WO2000073798A1

WO2000073798A1 - Vesicle containing polymers and sensor detection methods based thereon

Info

Publication number: WO2000073798A1
Application number: PCT/EP2000/004491
Authority: WO
Inventors: Dietmar KRÖGER; Horst Vogel; Michael Pawlak
Original assignee: Zeptosens Ag
Priority date: 1999-05-27
Filing date: 2000-05-18
Publication date: 2000-12-07
Also published as: JP2003501631A; EP1180242A1; AU5395000A

Abstract

The invention relates to a functionalized, polymer-reinforced (sterically stabilized) vesicle which comprises a biological, biochemical or synthetic identifying element for identifying and binding a ligand. In addition, the inventive vesicle optionally contains labels which serve as signal-generating constituents in a bioanalytical detection method. The invention also relates to a method for producing said vesicle and to the use thereof in detection methods.

Description

VESICLE CONTAINING POLYMER AND SENSOR DETECTION METHODS BASED ON IT

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. There is therefore a need for the development of 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

A) a vesicle

B) one or more polymer molecules bound to the inner and / or outer surface of the vesicle

C) at least one biological or biochemical or synthetic recognition element bound or adsorbed to the vesicle or bound or adsorbed to the polymer molecule for recognizing and binding a ligand.

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. For certain applications in which energy transfer is to be used to improve the differentiation of the specific signal from the background signals, it can be advantageous if several luminescence or fluorescence labels of different emission wavelengths and the same or different ones are used as signal-generating components Excitation wavelength can be used. The 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

A) the lipid molecules required for vesicle production by dialysis,

B) one or more polymer molecules to be bound to the inner and / or outer shell of the vesicle and

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. In particular, the analyte can be detected by means of an optical signal change. It is preferred that 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.

It is preferred if 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.

In particular, 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).

It can be advantageous if there is another 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 μm, 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.

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.

By spatially selective application of biological or biochemical or synthetic detection elements on the sensor platform, it is possible to generate spatially separated measuring areas (d). In contact with a luminescent analyte or a luminescent-labeled analogue of the analyte competing with the analyte for binding to the immobilized recognition elements or another luminescent-labeled binding partner in a multi-stage assay, these luminescent molecules will only selectively bind to the surface of the sensor platform in the measuring areas, which the areas are defined that are occupied by the immobilized recognition elements.

For many applications it is advantageous if 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. However, if, for example, you want to couple the excitation light of different light sources with a clearly different wavelength, 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 . For example, to reduce very different Coupling angle can therefore be advantageous if 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.

In order to generate the strongest possible evanescent field on the surface of the optically transparent layer (a), it is desirable that 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.

For example, the first optically transparent layer (a) can consist of Ti0 ₂ , ZnO, Nb ₂ θ ₅ , Ta ₂ θ 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.

In addition to the refractive index of the wave-guiding optically transparent layer (a), its thickness 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. Preferred are 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. It is clear that the discrete mode character of the guided light only refers to the transverse modes. These requirements mean that the thickness of the first optically transparent layer (a) is advantageously 40 nm to 300 nm. The thickness of the first optically transparent layer (a) is very particularly advantageously 70 nm to 160 nm.

Given predetermined refractive indices of the waveguiding, optically transparent layer (a) and the adjacent layers, 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. To increase the coupling efficiency, coupling the first diffraction order is advantageous. In addition to the height of the diffraction order, the grating depth is decisive for the level of the coupling efficiency. In principle, the coupling efficiency increases with increasing grid depth. Since the process of decoupling is completely reciprocal for coupling, 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.

It is further preferred that the ratio of the modulation depth to the thickness of the first optically transparent layer (a) is equal to or less than 0.2.

To enhance luminescence or to improve the signal-to-background ratio, it can furthermore be advantageous if 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

Designs of at least one detector for detecting the light emanating from the at least one or more measuring ranges (d) on the sensor platform.

It is advantageous if 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).

In the case of insufficient intensity of a single light source or the need for light sources of different emission wavelengths, for example for biological applications, it is advantageous if two or more coherent light sources with the same or different emission wavelength are used as excitation light sources.

In order to record the signals separately from a large number of measuring ranges, it is preferred that 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.

In the optical system according to the invention according to one of the embodiments mentioned, 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.

For a number of applications, it is advantageous if the excitation light is irradiated in pulses with a duration of between 1 fsec and 10 minutes.

For kinetic measurements or for the discrimination of rapidly decaying fluorescence of fluorescent contaminants in the sample or in materials of components of the optical system or the sensor platform itself, it can be advantageous if the emission light from the measurement areas is measured in a temporally resolved manner.

It is also possible for the excitation light to 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. Another possible embodiment is that the sensor platform is moved between steps of sequential excitation and detection. In this case, 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, an optical system 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.

The samples and any additional reagents can be supplied in parallel or crossed microchannels, under the influence of pressure differences or electrical or electromagnetic potentials.

For the quantitative and / or qualitative determination of the analyte in the bioanalytical detection method according to the invention, it is also possible that 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.

In the aforementioned methods, it is possible that (1) the isotropically emitted luminescence or (2) coupled into the optically transparent layer (a) and coupled out via the grating structure (c) luminescence or luminescence of both components (1) and (2) simultaneously be measured.

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.

However, 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.

Up to now, lipid vesicles with their double-layer membranes have mainly been used in two ways.

On the one hand, 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.

On the other hand, lipid vesicles are used commercially in the pharmaceutical business as transport systems of therapeutically active reagents for the targeted therapeutic treatment of diseases. In the following, the term "vesicle" is used exclusively, regardless of the different use of both terms in the literature. In general, vesicle-forming lipids contain amphiphatic lipids with hydrophobic and polar groups. In the following, the hydrophobic components are referred to as "lipid chains" and the hydrophilic or polar components as "polar head groups". In an aqueous environment, such lipid molecules can spontaneously form double-layer structures, such as lipid vesicles.

The structure and characterization of such vesicles is described in standard textbooks, for example in R. B. Gennis: "Biomembranes", Advanced Texts in Chemistry (editor: C. R. Cantor), Springer, Heidelberg 1989.

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. Other naturally occurring 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.

Many of the naturally occurring lipids mentioned above can also be chemically synthesized.

There are also synthetically produced 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.

An overview of a wide range of synthetic lipids is given in J.-H. Furhop and J. Köning: "Membranes and Molecular Assemblies: The Synkinetic Approach", Monographs in Supramolecular Chemistry (Editor: JF Stoddart), The Royal Society of Chemistry, 1994. 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 (MLV's) 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 . Depending on the production method, such as stirring, shaking, sonication, and the type of lipid, the ionic strength or the concentration, to name just a few parameters, MLVs of different sizes are formed.

Examples of further production methods are the “solvent spherule method”, the reverse phase method or vesicle fusion by freezing / thawing or dehydration / rehydration (see, for example, D. D. Lasic, Biochem. J. 256 (1988) 1-11).

Small unilamellar vesicles (small unilamellar vesicles SUVs) 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.

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. For the biological or biochemical reagent according to the invention, 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. It should be mentioned in particular that 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. Ideally, the vesicle-bound polymers have a high conformational flexibility and enable a high transition rate between different conformations, which corresponds to a high entropy. When such 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. This leads to a reduced (non-specific) binding to other particles or macroscopic surfaces. This effect is known as "entropic shielding" (D. D. Lasic and F. Martin: "Stealth Liposomes", CRC Press, Boca Raton, 1995). These 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:

(1) Electrostatic interactions between positively (or negatively) charged portions of the polymer and negatively (or positively) charged groups on the vesicle surface. The electrical charges of the vesicle 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.

(2) By covalent binding of the polymer molecule to the polar head groups of the lipid molecules, which serve as anchors for insertion into the lipid bilayer of the vesicle. 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. 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.

(3) Other suitable 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. 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.).

Various hydrophilic polymers are suitable for binding to vesicles, some examples of which are described below.

(l) Uncharged polymers

The use of polyethylene glycols and their derivatives with

"Stealth Vescicles" is described in DD Lasic and F. Martin: "Stealth Liposomes ", CRC Press, Boca Raton, 1995. In 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.

(2) Electrically charged 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.

(3) carbohydrates and their derivatives

Examples are chitin / chitosan dextran, starch and similar polymers, as they are named in US 5891468.

(4) Dendritic hydrophilic polymers

Typical examples of this class are described in G.R. Newkome, C.N. Moorefield and F. Vögtle: "Dendritic Molecules", Verlag Chemie (1996).

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). In this application, the vesicles mainly serve as carriers of water-insoluble membrane proteins and receptors, such as ion channel receptors (e.g. nAchR = acetylcholine receptor, 5HT3 receptor, glutamate receptor, GABA receptor), G-protein coupled receptors (e.g. neurokinin receptors, chemokine receptors Receptors, ß-adrenergic receptors), membrane enzymes (e.g. tyrosine kinases, adenylate cyclases) and membrane transporters (e.g. ATPases) The other main area of application relates to the use of vesicles as carriers or for the administration of therapeutic agents for the targeted treatment of diseases. The use of vesicles for this application has been known for more than 20 years. Most early applications are based on so-called conventional vesicles, which are typically composed of a few neutral or negatively charged different lipids (mostly phospholipids) and / or cholesterol. 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.

In order to bring the vesicles more precisely to the tissue where they are to be applied, antibodies or antibody fragments (F _AB ) are bound to the vesicle as specific recognition elements. After in-vivo administration, 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. A polymer such as polyethylene glycol (PEG), used to form SSVs (sterically stabilized vesicles), can also be used as this spacer, so that the antibody is bound at the end of the polymer chain. 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.

For example, 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. In the special case, 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. In a broader sense, 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.

In a further variant, US 589 1468, 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. In 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. Among other things, Peptides, cell poisons, nucleic acids, antigens and drugs are described. In successor patents, the extension to PEG as a polymer was introduced (WO 98/51336) and the range of applications was expanded further, e.g. an interleukin 2 peptide (IL2) as a ligand for IL2 T-cell membrane receptor recognition (WO 99/07324).

The 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 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. In particular, 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. Instead of binding to a macroscopic, solid surface, the specific bio-recognition can also be carried out by binding to a secondary vesicle that carries the corresponding receptors. In any case, 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.

As a further application, the use of vesicles for signal amplification in bioanalytical detection methods has been described by some groups. SA Choquette et al .: "Planar waveguide immunosensor with fluorescent liposome amplification", Analytical Chemistry 64 (1992) 55-60, describes vesicles in fluorescence-based immunoassays on planar waveguides. 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. At the same time, the vesicles in the hydrophilic interior are loaded with a large number of carboxy-fluorescein molecules as fluorescent labels. In the absence of theophylline in a sample to be examined, a maximum, very high fluorescence signal is therefore observed. However, the stability of these vesicles is not enhanced by polymers, so that there is a certain amount of leakage encapsulated fluorescence label, depending on the specific composition of the sample, is to be expected.

In surface-bound detection methods for an analyte, 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. In particular in the case of environment-sensitive biological interactions, such as, for example, with membrane receptors, it is essential to maintain the native conformation of the recognition element in order to maintain the specific binding ability. At the same time, it is important to minimize non-specific binding to the surface. 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.

In a particular embodiment of this invention, 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. Any "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. In order to ensure sufficient access for the ligand or analyte, it is preferred that 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.

For example, 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.

To minimize non-specific binding of the reconstituted vesicles as described above, various parameters such as the composition of the lipid mixture, the charge of the vesicle or the modified sensor surface, addition of detergents and ionic strength of the buffer were varied. Even if all of these parameters make a certain contribution to reduce the non-specific binding, the remaining non-specific binding was still too high in the end result.

In contrast, the incorporation of PEG lipids into the vesicles led to a surprising, strong reduction in non-specific binding. The specific binding signal also decreases due to steric hindrance. The end result, however, is a significant improvement in the ratio of specific to unspecific signal, which is the decisive factor for measurement technology. The influence of the incorporation of the polymer into the vesicles on the observed binding signals can be explained as follows: The polymer forms a steric barrier around the membranes and thus leads to the observed reduction in non-specific binding. PEG lipids with different chain lengths of PEG (with molecular weights of 750, 2000 and 5000 Daltons) were used.

The following example explains the invention in more detail.

Example instrumentation

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 μL / min at 25 ° C.

a)

Functionalization of sensor surfaces

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. For this purpose, a solution with 1 mM 16-mercaptohexadecanoic acid and 1.5 mM 11-mercaptoundekanol (from a mixture of 40 μL stock solution of 4 mM 16-mercapto-hexadecanoic acid in 1: 1 water / ethanol and 6 mM 11-mercaptoundekanol in ethanol with 80 μL Ethanol) in 1: 7 water / ethanol in aliquots of 30 μL 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.

After inserting the SPR chip into the SPR apparatus, 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 μL / min.

Then 20 μL of 0.2 M / V ethyl iV dimethylaminopropyl carbodiimide (EDC) / 0.05 M V hydroxysuccinimide (NHS) at a reduced flow rate of 5 μL / min, followed by an injection of 50 μL of a streptavidin solution in 1: 3 water / HBS (250 μg (mL) and a further injection of 35 μL ethanolamine solution (1 M, pH 8.5), each with a soot rate of 5 μL / min.Finally, α-bungarotoxin (BgTx) was added the surface was bound by adding a solution of biotinylated α-bungarotoxin (biot-BgTx) in HBS (50 μg / mL) for two minutes. b)

Reconstitution of Nicotine Acetylcholine Receptors (nAchR) in Lipid

Vesicles

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)) After evaporation of the solvent, the deposited lipid film was dissolved again with the addition of 480 μL buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, pH 7.4) and 270 μL ten percent (w / w) CHAPS to obtain a final lipid concentration of 2 mg / mL after subsequent sonication, 250 μL enriched receptor membranes (1.9 mg / mL) were added and the resulting suspension was centrifuged after shaking to remove undissolved material Dialysis of the supernatant against a 650-fold buffer volume (500 mM NaCl, 10 mM HEPES, 3 mM EDTA, 1 mM NaN ₃ , pH 7.4) in a "slide-a-lyser", first for 30 minutes at room temperature, then 8 to For 15 hours and finally again for 2 to 4 hours, replacing the dialysis buffer with each step. The dialyzed vesicles were kept for up to two days and centrifuged again before performing SPR measurements to remove aggregates formed from non-reconstituted receptors. Unless otherwise stated, all steps were carried out at 4 ° C.

The concentrations of the receptors incorporated into the vesicles were determined by measuring the optical density at 280 nm (ε = 450,000 M ^" ^ m ^{" 1} ) in the presence of a large excess of CHAPS in order to dissolve the vesicles and to reduce light scattering effects. Assuming that the observed absorption was caused exclusively by nAchR, typical receptor concentrations of 350 nM were determined. A narrow size distribution of the vesicles, with a diameter of 18-20 nm, was determined by means of quasi-elastic light scattering according to standard methods. With a typical surface area of 60 Ä ² per lipid molecule, it was estimated that on average one vesicle each contained 4000 lipid molecules and 0.5 receptors. Nonspecific binding of nAchR vesicles

(1) Four solutions of nAchR vesicles (solutions (1) - (3) without, (4) with 2% PEG lipids) were prepared according to the procedure described above. 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. After 30 minutes 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.

To reduce the non-specific binding of lipids, the sensor surface was pretreated with a mixture with a molar composition of 70% DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine), 10% DOPG (1,2-dioleoyl-sn- glycero-3-phosphatidylglycerol), 10% chol, 3% l, 2-dimyristoyl-5 «-glycero-3-phosphatidic acid [poly (ethylene glycol)] ester (M _r PEG = 750, DMPA-PEG ₇₅ 0), 3% l-palmitoyl-2-oleoyl-5 «-glycerophosphatoethanolamine- V ^r - [poly (ethylene glycol)] (M _r PEG = 2000, POPE-PEG ₂₀ oo) and), 3% 1- palπütoyl-2-oleoyl- 5 / ι-glycerophosphatoethanolamine-N- [poly (ethylene glycol)] (M _r PEG = 5000, POPE-PEG50 ₀₀ ) in a buffer of 150 mM NaCl, 10 mM HEPES, 3 mM EDTA, 1 mM NaN ₃ at pH 7.4.

(2) To determine the non-specific binding of the vesicles to the sensor surface modified with BgTx as the immobilized recognition element, all binding sites of nAchR for BgTx on the vesicles were saturated with a large excess of BgTx and then in solution of the corresponding dialysis buffer the binding of the saturated vesicles to the BgTx-modified sensor surface measured as a signal for non-specific binding. The specific binding signal was then determined as the difference between the total binding signal with non-BgTx-saturated vesicles and the non-specific signal. The results are summarized in Table 1. Table 1:

In the present example, a maximum ratio of specific to non-specific signal was observed for PEG lipids with a PEG molecular weight of 750 to 2000 daltons. The proportion of PEG lipids in the total number of vesicle-forming lipids was also varied in a range from 0% to 10%. This showed a tendency that, especially in the case of longer-chain polymers, a high proportion leads to an adverse reduction in the specific binding signal due to steric hindrance. According to this invention, 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.

Claims

claims

1. A biological or biochemical reagent, characterized in that it

A) a vesicle,

B) one or more polymer molecules bound to the inner and / or outer surface of the vesicle and

C) comprises at least one biological or biochemical or synthetic recognition element bound or adsorbed to the vesicle or bound or adsorbed to the polymer molecule for recognizing and binding a ligand.

2. Biological or biochemical reagent according to claim 1, characterized in that at least one biological or biochemical or synthetic recognition element is selected 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 especially membrane receptors.

3. Biological or biochemical reagent according to one of claims 1 -

2, characterized in that at least one biological or biochemical or synthetic recognition element is associated with or integrated into the surface of the vesicle or is bound to the polymer or to the lipids of the vesicle.

4. Biological or biochemical reagent according to one of claims 1 -

3, characterized in that a water-soluble polymer is used as the stabilizing polymer.

5. Biological or biochemical reagent according to one of claims 1 -

4, characterized in that a hydrophilic polymer from the group consisting of polyethylene glycols, preferably with a molecular weight of 750 to 2000 daltons, polypeptides, polysulfoxides, carbohydrates such as dextrans and dendritic hydrophilic polymers, is used as the stabilizing polymer.

6. Biological or biochemical reagent according to one of claims 1 -

5, characterized in that the vesicle is unilamellar.

7. Biological or biochemical reagent according to one of claims 1 -

6, characterized in that the vesicle comprises lipids from the group consisting of naturally occurring lipids such as glycerophospholipids, such as, for example, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, sphinogomyelin, glycolipids, sterols such as, for example, cholesterol, cardiol occurring lipids, for example those with hydrocarbon chains coupled to ether groups, branched hydrocarbon chains or hydrocarbon chains with aliphatic ring structures or bipolar lipids, artificial lipids, such as positively charged lipids, such as, for example, dodecylammonium bromide (DODAB) or boloamphiphiles, as well as artificial lipids, which fluorinated or halogenated chains are fluorinated or activated or contain polymerizable groups.

8. Biological or biochemical reagent according to one of claims 1 -

7, characterized in that it additionally comprises a label as a signal-generating component for detection in a bioanalytical detection method, the label as a signal-generating component preferably being associated with the vesicle.

9. A biological or biochemical reagent according to claim 8, characterized in that the additional at least one label as a signal-generating component is located in the interior of the vesicle or is bound to the outer shell of the vesicle directly or via a spacer or via the polymer.

10. Biological or biochemical reagent according to claim 8, characterized in that the additional one label as a signal-generating component is selected from the group consisting of ESR or NMR spin labels, mass labels, electrochemical labels or in particular luminescence labels or fluorescence labels.

11. Biological or biochemical reagent according to claim 8, characterized in that a plurality of similar luminescence or fluorescent labels are associated with the vesicle as signal-generating components or several luminescence or FTuorescence labels of different emission wavelengths and the same or different excitation wavelengths are associated with the vesicle.

12. Biological or biochemical reagent according to claim 10 or 11, characterized in that luminescent or fluorescent nanoparticles are used as luminescent or fluorescent labels.

13. A method for producing a biological or biochemical reagent according to any one of claims 1-12, characterized in that

A) the lipid molecules required for vesicle production by dialysis,

C) brings together at least one biological or biochemical recognition element to be bound or adsorbed to the vesicle or to be bound or adsorbed to a polymer molecule for the recognition and binding of a ligand in sequential mixing and evaporation steps.

14.Bioanalytical detection method using a biological or biochemical reagent according to any one of claims 1-12.

15. Bioanalytical detection method according to claim 14 under

Use of a biological or biochemical reagent according to one of claims 1-12, characterized in that the detection of the analyte by means of a method selected from the group consisting of electrochemical detection, impedance spectroscopy, electron spin resonance, nuclear magnetic resonance, oscillating quartz measurements and in particular the detection of optical signal changes or a combination this procedure is done.

lό.Bioanalytical detection method according to claim 15, characterized in that the determination of the optical signal change with the aid of an optical sensor serving as a sensor platform, the optical sensor preferably being selected from the group consisting of optical waveguide sensors or surface plasmon sensors.

17.Bioanalytical detection method according to one of claims 15 - 16, characterized in that 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 or in particular a luminescence or fluorescence generated in the near field of the sensor.

18. Bioanalytical detection method according to one of claims 16 - 17, characterized in that a planar or fibrous waveguide is used as the sensor platform.

19.Bioanalytical detection method according to claim 18, characterized in that a planar thin-film waveguide is used as the sensor platform, which comprises a first optically transparent layer (a) on a second optically transparent layer (b).

20. Bio-analytical detection method according to claim 19, characterized in that the excitation light is coupled into the planar thin-film waveguide serving as a sensor platform via one or more gratings (c).

21. Bioanalytical detection method with a sensor platform according to one of claims 16 - 20, characterized in that between the optically transparent layers (a) and (b) and in contact with layer (a) there is a further optically transparent layer (b ') lower refractive index than that of layer (a) and a thickness of 5 nm - 1000 μm, preferably 10 nm - 1000 nm.

22. Bioanalytical detection method with a sensor platform according to one of claims 16-21, characterized in that an adhesion-promoting layer (f) is applied to the optically transparent layer (a) for the immobilization of biological or biochemical detection elements (e).

23. Bioanalytical detection method with a sensor platform according to one of claims 19 to 22, characterized in that the thin-film waveguide serving as the sensor platform comprises several measuring ranges (d) for the simultaneous or sequential determination of one or more analytes from one or more samples.

24. Bio-analytical detection method with a sensor platform according to claim 23, characterized in that spatially separated measuring areas (d) are generated by spatially selective application of biological or biochemical or synthetic detection elements on said sensor platform.

25. Bioanalytical detection method with a sensor platform according to one of claims 20 to 24, characterized in that the grating structure (c) has a spatially varying periodicity perpendicular or parallel to the direction of propagation of the excitation light coupled into the optically transparent layer (a).

26. Bioanalytical detection method with a sensor platform according to one of claims 19 to 25, characterized in that the material of the second optically transparent layer (b) consists of glass, quartz or a transparent thermoplastic from the group consisting of polycarbonate, polyimide or polymethyl methacrylate is formed.

27. Bioanalytical detection method with a sensor platform according to one of claims 19 - 26, characterized in that the refractive index of the first optically transparent layer (a) is greater than 2.

28.Bioanalytical detection method with a sensor platform according to one of claims 19 to 27, characterized in that the thickness of the first optically transparent layer (a) is 40 nm to 300 nm.

29. Bioanalytical detection method with a sensor platform according to one of claims 20-28, characterized in that the grating (c) has a period of 200 nm - 1000 nm and the modulation depth of the grating (c) 3 nm to 100 nm, preferably 10 nm is up to 30 nm.

30.Bioanalytical detection method with a sensor platform according to claim 29, characterized in that the ratio of the depth of modulation to the thickness of the first optically transparent layer (a) is equal to or less than 0.2.

31.Bioanalytical detection method with an optical system for determining one or more luminescences, with at least one excitation light source of a sensor platform according to one of claims 19 to 30, at least one detector for detecting the light emanating from the at least one or more measuring ranges (d) on the sensor platform.

32. Bioanalytical detection method with a sensor platform according to one of claims 20 to 31, characterized in that the excitation light emitted by the at least one light source is coherent and is radiated onto the one or more measuring ranges at the resonance angle for coupling into the optically transparent layer (a) becomes.

33. Bioanalytical detection method according to one of claims 17 - 32, characterized in that at least one spatially resolving detector is used for the detection.

34.Bioanalytical detection method according to one of claims 17 - 33, characterized in that between the one or more excitation light sources and the sensor platform and / or between said sensor platform and the one or more detectors optical components from the group are used, which are of lenses or lens systems for shaping the transmitted light bundles, planar or curved mirrors for deflection and, if necessary, additionally for shaping light bundles, prisms for deflecting and possibly for spectrally dividing 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 beams or polarization-selective elements to choose from discrete directions of polarization of the excitation or luminescent light are formed.

35. Bioanalytical detection method according to one of claims 17 to 34, characterized in that the excitation light is irradiated onto and the emission light is detected from one or more measuring ranges sequentially for individual or several measuring ranges.

36.Bioanalytical detection method according to claim 35, characterized in that sequential excitation and detection takes place using movable optical components, which is formed from the group of mirrors, deflection prisms and dichroic mirrors.

37. Bioanalytical detection method according to claim 35 or 36, characterized in that the sensor platform is moved between steps of sequential excitation and detection.

38. Bioanalytical detection method according to one of claims 17 - 37, characterized in that 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 analog of the analyte or in a multi-stage assay on one the binding partner of the analyte or the biological or biochemical or synthetic recognition elements used are bound.

39.Bioanalytical detection method according to one of claims 17 - 38, characterized in that (1) the isotropically emitted luminescence or (2) coupled into the optically transparent layer (a) and coupled out via the lattice structure (c) or luminescence of both components ( 1) and (2) can be measured simultaneously.

40.Bioanalytical detection method according to at least one of the

Claims 14 - 39 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.

41. Bioanalytical detection procedure according to at least one of the

Claims 14 - 40, characterized in that the samples to be examined naturally occurring body fluids such as blood, serum, plasma, lymph or urine or egg yolk or optically cloudy liquids or surface water or soil or plant extracts or bio or synthesis process broths or taken from tissue parts of living or deceased organisms are.