce non-specific binding to the surface.
  • the biological or biochemical reagent according to the invention contains hydrophilic polymer molecules which are bound to the inner and / or outer surface of the vesicle.
  • the hydrophilic polymer is bound to the vesicle to create a hydrophilic shell. This shell can be viewed as a steric barrier to the diffusion of molecules into or out of the vesicle.
  • Such vesicles are therefore also referred to as sterically stabilized vesicles.
  • the vesicle-bound polymers have a high conformational flexibility and enable a high transition rate between different conformations, which corresponds to a high entropy.
  • a vesicle approaches or even binds to a particle or a macroscopic surface, such as the surface of a sensor
  • the reduction in the conformational freedom of the polymer corresponds to a loss of entropy.
  • vesicles can evade the mononuclear phagocyte system and are also called “stealth vesicles”. They are used to transport active pharmaceutical ingredients in an organism. In the understanding of this invention, such vesicles are referred to as “sterically stabilized vesicles” (SSV's).
  • the polymer molecule can be attached to the vesicle surface by one of the following methods or a combination of these methods:
  • Electrostatic interactions between positively (or negatively) charged portions of the polymer and negatively (or positively) charged groups on the vesicle surface can be, for example, from the polar lipid head groups or others suitable compounds, such as natural or artificial polypeptides, in or on the vesicle double layer.
  • Suitable functional groups on lipids are amino groups, e.g. Diacylglycerophosphatidylethanolamine, or SH groups or OH groups of natural or synthetic lipid molecules.
  • the polymers can be bound either directly to the lipid molecules or via spacer molecules between the lipid molecules or lipid-like molecules. Some of these compounds are commercially available with active head groups, which are, for example, reactive towards amines or thiols.
  • succinimide derivatives examples include succinimide derivatives, pyridinylthio derivatives or maleimide derivatives, and some are sold, for example, by Avanti Polar Lipids, Alabaster, USA. Further examples not commercially available were described in G. Brink et al., Biochem. Biophys. Acta 1196 (2, 1994) 227-230.
  • anchoring molecules are naturally occurring transmembrane proteins or membrane-spanning polypeptides or membrane-associated proteins or polypeptides or their synthetically produced analogs. Said membrane proteins or polypeptides are also referred to as the hydrophobic core ("core") of the membrane, for example in the case of membrane-spanning or transmembrane proteins or polypeptides.
  • core hydrophobic core
  • Peripheral protein or polypeptide molecules are connected to the membrane primarily through ionic interactions, hydrogen bonds or hydrophobic interactions, and in some cases via lipid anchors which are covalently bound to a protein molecule (see L. Stryer: "Biochemistry", 4th edition, Freeman ( 1995) p. 275 ff.).
  • hydrophilic polymers are suitable for binding to vesicles, some examples of which are described below.
  • Stepth Vescicles is described in DD Lasic and F. Martin: “Stealth Liposomes ", CRC Press, Boca Raton, 1995.
  • US 5534241 US 5770222 and US 5891468 vesicle-bound polyglycols or similar hydrophilic polymers are described as part of diblock copolymers consisting of hydrophilic and hydrophobic polymers, to the hydrophobic polymers and in to enclose and shield active substances enclosed in the vesicles and to release them under suitable physiological conditions by opening the hydrophilic polymers.
  • Negatively or positively charged or zwitterionic polymers can be used. Typical examples are polypeptides and polysulfoxides, which, like further examples, are mentioned in US 5770222.
  • Examples are chitin / chitosan dextran, starch and similar polymers, as they are named in US 5891468.
  • lipid vesicles There are two main uses for lipid vesicles.
  • the first area of application is the use of lipid vesicles as model systems for biological membranes, especially in the field of basic research (see, for example, RB Gennis: "Biomembranes", Advanced Texts in Chemistry (publisher: CR Cantor), Springer, Heidelberg (1989).
  • vesicles as carriers or for the administration of therapeutic agents for the targeted treatment of diseases.
  • membrane enzymes e.g. tyrosine kinases, adenylate cyclases
  • membrane transporters e.g. ATPases
  • these conventional vesicles When used for drug delivery, these conventional vesicles have a very short circulation time in the blood. When administered in vivo, they show a strong tendency towards rapid accumulation in the phagocytic cells of the mononuclear phagocytic system. In order to overcome the disadvantageous short circulation time, hydrophilic polymers were bound to the vesicle, as already mentioned above.
  • antibodies or antibody fragments are bound to the vesicle as specific recognition elements.
  • these vesicles circulate in the bloodstream and bind to the corresponding antigen, which occurs specifically in the target tissue, by means of the antibodies bound to them, which leads to a concentration of the vesicles and thus also the active substances transported by them in this tissue.
  • These vesicles are called immuno-liposomes.
  • the antibodies can be bound to the lipids of the vesicle either directly or via so-called spacer molecules.
  • PEG polyethylene glycol
  • Other known examples include binding the antibody directly (or via a short spacer molecule) to a lipid of an SSV such that the antibody is within the hydrophilic polymer shell.
  • WO 94/21235 claims liposome compositions for patient treatment which have an outer, hydrophilic layer of PEG (molecular weight 1000-10000 Dalton) and which have covalently bound recognition elements, so-called effectors.
  • the PEG envelope is used to prevent the vesicles from breaking down in the bloodstream.
  • the effectors consist of small to medium-sized immunodetection elements, namely F AB fragments, Glycoproteins, cytokines, polysaccharides or various peptides or peptide hormones.
  • WO 97/35561 claims biologically activated liposomes stabilized with a water-soluble polymer shell for therapeutic use.
  • Biological, amphiphilic recognition elements in active form are non-covalently linked to the polymer-stabilized liposomes, i.e. via physisorption.
  • the recognition element is a peptide, a 'growth hormone releasing factor'.
  • Unilamellar liposomes with a diameter of less than 300 nm, produced by extrusion, are preferred.
  • PEG is preferably used as the polymer, which is present in the form of a covalently coupled PEG lipid in the liposome-forming membrane.
  • a liposome composition for diagnostic purposes is claimed, which additionally contains labels for detection purposes. Fluorescence markers, radioactive labels, dyes and components for signal amplification in nuclear magnetic resonance are given as detectable markers. The method and position of attaching the labels to the vesicle are not specified.
  • polymer-stabilized vesicles for therapeutic use are claimed in which ligands for the detection of cell receptors are bound at the end of the polymer chains. After target recognition via ligand-receptor interaction and subsequent fusion of the vesicle with the target membrane, the vesicle should also have the function of releasing the hydrophilic polymer chains.
  • WO 97/33618 i.a. Vesicles for intracellular drug delivery especially for therapeutic purposes of cancer therapy, which is mediated by T-lymphocytes, claimed, wherein the recognition elements are attached via a combination spacer-polymer-spacer. HPMA copolymer is used as the polymer.
  • vesicles described above and their areas of application have hitherto exclusively related to therapeutic use with the main function of a vesicle as a carrier or transport vehicle.
  • the use of vesicles in the bioanalytical field has only been described occasionally.
  • WO 97/39736 describes vesicles as reagents for immunoassays in order to analyze patient samples.
  • Vesicles with associated ligands for the detection of analyte molecules are generally claimed here, where the ligands can consist of proteins, peptides, antibodies or fragments thereof as well as nucleic acids.
  • the ligands are smaller hapten molecules for immuno-recognition, which are covalently linked to the vesicle in particular in the form of hapten lipids.
  • Signal detection in the assay is carried out by binding the vesicle reagent to a hard surface equipped with the appropriate biological receptors in the form of beads, particles or a microtiter plate wall.
  • the signal is generated by attaching a secondary label in solution to the liposome after analyte detection, for example in a sandwich assay, the label being a fluorescent or luminescent molecule, a dye, or a signal-generating enzyme, for example an alkaline phosphatase.
  • the specific bio-recognition can also be carried out by binding to a secondary vesicle that carries the corresponding receptors.
  • the specific ligand-receptor binding should be maintained during the analysis in the presence of the vesicle. It should be noted that in the case described, no additional polymers are used and described for stabilizing the liposome.
  • vesicles for signal amplification in bioanalytical detection methods has been described by some groups.
  • a competitive assay method is described in which vesicle-bound theophyillin molecules compete with free theophylline molecules in solution as analyte for binding to anti-theophylline antibodies immobilized on the waveguide surface.
  • the vesicles in the hydrophilic interior are loaded with a large number of carboxy-fluorescein molecules as fluorescent labels.
  • a partner of an affinity system is immobilized on a solid surface, for example a chemical or biochemical sensor, in order to specifically recognize and bind the analyte from a sample in the subsequent detection method.
  • the functionalization of the surface is of crucial importance.
  • environment-sensitive biological interactions such as, for example, with membrane receptors
  • a variety of techniques are known for the detection of analytes on a solid surface.
  • optical detection methods which are based on interactions in the surface-bound near field of the sensor, are particularly important because of minimal interference with the binding processes.
  • Such optical near-field methods are known, for example, as refractive or luminescence-based detection methods in the evanescent field of a waveguide or in the field of the depth of penetration of a surface plasmon produced in a thin metal film (surface plasmon resonance, SPR).
  • Fluorescence-based detection methods by fluorescence excitation in the evanescent field of planar waveguides are described, for example, in WO 95/33197 and WO 95/33198 for discrete analyte determinations and in WO 96/35940 for the simultaneous determination of several analytes.
  • lipids and amphiphilic or hydrophilic polymers are converted into sterically stabilized vesicles which comprise a biological or biochemical or synthetic recognition element for recognizing and binding a ligand.
  • a biological or biochemical or synthetic recognition element refers to any biological molecule or molecular complex or artificial, chemical or genetic modification, or synthetic molecule or molecular complex that is specific to any other Connection binds.
  • Biological or biochemical or synthetic recognition elements are preferably used from the group consisting of antibodies, antibody fragments, nucleic acids or nucleic acid analogs, DNA, RNA, enzymes, natural and synthetic polypeptides, histidine tag components and membrane receptors.
  • Said other compound can be ions, atoms, clusters of atoms, molecules, molecular clusters, any synthetic or biological compounds or parts thereof, which are specifically recognized and bound by said recognition element.
  • the biological or biochemical or synthetic recognition elements are associated with or integrated into the surface of the vesicle or are bound to the polymer or to the lipids of the vesicle.
  • the specific recognition and binding of said compound by the identified recognition element can be part of a bioanalytical detection method and thus be a further subject of the invention.
  • said compound can be an analyte to be detected in a sample.
  • the detection can be carried out by determining the change in a physically measurable quantity as a result of the binding of the analyte itself or an analogue of the analyte (for example in a competitive detection method) or in a multi-stage binding process in which the analyte or its analogue is bound in a partial step will be done.
  • a physically measurable quantity can be changed, for example, by luminescence after excitation of light or signals from an ESR or NMR label after excitation, or by changing the molecular mass adsorbed on a surface.
  • the detection can take place in free solution or on a solid surface, for example the surface of a sensor.
  • the bioanalytical detection method was carried out using a commercial SPR apparatus (Biacore 1000, Upsala, Sweden). Unless otherwise stated, the process was carried out under constant flow at a flow rate of 5 ⁇ L / min at 25 ° C.
  • Monomolecular layers were produced by self-assembly (self-assembled monolayers, SAM's) of 16-mercapto-hexadecanoic acid and 11-mercaptoundekanol on the pure gold surface of SPR sensor chips Jl (Biacore) outside the device in a chamber saturated with ethanol Avoid evaporation of the solvent.
  • SAM's self-assembled monolayers
  • a solution with 1 mM 16-mercaptohexadecanoic acid and 1.5 mM 11-mercaptoundekanol (from a mixture of 40 ⁇ L stock solution of 4 mM 16-mercapto-hexadecanoic acid in 1: 1 water / ethanol and 6 mM 11-mercaptoundekanol in ethanol with 80 ⁇ L Ethanol) in 1: 7 water / ethanol in aliquots of 30 ⁇ L added to the sensor surface. After 30 minutes, the solution was renewed, resulting in the formation of a mixed monomolecular self-assembled layer (SAM) after 80 to 100 minutes.
  • SAM monomolecular self-assembled layer
  • the SAM was functionalized using the following procedure: The sensor surface was firstly coated with HEPES-buffered saline (HBS: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20, pH 7.4) flushed at a flow rate of 10 ⁇ L / min.
  • HBS HEPES-buffered saline
  • Lipids dissolved in chloroform were mixed in the desired ratio (typically 80 mol% l, 2-dioleoyl-s “-glycero-3-phosphatidylchorine (DOPC), 10" cholesterol (chol), and 10% l, 2-dioleoyl-j "- Glycero-3-phosphatidylglycerol (DOPG))
  • DOPC 2-dioleoyl-s "-glycero-3-phosphatidylchorine
  • chol cholesterol
  • DOPG 2-dioleoyl-j "- Glycero-3-phosphatidylglycerol
  • solutions (1) to (3) were dialyzed against a 650-fold buffer volume (10 mM HEPES, 3 mM EDTA, 1 mM NaN 3 , pH 7.4) at room temperature.
  • the dialysis buffers for (1) and (2) additionally contained 500 mM NaCl, for (3) additionally 500 mM NaCl and CHAPS in a concentration corresponding to one tenth of the kitic micellar concentration (CMC), ie approx. 0.5 mM.
  • CMC kitic micellar concentration
  • the buffers were renewed and the ionic strength of the buffer (1) was reduced from 500 mM NaCl to 300 mM NaCl. After 5 hours of further dialysis at 4 ° C., the buffers were renewed again, the ionic strength of the buffer (1) being reduced further to 150 mM NaCl. Dialysis was completed after 10 hours at 4 ° C.
  • the optimal selection of the polymer chain length and the optimal proportion of polymer-coupled lipids depends on the size of the vesicle-associated biological or biochemical or synthetic recognition element and the accessibility for the analyte to be bound, with a tendency to prefer relatively long-chain polymers and / or a high proportion of the total number of lipid molecules in the case of large recognition elements or relatively short-chain polymers and / or a small proportion in the case of small recognition elements.

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Abstract

L'invention concerne une vésicule (stabilisée par voie stérique) fonctionnalisée, renforcée par polymère, qui comprend un élément d'identification biologique ou biochimique ou synthétique s'utilisant pour identifier et lier un ligand, ainsi qu'éventuellement des marqueurs comme constituants générateurs de signaux dans un procédé de mise en évidence bioanalytique. L'invention concerne en outre un procédé pour préparer et utiliser cette vésicule dans des procédés de mise en évidence.
EP00938651A 1999-05-27 2000-05-18 Vesicule contenant des polymeres et procedes de mise en evidence par detecteur, fondes dessus Withdrawn EP1180242A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CH99099 1999-05-27
CH99099 1999-05-27
PCT/EP2000/004491 WO2000073798A1 (fr) 1999-05-27 2000-05-18 Vesicule contenant des polymeres et procedes de mise en evidence par detecteur, fondes dessus

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EP1180242A1 true EP1180242A1 (fr) 2002-02-20

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EP (1) EP1180242A1 (fr)
JP (1) JP2003501631A (fr)
AU (1) AU5395000A (fr)
WO (1) WO2000073798A1 (fr)

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AU2002325120A1 (en) * 2001-09-10 2003-03-24 Celator Technologies Inc. Unilamellar vesicles stabilized with short chain hydrophilic polymers
WO2005084210A2 (fr) * 2004-02-27 2005-09-15 Hitachi Chemical Research Center, Inc. Sondes de detection multiplex
US20070218453A1 (en) * 2004-03-31 2007-09-20 Katsuyuki Tanizawa Sensing Tool
GB0512769D0 (en) * 2005-06-23 2005-07-27 Avacta Ltd Ratiometic multicolour fluorescence correlation screening; agents for use therewith and methods of production thereof
GB0701444D0 (en) * 2007-01-25 2007-03-07 Iti Scotland Ltd Detecting analytes
EP2422194B1 (fr) 2009-04-20 2014-12-17 Agency For Science, Technology And Research Système vésiculaire et ses utilisations
KR20130041775A (ko) * 2010-03-31 2013-04-25 세키스이 메디칼 가부시키가이샤 내인성 리포단백질의 영향 회피방법 및 시약
JP6044920B2 (ja) * 2012-03-27 2016-12-14 国立研究開発法人農業・食品産業技術総合研究機構 酸化ｌｄｌ受容体に作用するリポソーム

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DE69120883T2 (de) * 1990-09-13 1997-03-06 Wako Pure Chem Ind Ltd Verfahren zur Bestimmung von Antistreptolysin-O
WO1993010226A1 (fr) * 1991-11-19 1993-05-27 North Carolina State University Dosage de diagnostic immunologique au moyen de liposomes portant des marqueurs sur leur surface exterieure
JP2711974B2 (ja) * 1993-02-03 1998-02-10 日水製薬株式会社 免疫凝集反応試薬及び免疫分析方法
EP0914094A4 (fr) * 1996-03-28 2000-03-01 Univ Illinois Materiaux et procedes destines a la preparation de compositions de liposomes ameliorees
TW520297B (en) * 1996-10-11 2003-02-11 Sequus Pharm Inc Fusogenic liposome composition and method

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AU5395000A (en) 2000-12-18
WO2000073798A1 (fr) 2000-12-07

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