JP2003501631A - Functionalized vesicles - Google Patents

Abstract

  (57) [Summary] The present invention relates to functionalized, polymer-reinforced (sterically stabilized) vesicles, characterized in that they comprise biological, biochemical or synthetic identification elements for ligand identification and binding. . Further, the vesicles of the present invention optionally include a label that serves as a signal-generating component in a bioanalytical detection method. The present invention also relates to a method for producing the vesicle and its use in a detection method.

Description

Detailed Description of the Invention

The subject of the present invention is a functionalized, polymer-reinforced (sterically stabilized) vesicle comprising a biological or biochemical recognition element for ligand recognition and binding, a process for its preparation. And its application.

There is a need for the development of reagents for bioanalytical applications both in solution and on solid surfaces, which reagents serve as recognition elements for the measurement of ligands that specifically bind to them. When used, it preserves the functionality and native conformation of biological molecules such as membrane receptors, and the reagent simultaneously minimizes nonspecific interactions with this recognition element. Is.

The subject of the invention is A) vesicles, B) one or more polymer molecules bound to the inner and / or outer surface of the vesicles, and C) for the recognition and binding of ligands, A biological or biochemical reagent, characterized in that it comprises at least one biological or biochemical or synthetic recognition element, which recognition element is bound or adsorbed to a vesicle or a polymer molecule. Is.

Yet another subject of the present invention is a functionalized, polymer-reinforced (sterically stabilized) vesicle, characterized in that it further comprises a label as a signal-generating component in a bioanalytical detection method. (Wherein the labels are ESR or NMR labels, mass labels,
It can be selected from the group comprising electrochemical labels, luminescent labels or fluorescent labels). At least one label as a signal-generating component may be located inside the vesicle, or may be attached to the surface of the vesicle either directly or by a spacer or by a polymer. The vesicles may also be associated with a number of similar labels, especially similar luminescent or fluorescent labels. This is because when multiple luminescent or fluorescent labels with different emission wavelengths and similar or different excitation wavelengths are used,
Energy transfer may be advantageous for certain applications where it is used to improve the difference between specific binding signal and background. The luminescent or fluorescent label may be a conventional luminescent or fluorescent label, ie a so-called semiconductor-based luminescent or fluorescent nanoparticle (WCW Chan and S. Nie, “Quantum dot bioconjugates.
for ultrasensitive nonisotopic detection ”, Science 281 (1998) 2016-201
8).

Yet another subject of the invention is their application in methods for the production of said functionalized vesicles and in bioanalytical detection methods. These methods can be selected from the group comprising optical signal changes, electrochemical detection, impedance spectroscopy, electron spin resonance, nuclear spin resonance, crystal balance measurements, or a combination of these methods.

Yet another subject of the invention is a process for the production of the biological or biochemical reagents according to the invention, which comprises A) a lipid molecule necessary for the production of vesicles by dialysis, B) of the vesicles and And / or one or more polymer molecules to be attached to the outer surface,
And C) Sequential mixing and evaporation of at least one biological or biochemical recognition element for recognition and binding of the ligand, which recognition element is to be bound or adsorbed to the vesicle or polymer molecule. It is a method of combining them in the process.

Yet another subject matter of the invention is a bioanalytical detection method using the biological or biochemical reagents of the invention. In particular, the analyte can be detected by a change in optical signal. The optical signal change is preferably detected by an optical sensor that acts as a sensor platform. Preferably, the optical sensor platform is selected from the group comprising optical waveguide sensors and surface plasmon resonance sensors.

In a preferred embodiment, the change in the optical signal to be detected is based on the change in the effective refractive index in the near field of the sensor surface. In another preferred embodiment,
The optical signal change to be detected is based on the luminescence or fluorescence produced in the near field of the sensor.

In particular, planar or fiber-type waveguides can be used as sensor platforms. In a preferred embodiment, a planar thin film waveguide is used, characterized in that it comprises a first optically transparent layer (a) on a second optically transparent layer (b).

An additional subject of the invention is the biological or inventive method according to the invention, in which the excitation light is coupled by means of one or several gratings (c) to a thin-film waveguide acting as a sensor platform. A bioanalytical detection method that uses biochemical reagents.

Lower refractive index than layer (a) and 5 nm to 1000 μm, preferably 10 nm
A further optically transparent layer (b ') of ~ 1000 nm thickness comprises layers (a) and (
It would be advantageous if it was located between b) in contact with layer (a). The purpose of this intermediate layer is to reduce the surface roughness underneath layer (a) or to create a vanishing field (evanescent field) for the guided light in layer (a) to one or more underlying layers. Reduce permeation, or improve the adhesion of layer (a) to one or more underlying layers, or reduce heat-induced stress in the sensor platform, or layers to underlying layers The chemical isolation of the optically transparent layer (a) from the layer below by sealing the micropores of (a).

There are many methods for the deposition of biological or biochemical recognition elements on the optically transparent layer (a). This can be achieved, for example, by physical adsorption or electrostatic interaction. And in general, the orientation of the recognition element is a statistical property at random. In addition, there is a risk that some of the immobilized recognition elements will be washed away by changing the composition of the analyte-containing sample or reagents applied to the assay. Therefore, the application of an adhesion promoting layer (also called anchoring layer) (f) on the optically transparent layer (a) may be advantageous for the immobilization of biological or biochemical recognition elements. The fuser layer should also be optically transparent. In particular, the anchoring layer should not exceed the depth of penetration of the vanishing field of the waveguiding layer (a) into the adjacent medium. Therefore, the fixing layer should have a thickness of less than 200 nm, preferably less than 20 nm.

Yet another subject of the invention is that a thin-film waveguide, which acts as a sensor platform, provides several measurement areas for the simultaneous or sequential measurement of one or more analytes in one or more samples. A bioanalytical detection method using a sensor platform, which comprises:

The laterally separated measuring regions (d) can be produced by laterally selective deposition of biological or biochemical or synthetic recognition elements on the sensor platform. When contacted with a luminescent analyte, or an analogue of a luminescent labeled analyte, which competes with the analyte for binding to the immobilized recognition element, these luminescent molecules are bound by the measurement region (immobilized recognition element). (Limited by the area occupied by) and selectively binds only to the surface of the sensor platform.

It is advantageous for many applications if the grating structure (c) is a diffraction grating with a constant period. The resonance angle is constant over the entire range of the lattice structure because of the incoupling of the excitation light into the measurement region by the lattice structure (c). However, if, for example, excitation light from different light sources of significantly different wavelengths is coupled, the associated resonance angles for incoupling may be even more significantly different, and thus the optical system housing. It requires the use of additional elements for adjustment in the sensor platform or results in a geometrically very unfavorable coupling angle. Thus, for example, it would be advantageous if the grating structure (c) was a multiple diffraction grating, in order to avoid very different coupling angles.

The material of the second optically transparent layer (b) may be characterized as comprising glass, quartz, or a transparent thermoplastic of the group comprising polycarbonate, polyimide or polymethylmethacrylate.

The index of refraction of the optically transparent layer (a) as a waveguide is due to the creation of the strongest possible evanescent field on the surface of the optically transparent layer (a). , Must be significantly higher than the refractive index of the adjacent layers. First optically transparent layer (a)
It is particularly advantageous if the index of refraction is greater than 2.

The first optically transparent layer (a) is, for example, TiO ₂ , ZnO, Nb ₂ O ₅ , T
It may include a ₂ O ₅ , HfO ₂ , or ZrO ₂ . The first optically transparent layer is Ti
It is particularly preferable if it contains O ₂ or Ta ₂ O ₅ .

Besides the index of refraction of the optically transparent layer (a) as a waveguide, its thickness is such that the vanishing field is as strong as possible at the interface of this layer to the adjacent layer with a low index. Is the second essential parameter for the production of Where the strength of the vanishing field is
As long as the layer thickness is sufficient to guide at least one mode at the excitation wavelength, it will increase as the thickness of the waveguiding layer (a) decreases. Here, the minimum "cutoff" wavelength for guiding a mode depends on the wavelength of this mode, and is longer for long wavelength light than for short wavelength light. However, the choice of preferred layer thickness sets a lower limit because the unwanted propagation loss is also significantly increased by the "cutoff" wavelength approach. Preference is given to the layer thickness of the optically transparent layer (a) allowing only 1 to 3 modes of waveguiding at a given excitation wavelength; particular preference is given to this excitation wavelength. The thickness of the layer that results in a single mode waveguide. It has to be understood here that the nature of the discrete modes of guided light applies only to transverse modes.

As a result of these requirements, the thickness of the first optically transparent layer (a) is preferably between 40 nm and 300 nm. The thickness of the first optically transparent layer (a) is 70 nm
160 nm is particularly advantageous.

For a given index of refraction of the optically transparent layer (a) and the adjacent layers as waveguides, the resonance angle for the incoupling of the excitation light is the diffraction order incoupling, It depends on the excitation wavelength and the grating period. In order to increase the incoupling efficiency, incoupling of the first diffraction order is advantageous. The incoupling efficiency, apart from the number of diffraction orders, is mainly measured by the grating depth. In principle, the coupling efficiency increases with increasing grating depth. However, the out-coupling process is just the opposite of the in-coupling process, so that the out-coupling efficiency rises at the same time, resulting in a measurement area (d) located on or close to the lattice structure (c). It is optimal for the excitation of the luminescence at, but this optimal depends on the shape of the measurement area and the shape of the emitted excitation beam. Due to such boundary conditions, if the grating (c) has a period of 200 nm to 1000 nm and a grating modulation depth of 2 nm to 100 nm, preferably 10 nm to 30 nm,
It is advantageous.

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

For amplification of the luminescence or improvement of the signal to background ratio, optionally on an additional dielectric layer (eg of silica or magnesium fluoride) with a lower refractive index than layer (a), preferably gold. Alternatively, a thin metal layer of silver is used as an optically transparent layer (a
) And an immobilized biological or biochemical recognition element, this would also be advantageous (where the thickness of the metal layer and optionally of the additional intermediate layer depends on the excitation wavelength and / or Or selected so that surface plasmons can be excited at the luminescence wavelength).

Yet another subject matter of the invention is an optical system for the measurement of one or more luminescence, comprising: at least one excitation light source, a sensor platform according to any one of the embodiments described above, A system characterized in that it comprises at least one detector for the measurement of light from at least one or more measurement areas (d) on the sensor platform.

Here, the excitation light from at least one light source is emitted towards one or more measurement areas at a resonance angle for coupling to the coherent and optically transparent layer (a). It is advantageous if done.

Two or more coherent light sources with similar or different emission wavelengths are used in case of insufficient intensity of a single light source or when light sources with different emission wavelengths are needed, eg for biological applications It is advantageous if you do.

In order to separately record the signals from the multiple measurement areas, it is preferred to use at least one laterally dividing detector for signal detection.

In an optical system of the invention according to any one of the embodiments described above, a lens or lens system for shaping the transmitted light bundle, a flat or curved mirror for deflecting the light bundle and optionally additional shaping, a light bundle Prism for the deflection and optionally spectral separation, a dichroic mirror for spectrally selective deflection of a part of the beam, a neutral density filter for adjusting the transmitted light intensity, a spectrally selective part of the beam. A group of optical components comprising an optical filter or monochromator for transmission or a polarization selection element for selection of discrete polarization directions of the excitation or luminescence light, between one or more excitation light sources and the sensor platform, and / or The sensor platform and 1
It may be located between one or more detectors.

For many applications it is advantageous if the excitation light is emitted in pulses with a time between 1f seconds and 10 minutes.

The emission from the measurement area is recorded in a time-divided manner for kinetic measurements or for the identification of rapidly decaying fluorescence from fluorescent contaminants in the material of the sample or part of the optical system or in the sensor platform itself. It would be advantageous if done.

The emission of the excitation light and the detection of the emission from one or more measurement areas can also be carried out sequentially for single or multiple measurement areas. Here, the sequential excitation and detection can be performed by using a group of movable optics including mirrors, deflecting prisms and dichroic mirrors. In another possible embodiment, the sensor platform is moved during the sequential steps of excitation and detection. In this case, the one or more excitation light sources and the components used for signal detection may be fixed at a distance.

Yet another subject of the invention is a complete analytical system for the determination of one or more analytes by means of luminescence detection in at least one sample at one or more measurement areas on a sensor platform. An optical sensor platform according to any of the above embodiments, an optical system according to any of the above embodiments, and for contacting one or more samples with a measurement region on the sensor platform. The optical film waveguide (layered waveguide) is also provided together with the above-mentioned supply means.

Here, the supply of sample and optional additional reagents can be carried out in parallel or crossed microchannels by the application of a pressure difference or an electric potential or an electromagnetic potential.

For the quantification and / or qualitative determination of the analyte, the bioanalytical detection method of the invention also comprises one or more luminescent or fluorescent labels for the detection of the analyte.
It binds to the analyte, or to the analyte analogue in the competitive assay, or to the binding partner of the analyte or one of the applied biological, biochemical or synthetic recognition elements in the multi-step assay. It is also possible.

In the above method, (1) isotropically emitted luminescence or (2) is optically coupled to the optically transparent layer (a) and is outcoupled by the lattice structure (c). The existing luminescence or the luminescence of both (1) and (2) can be measured simultaneously.

The bioanalytical detection methods of the present invention include antibodies or antigens, receptors or ligands, chelating agents or “histidine-tag moieties”, oligonucleotides, DNA or RNA chains, DNA or RNA analogs, enzymes, enzymes. It is used for the simultaneous or sequential quantitative or qualitative measurement of one or more analytes of the group comprising cofactors or inhibitors, lectins and carbohydrates.

The sample to be tested may be a natural body fluid such as blood, serum, plasma, lymph or urine or egg yolk. However, the sample to be examined may also be an optically cloudy liquid, surface water, soil or plant extracts, broth of bio- or synthetic processes. The sample to be examined may also be taken from living or dead organism tissue.

Lipid vesicles with bilayer membranes have been used to date, mainly due to two aspects. First, lipid vesicles have been applied in research as a model system for biological membranes. Here, the vesicles act as a carrier system for water-insoluble membrane proteins and receptors. Secondly, in pharmaceutical applications, lipid vesicles are industrially applied as carrier systems for therapeutically active reagents for the specific treatment of diseases.

In the following, the term “vesicle” is used exclusively, irrespective of the different use of both terms in the literature (“vesicle” and “liposome”).

Vesicle-forming lipids are generally characterized in that they include amphipathic lipids having hydrophobic and polar groups. In the following, hydrophobic moieties are designated as "lipid chains" and hydrophilic or polar moieties are designated as "polar head groups". In an aqueous environment, such lipid molecules are able to spontaneously form bilayer structures, such as lipid vesicles.

The structure and properties of such vesicles can be found in standard textbooks such as RB Gennis: “B
iomembranes ”, Advanced Texts in Chemistry” (editor: CR Cantor), Spri
nger, Heidelberg, 1989.

The lipid molecule described above is also a region formed by the head group of a vesicle in which the lipid chain is in the hydrophobic interior of the lipid bilayer and in which the polar head group resides, in the already expanded bilayer or vesicle. It can be easily incorporated as it is incorporated into. Vesicle-forming lipids are preferably characterized in that they contain two hydrocarbon chains of varying length, with typical lengths of 14 to 22 carbon atoms, including for example alkyl chains. .

Such lipid molecules can be isolated, for example, from the biological membranes of various organisms and are called “natural lipids”. Typical examples are glycerophospholipids such as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, sphingomyelin. Other natural lipids include glycolipids, sterols (eg, cholesterol), cardiolipin, plasmalogens, and lipids present in archaebacteria (eg, those with hydrocarbon chains attached to ether groups, those with branched hydrocarbon chains, or So-called bipolar lipids (having an alicyclic structure and a hydrocarbon chain) or lipid molecules extending across the lipid bilayer (for a detailed description, see, for example, RBG).
ennis: “Biomembranes”, Advanced Texts in Chemistry ”(editor: CR Can
tor), Springer, Heidelberg, 1989). Yet another example includes synthetic lipids such as those commercially available from, for example, Avanti Polar Lipids (Alabaster, USA).

Many of the natural lipids can also be chemically synthesized. Furthermore, there are synthetically produced lipids that do not belong to the class of natural lipids. Both in the lipid chain and in the polar head groups, these may contain groups which can be activated or polymerized. Yet another example is positively charged lipids such as dodecyl ammonium bromide (DODAB), or boroamphiphiles, and lipids containing fluorinated or halogenated lipid hydrocarbon chains.

An overview of broad-spectrum synthetic lipids is provided by J.-H. Furhop and J. Koening: “Membrane.
s and Molecular Assemblies: The Synkinetic Approach ”, Monographs in Sup
ramolecular Chemistry (editor: JF Stoddard), The Royal Society of Chem
Given to istry, 1994. Synthetic lipids are ideally suited for the formation of vesicles as pure compounds, as a mixture of various synthetic lipids, or as a mixture containing synthetic and natural lipids.

Vesicles typically have the following protocol (eg, F. Szoka and D. Papahadjopo
ulos, Rev. Biophys. Bioeng. 9 (1980), 467-508).
Formed by one or a combination.

Multilamellar vesicles (MLV) are typically prepared by adding water or an aqueous buffer solution to a lipid film pre-deposited from a lipid solution in an organic solvent into a suitable container by evaporation of the solvent. Such as dried lipid films or powders by rehydration under various conditions. Depending on manufacturing methods such as stirring, shaking, sonication, and depending on the nature, ionic strength and concentration of lipids, MLVs of different sizes are formed to mention a few parameters. .

Yet another example of a method of manufacture is the “solvent spherule method”, the reverse phase method or vesicle fusion by freeze / thaw or dehydration / rehydration (eg DD.
Lasic, Biochem. J. 256 (1988), 1-11).

Small unilamellar vesicles (SUVs) are ML by sonication or filtering of MLVs
Formed by extrusion of V. In the latter case, the vesicle diameter is typically of the same order of magnitude as the pore size of the applied filter. Another very useful manufacturing method is the surfactant dilution method. Here, the lipids or MLVs are solubilized in the presence of detergent, and the detergent is removed by dilution, dialysis, chromatography, adsorption, ultrafiltration or centrifugation, and finally SUV It is formed.

Large unilamellar vesicles (LUV) are typically by removal of detergent, or by injection methods, where lipids dissolved in an organic solvent are injected into water or an aqueous buffer, or vice versa. It is formed by the phase evaporation method. In the latter case LUVs are formed by the ejection of droplets of organic solvent, in which the lipid is dissolved and dispersed in the aqueous phase.

Very large vesicles can be formed, for example, by the vibrationless swelling of homogeneously dried lipid films in aqueous solution.

Vesicles with a diameter of 20 to 1000 nm, in particular 50 to 400 nm, are preferred for the biological or biochemical reagents according to the invention. Most preferred are vesicles with a diameter of 50-200 nm.

The polymers mentioned under B) are used both for the stabilization of vesicles and the reduction of non-specific binding to the surface. In particular, it should be mentioned that the biological or biochemical reagents according to the invention comprise hydrophilic polymer molecules, which are bound to the inner and / or outer surface of the vesicle. This hydrophilic polymer is attached to the vesicles to create a hydrophilic shell. This shell can be considered as a steric barrier to the diffusion of molecules into and out of vesicles. Therefore, such vesicles are also referred to as "sterically stabilized vesicles." In the ideal case, the vesicle-bound polymer has a high degree of conformational flexibility, allowing for rapid entropy changes and rapid changes between different conformations. Upon the approach or even binding of such vesicles to particles or the macroscopic surface (eg, the surface of the sensor), a reduction in the polymer's conformational freedom corresponds to a loss of entropy. This reduces (non-specific) binding to other particles or macroscopic surfaces. This effect is called "entropy masking" (DD Lasic and F. Marti
n: “Stealth Liposomes”, CRC Press, Boca Raton, 1995). Such vesicles can escape the mononuclear phagocyte system, the "Stealth Vesicles".
Also called. These have applications for the transport of drugs in living organisms. In the context of the present invention, such vesicles are referred to as "sterically stabilized vesicles" (SSV).

The polymer molecule can be attached to the vesicle by one or a combination of the following methods: (1) a positively (or negatively) charged portion of the polymer and a negative (or positive) portion of the vesicle surface. To)
Electrostatic interaction between charged groups. The vesicle charge may arise, for example, from polar lipid head groups, or other suitable compounds in or on the vesicle bilayer, such as natural or artificial polypeptides.

(2) Covalent attachment of the polymer molecule to the polar head group of the lipid molecule, which serves as an anchor for the incorporation of vesicles into the lipid bilayer. Suitable functional groups for lipids are amino groups such as diacylglycerophosphatidylethanolamine, or SH or OH groups of natural or synthetic lipid molecules. The polymer is directly attached to the lipid molecule
And with spacer molecules between lipid molecules or lipid-type molecules can be used both. Some such compounds with active head groups are commercially available and they are reactive with, for example, amines or thiols. Examples include succinimide derivatives, pyridinylthio derivatives or maleimide derivatives,
And part of it, for example, Avanti Polar Lipids
s) (Alabaster, USA). Further, non-commercial examples include G. Brink et al., Biochem. Biophys. Acta 1196 (2, 1994) 227-230.
It is described in.

(3) Other suitable anchor molecules are natural transmembrane proteins or membrane spanning polypeptides, or membrane bound proteins or polypeptides, or synthetic analogues thereof. The membrane protein or polypeptide is also referred to as the hydrophobic core of the membrane, eg in the case of membrane spanning or transmembrane proteins or polypeptides. Exogenous protein or polypeptide molecules associate with the membrane primarily by ionic, hydrogen-bonding, or hydrophobic interactions, and in some cases by lipid anchors that are covalently attached to the protein molecule ( L. Stryer: “Bio
chemistry ”, 4th edition, Freeman (1995) 275 pp.).

A variety of hydrophilic polymers are suitable for attachment to vesicles, some examples of which are described below. (1) The use of polyethylene glycol and its derivatives with uncharged polymer "stealth vesicles"
DD Lasic and F. Martin: “Stealth Liposomes”, CRC Press, Boca Raton, 199.
It is described in 5. In U.S. Pat.Nos. 5,534,241, 5,770,222, and 5,891,468, vesicle-bound polyethylene glycol or similar hydrophilic polymers consist of hydrophilic and hydrophobic polymers to confine the hydrophobic polymer and drug encapsulated in the vesicles. Described above as part of a diblock copolymer for the purpose of releasing these hydrophilic polymers and drugs by releasing the hydrophilic polymer under appropriate physiological conditions.

(2) Charged Polymer A negatively or positively charged polymer or a zwitterionic polymer can also be used. Typical examples are polypeptides and polysulfoxides, and these are mentioned, for example, in US Pat. No. 5,891,468.

(3) Carbohydrates and derivatives thereof Examples include chitin / chitin-dextran, starch and US Pat. No. 5,891,
There is a similar polymer referred to in 468.

(4) Dendritic Hydrophilic Polymers Typical examples of this class are GR Newkome, CN Moorefirled, and F. Voegtle: “D.
endritic Molecules ", Verlag Chemie (1996).

There are two major areas of application for lipid vesicles. The first area of application is mainly the use of lipid vesicles as a model system for biological membranes in basic research (eg RB Gennis: “Biomembranes”, Advanced Texts in Chemistry (ed.
itor: CR Cantor), Springer, Heidelberg (1989)). In the applications described in this reference, vesicles are mainly ion channel receptors (eg nAchR = acetylcholine receptors, 5HT3 receptors, glutamate receptors, GABA receptors), G protein coupled receptors (eg. , Water-insoluble membrane proteins and receptors such as neurokinin receptors, chemokine receptors, β-adrenergic receptors), membrane enzymes (eg tyrosine kinases, adenyl cyclases), and membrane transporters (eg ATPases) Used as a carrier for the body.

Another main area of application relates to the use of vesicles as a carrier or for the administration of drugs for the treatment of certain diseases. The use of vesicles for this application has been known for over 20 years. Many of the early applications were typically some neutral or negatively charged,
It is based on so-called conventional vesicles, which consist of different lipids (mainly phospholipids) and / or cholesterol. During use for drug administration, these conventional vesicles have a very short circulation time in the blood. During in vivo administration, they show a strong tendency to rapidly accumulate in phagocytes of the mononuclear phagocyte line. To overcome the disadvantageous short circulation time, hydrophilic polymers have been attached to the vesicles, as already mentioned above.

In order to direct the vesicle to the tissue to which it is more specifically applied, as a specific recognition element,
An antibody or antibody fragment (F _AB ) is bound to the vesicle. After in vivo administration, these vesicles circulate in the bloodstream and are transported to the tissue by vesicles and vesicles by binding to the target antigen that is specifically present in the target tissue by associated antibodies. The drug to be concentrated. Therefore, these vesicles are called immunoliposomes. Here, the antibody can be bound to the lipids of the vesicles directly or by means of so-called spacer molecules. Polymers such as polyethylene glycol (PEG), which are applied for the formation of SSVs (sterically stabilized vesicles), can also be used as spacers, and thus the antibody at the end of the polymer chain. Binding occurs. Other known examples include direct attachment of the antibody to the lipid of SSV (or its attachment by a short spacer molecule) such that the antibody is located within the hydrophilic polymer shell.

For example, a liposome composition for the treatment of a patient having an outer hydrophilic layer of PEG (molecular weight 10,000 to 100,000 Da) and a recognition element (so-called effector) covalently bound to them is described in WO Claimed at 94/21235. This patent application claims a shell of PEG to prevent the degradation of vesicles in the bloodstream. Effectors consist of small and medium sized immunological recognition elements, ie, _Fab fragments, glycoproteins, cytokines, polysaccharides, or various peptides and peptide hormones. In WO 97/35561 biologically active liposomes stabilized by a water-soluble polymer shell are claimed for therapeutic use. Here, the active biological amphipathic recognition element is associated non-covalently, ie by physical adsorption, with the polymer-reinforced liposome. In certain cases, the recognition element is the peptide "growth hormone releasing factor". Diameter 3 produced by extrusion
Unilamellar liposomes of less than 00 nm are preferred. As the polymer, preferably
PEG is used which is provided as a covalently attached PEG lipid in a liposome-forming membrane. In the broader sense, lipid compositions that further include a label for detection purposes are claimed for diagnostic purposes. Fluorescent markers, radioactive labels, dyes, and components for signal amplification in nuclear spin resonance are listed as detectable markers. The method and location of binding of the label to the vesicles has not been specified.

In another variation (US Pat. No. 5,891,468), polymer-reinforced vesicles containing ligands for recognition of cellular receptors attached to the ends of polymer chains are claimed for therapeutic applications. Has been done. The vesicles have the additional function of releasing hydrophilic polymer chains after successful target recognition by ligand-receptor interactions and subsequent fusion of the vesicles with the target membrane. In addition to others, WO 97/33618
Claims vesicles delivered by T lymphocytes, particularly for intracellular drug administration for therapeutic purposes in the treatment of cancer, wherein the recognition element is linked by a spacer-polymer-spacer type combination. Has been). Specifically, HPMA
Copolymers are used as polymers. Peptides, cytotoxins, nucleic acids, antigens, and drugs have been described as recognition elements. Later patents used PEG as the polymer.
(WO 98/51336), the extension was introduced, and in the following, the range of application was expanded (eg interleukin 2 peptide (IL2) to IL2).
It is used as a ligand for T cell membrane receptor recognition (WO 99/07324)).

The areas of vesicles and their applications mentioned above have heretofore been exclusively concerned with therapeutic applications, due to the main function of the vesicles as a carrier or transport vehicle. There are only isolated reports on the use of vesicles in the field of bioanalysis. WO 97/39736 describes vesicles as reagents for immunoassays, for example for the analysis of samples from patients. This patent application claims ligand-associated vesicles for detection of analyte molecules, where the ligand is a protein,
Peptides, antibodies or fragments thereof, and nucleic acids). Specific ligands are small hapten molecules for immunological recognition, and these are covalently attached to vesicles, especially in the form of hapten lipids. The signal detection in the assay is performed by the binding of the vesicle reagent to a solid surface in the form of beads, particles or walls of a microtiter plate provided with the respective biological receptor. The signal generation in the assay is established after successful recognition of the analyte, eg in the sandwich assay by the binding of a secondary label in solution to the liposome, where the label is a fluorescent or luminescent molecule, a dye. , Or a signal-generating enzyme such as alkaline phosphatase). Instead of binding to the macroscopic solid phase surface, specific biorecognition can occur by binding to a second vesicle with its respective receptor. In each case, specific ligand-receptor binding in the presence of vesicles must be preserved throughout the assay. Note that this reference does not use or describe additional polymers for stabilizing the liposomes.

As yet another application, the use of vesicles for signal amplification in bioanalytical detection methods has been reported by several groups. For example, the use of vesicles in fluorescence-based immunoassays on planar waveguides has been demonstrated by SA Choquette: “Planar wavegui
de immunosensor with fluorescent liposome amplification ”, Analytical Ch
Reported in emistry 64 (1992) 55-60. Here, a competitive assay is described in which a vesicle-bound theophylline molecule in solution competes with a free theophylline molecule as an analyte for binding to an anti-theophylline antibody immobilized on the waveguide surface. There is. The hydrophilic inner surface of the vesicles is simultaneously loaded with multiple carboxyfluorescein molecules as fluorescent labels. Thus, a maximum, very strong fluorescent signal is observed when theophylline is absent in the sample to be analyzed. However, the stability of vesicles is not enhanced by the polymer, so that depending on the particular composition of the sample, some leakage of encapsulated fluorescent label should be expected.

In a surface-restricted method for the measurement of analytes, one partner of the affinity system may be used in order to specifically recognize and bind the analyte from the sample in a subsequent measurement method, eg a biochemical sensor. The chemical substance is immobilized on the solid surface. For this reason, surface functionalization is essentially important. Especially in the case of biological interactions that are sensitive to its environment, for example at membrane receptors, the conservation of the native conformation of the recognition element is important for maintaining its specific binding capacity. At the same time, it is important to minimize non-specific binding to the surface. Various methods are known for the determination of analytes on solid surfaces. Here, optical analysis methods based on the near-field interaction of surface-limited sensors are of particular importance because of the minimal hindrance of binding events. For example, in the vanishing field of a waveguide,
Alternatively, an analysis method based on refraction or luminescence in the penetration depth of the surface plasmon field generated in the metal thin film (surface plasmon resonance, SPR) is known as such an optical near-field method.

Fluorescence-based analysis methods by fluorescence excitation in the quenching field of a planar waveguide are described, for example, in WO 95/33197 and WO 95/33198 for the measurement of a single analyte, and in the simultaneous measurement of multiple analytes. Are described in WO 96/35940.

In certain embodiments of the invention, the lipid and the amphipathic or hydrophilic polymer are sterically stable, including a biological or biochemical or synthetic recognition element for ligand recognition and binding. It can be transformed into vesicles. Here, “biological or biochemical or synthetic recognition element” means any biological molecule or biological molecule complex or synthetic molecule or synthetic substance that specifically binds to any other compound. Means a molecular complex. Preferably, antibody, antibody fragment, nucleic acid or nucleic acid analog, DNA, R
Biological or biochemical or synthetic recognition elements of the group comprising NA, enzymes, natural and synthetic polypeptides, histidine-tag-components, and membrane receptors are used. The other compound is an ion, atom, or atom that is specifically recognized and bound by the recognition element.
It may be a cluster of atoms, a molecule, a cluster of molecules, any synthetic or biological compound or part thereof. A biological or biochemical or synthetic recognition element is associated with or incorporated into the surface of the vesicle, or provided with a polymer or vesicle to provide sufficient accessibility to the ligand or analyte. Preferably bound to the lipid of

The specific recognition and binding of the compound by the recognition element is part of a bioanalytical assay and thus may be another subject of the invention.

For example, the compound may be the analyte in the sample to be measured. here,
The measurement may be by measuring the change in a physically measurable parameter caused by the binding of the analyte itself or an analogue of the analyte (eg in a competitive assay), or a multi-step binding event (where the analyte is Wright or an analogue thereof may be combined in one of the substeps). A change in a physically measurable parameter occurs, for example, by luminescence after luminescence excitation, or by a signal from the ESR or NMR-label after its excitation, or by a change in the molecular mass adsorbed on the surface. sell. The measurement can be carried out in free solution or on a solid surface, for example on the surface of the sensor.

In order to minimize the non-specific binding of the reconstituted vesicles described above, the composition of the lipid mixture, the charge of the vesicle or modified sensor surface, the addition of detergent, and the ionic strength of the buffer may be varied. Various parameters were changed, such as All these parameters can contribute to a reduction in non-specific binding to some extent, but the final residual residual non-specific binding was still too high.

In contrast, incorporation of PEG lipids into vesicles resulted in a surprisingly strong reduction in non-specific binding. In fact, there is also a decrease in specific binding signal due to steric hindrance. However, the net result is a marked improvement in the ratio of specific signal to non-specific signal, an essential parameter in the measurement technique. The effect of polymer incorporation on the observed binding signal can be explained as follows: The polymer forms a steric barrier around the membrane, and a reduction in nonspecific binding is observed. PEG lipids of various chain lengths of PEG (with molecular weights of 750, 2000 and 5000 daltons) were used.

[0075]   The invention is explained in more detail in the examples below.

Example Measurements Bioanalytical methods are based on commercially available SPR configurations (Biacore 1000,
Uppsala, Sweden). Unless otherwise stated, the method is 2
It was carried out at 5 ° C. and a constant flow rate of 25 μl / min.

A) Functionalization of the sensor surface SPR in a chamber saturated with ethanol to avoid solvent evaporation.
On the outside of the configuration, self-assembly of 16-mercapto-hexadecanoic acid and 11-mercapto-undecanol on the clean gold surface of SPR sensor chip J1 (Biacore) resulted in a monolayer (self-assembled monolayer, SAM). ) Was generated. Thus, a solution containing 1 mM 16-mercapto-hexadecanoic acid and 1.5 mM 11-mercapto-undecanol in water / ethanol 1: 7 (1: 1 water /
4 mM 16-mercapto-hexadecanoic acid in ethanol and 6 in ethanol
A stock solution of mM 11-mercapto-undecanol (40 μl each) and taken from a mixture of 80 μl ethanol) was deposited on the sensor surface in 30 μl aliquots. After 30 minutes, the solution was changed to form a mixed self-assembled monolayer (SAM) after 80-100 minutes.

After insertion of the SPR chip into the SPR configuration, the SAM was functionalized by the following method: The sensor surface was first HEPES buffered salt solution (HBS: 10 mM HEPE).
S, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20, pH
Washing according to 7.4) at a flow rate of 10 μl / min.

Next, 20 μL of 0.2 M N-ethyl-N′-dimethylaminopropyl-carbodiimide (EDC) /0.05 M N-hydroxysuccinimide (NHS) was supplied to the chip surface at a low flow rate of 5 μL / min, Subsequently 50 μl of a 1: 3 solution of streptavidin in water / HBS (250 μg / ml) and then 35 μl of ethanolamine solution (1M, pH 8.5) were each injected at a flow rate of 5 μl / min. Finally, a solution of biotinylated α-bungarotoxin (biot-BgTx) in HBS (
Α-bungarotoxin (BgTx) was bound to the surface by supplying 50 μg / ml) for 2 minutes.

B) Reconstitution of nicotine-acetylcholine receptor (nAchR) in lipid vesicles Lipids dissolved in chloroform were mixed in the desired ratio (typically 80 mol% 1,2-
Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 10
% Cholesterol (Chol), and 10% 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG)). After evaporation of the solvent, the deposited lipid film was washed with a buffer solution (10 mM HEPES, 150 mM Na).
Cl, 3 mM EDTA, pH 7.4) 480 μl and CHAPS (10% w / w) 2.
2 mg / ml after subsequent ultrasonic exposure by re-dissolving by adding 70 μl
A final lipid concentration of was obtained. Undissolved substances were removed by adding 250 μl of concentrated receptor membrane (1.9 mg / ml), stirring the resulting suspension, and centrifuging. "
650 times buffer solution (500m in slide-a-lyser)
M NaCl, 10 mM HEPES, 3 mM EDTA, 1 mM NaN ₃ , pH 7.
Dialysis of the supernatant against 4) (first 30 minutes at room temperature, then 8-15 hours and finally again 2-4 hours; dialysis buffer was changed for each step) to produce lipid vesicles. The dialyzed vesicles were stored for up to 2 days and centrifuged again before performing SPR measurements to remove aggregates formed from unreconstituted receptor. Unless otherwise stated, all steps were performed at 4 ° C.

The concentration of receptor incorporated into the vesicles was determined by measuring the optical density at 280 nm (ε = 450,000 M ⁻¹ cm ⁻¹ ) in the presence of a large excess of CHAPS to lyse the vesicles, And reduced the light scattering effect. A typical receptor concentration of 350 nM was determined, assuming that the observed absorption was exclusively caused by nAchR.
The narrow size distribution of vesicles 18-20 nm in diameter was determined by quasi-elastic light scattering according to the following standard method. It was estimated that one vesicle contained an average of 4000 lipid molecules and 0.5 receptors, based on a typical surface of 60Å ² per lipid molecule.

[0082] Non-specific binding of nAchR-vesicles (1) Four solutions of nAchR-vesicles (solutions (1) to (3) do not contain PEG, (
4) contained 2% PEG lipid) was prepared by the method described above. Solution (1)-(3
) Buffer capacity (10 mM HEPES, 3 mM EDTA, 1 mM NaN) ₃ , PH 7.4) at room temperature. Dialysis buffer for (1) and (2)
The solution further contains 500 mM NaCl, and the dialysis buffer for (3) contains an additional 500
mM NaCl and CHAPS correspond to 1/10 of the critical micelle concentration (CMC)
It was included at a concentration of about 0.5 mM. After 30 minutes, change the buffer and
The ionic strength of (1) decreased from 500 mM NaCl to 300 mM NaCl
. After dialysis at 4 ° C for 5 hours, the buffer solution was exchanged again.
The ionic strength was further reduced to 150 mM NaCl. After 10 hours at 4 ° C, dialysis
Finished.

[0083]   To reduce non-specific binding of lipids, the sensor surface was treated with 150 mM NaC.
1, 10 mM HEPES, 3 mM EDTA, 1 mM NaN₃Buffer consisting of (p
70% DOPC (1,2-dioleoyl-sn-glycero-3 in H7.4)
-Phosphatidylcholine), 10% DOPG (1,2-dioleoyl-sn-
Glycero-3-phosphatidylglycerol), 10% chol, 3% 1,
2-Dimyristoyl-sn-glycero-3-phosphatidic acid [poly (ethylene
Glycol)] Ester (M_r  PEG = 750, DMPA-PEG₇₅₀) 3%
  1-palmitoyl-2-oleoyl-sn-glycero-phosphatoethanol
Amine-N- [poly (ethylene glycol)] (M_r  PEG = 2000, PO
PE-PEG₂₀₀₀) And 3% 1-palmitoyl-2-oleoyl-sn-gly
Cello-phosphatoethanolamine-N- [poly (ethylene glycol)] (M _r   PEG = 5000, POPE-PEG₅₀₀₀) A mixture of 1 molar composition of
I understood

(2) Saturating all binding sites of nAchR on vesicles to BgTx with large excess of BgTx for measurement of non-specific binding of vesicles to sensor surface modified with BgTx as immobilized recognition element. And then binding of saturated vesicles to the surface of the BgTx-modified sensor was measured in each dialysis buffer solution as a signal of non-specific binding. The specific binding signal was then determined as the difference between the total binding signal and the non-specific signal obtained with vesicles not saturated with BgTx. The results are summarized in Table 1.

[0085]

[Table 1]

In this example, the maximum ratio of specific to non-specific signals is 750-2.
It was observed with a PEG lipid with a PEG molecular weight of 000 Daltons. The percentage of PEG lipids ranged from 0% to 10% as part of the total number of vesicle-forming lipids. Here, it was observed that a high percentage of PEG lipids led to a disadvantageous reduction of the specific binding signal, especially in the case of long-chain polymers, due to steric hindrance. According to the present invention, the optimal choice of polymer chain length and the optimal percentage of polymer-bound lipid is
It depends on the size of the biological or biochemical or synthetic recognition element associated with the vesicle and the accessibility to the bound analyte, and-in the case of large recognition elements-relatively long chain ( PEG) polymers and / or a high percentage of these polymers as part of the total number of lipid molecules, or-in the case of small recognition elements, relatively short chain (PEG) polymers and / or total number of lipid molecules. A low percentage of these polymers as part of the trend tended to be preferred.

[Procedure amendment]

[Submission date] November 28, 2001 (2001.11.28)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Title of invention

[Correction method] Change

[Contents of correction]

Title: Functionalized vesicles

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0064

[Correction method] Change

[Contents of correction]

For example, a liposome composition for the treatment of a patient having an outer hydrophilic layer of PEG (molecular weight 10,000 to 100,000 Da) and a recognition element (so-called effector) covalently bound to them is described in WO Claimed at 94/21235. This patent application claims a shell of PEG to prevent the degradation of vesicles in the bloodstream. Effectors consist of small and medium sized immunological recognition elements, ie, _Fab fragments, glycoproteins, cytokines, polysaccharides, or various peptides and peptide hormones. In WO 97/35561 biologically active liposomes stabilized by a water-soluble polymer shell are claimed for therapeutic use. Here, the active biological amphipathic component is associated non-covalently, ie by physical adsorption, with the polymer-reinforced liposome. In certain cases, the recognition element is
It is a peptide "growth hormone releasing factor". Diameter 300 produced by extrusion
Unilamellar liposomes of less than nm are preferred. As the polymer preferably PEG is used which is provided as a covalently attached PEG lipid in a liposome-forming membrane. In the broader sense, lipid compositions that further include a label for detection purposes are claimed for diagnostic purposes. Fluorescent markers, radioactive labels, dyes, and components for signal amplification in nuclear spin resonance are listed as detectable markers. The method and location of binding of the label to the vesicles has not been specified.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/532 G01N 33/532 A 33/543 595 33/543 595 (81) Designated country EP (AT, BE) , CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA , GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, S I, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Pauluk, Michael Germany, De-79725 Lofenburg, Andersbach Strasse 5 F term (reference) 2G043 AA03 BA16 CA03 EA01 EA02 FA06 HA01 HA02 HA03 HA05 HA07 HA09 JA01 JA02 2G059 AA05 BB12 CC16 EE04 EE05 EE07 EE12 FF12 GG03 JJ01 JJ13 JJ01 JJ13 JJ01 JJ13 JJ01 JJ13 JJ13 JJ01

Claims (41)

[Claims] 1. A) vesicles, B) one or more polymer molecules bound to the inner and / or outer surface of the vesicles, and C) at least one organism for recognition and binding of ligands. A biological or biochemical reagent comprising a biological, biochemical or synthetic recognition element, wherein the recognition element is bound or adsorbed to a vesicle or a polymer molecule. 2. A biological, biochemical or synthetic recognition element is an antibody, antibody fragment, nucleic acid or nucleic acid analogue, DNA, RNA, enzyme, natural and synthetic polypeptides, histidine-tag moieties and especially membranes. The biological or biochemical reagent according to claim 1, which is selected from the group comprising a receptor. 3. At least one biological or biochemical or synthetic recognition element associated with or incorporated into the surface of the vesicle, or attached to a polymer or lipid of the vesicle. A biological or biochemical reagent according to any one of claims 1 to 2. 4. The biological or biochemical reagent according to claim 1, wherein a water-soluble polymer is used as the stabilizing polymer. 5. A polyethylene glycol as a stabilizing polymer, which comprises:
Hydrophilic polymers of the group comprising polypeptides, polysulfoxides, carbohydrates such as dextran and dendritic hydrophilic polymers are used, preferably having a molecular weight of 750 to 2000 Daltons). Biological or biochemical reagents as described. 6. The biological or biochemical reagent according to any one of claims 1 to 5, wherein the vesicle is a unilamella. 7. The vesicles are glycerophospholipids (eg phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, sphingomyelin), glycolipids, sterols (eg cholesterol), cardiolipin, plasmalogen, Natural lipids such as lipids existing in archaebacteria (for example, those having a hydrocarbon chain bound to an ether group, those having a branched hydrocarbon chain or those having a hydrocarbon chain along with an alicyclic structure), or bipolar lipids, Synthetic lipids (eg positively charged lipids such as dodecyl ammonium bromide (DODAB), or boroamphiphiles)
And a fluorinated or halogenated lipid hydrocarbon chain or a lipid of the group comprising synthetic lipids containing an activating or polymerizing group, the biology according to any one of claims 1 to 6. Or biochemical reagents. 8. A signal generating component in a bioanalytical detection method,
The biological or biochemical reagent according to any one of claims 1 to 7, wherein the label is associated with vesicles. 9. An additional at least one label as a signal generating component,
9. A biological or biochemical reagent according to claim 8 which is located inside the vesicles or is attached directly or by a spacer or by a polymer to the surface of the vesicles. 10. The organism according to claim 8, wherein the additional label as a signal-generating component is selected from the group comprising ESR or NMR spin labels, mass labels, electrochemical labels or especially luminescent or fluorescent labels. Biological or biochemical reagents. 11. A large number of similar luminescent or fluorescent labels associated with vesicles, or a plurality of luminescent or fluorescent labels of different emission wavelengths and similar or different excitation wavelengths, are used as signal generating components. Associated with the cell,
The biological or biochemical reagent according to claim 8. 12. The biological or biochemical reagent according to claim 10 or 11, wherein luminescent or fluorescent nanoparticles are used as luminescent or fluorescent label. 13. A method for producing a biological or biochemical reagent according to any one of claims 1 to 12, wherein A) is a lipid molecule required for vesicle production by dialysis, and B) is a vesicle. One or more polymer molecules to be attached to the inner and / or outer surface,
And C) at least one biological or biochemical recognition element for recognition and binding of the ligand, wherein the recognition element is to be bound or adsorbed to the vesicle or polymer molecule. A method of combining in the mixing and evaporation steps. 14. A bioanalytical detection method using the biological or biochemical reagent according to any one of claims 1 to 12. 15. The analyte is selected from the group comprising electrochemical detection, impedance spectroscopy, electron spin resonance, nuclear spin resonance, quartz balance measurement, and especially detection of optical signal changes, or a combination of these methods. 15. The bioanalytical detection method according to claim 14, which uses the biological or biochemical reagent according to any one of claims 1 to 12, which is detected by the method according to claim 1. 16. Detection of optical signal changes is performed by an optical sensor acting as a sensor platform, wherein preferably the optical sensor is selected from the group comprising optical waveguide sensors and surface plasmon resonance sensors.
16. The bioanalytical detection method according to claim 15. 17. The optical signal change to be detected is based on a change in the effective refractive index of the sensor surface in the near field, or in particular a change in the luminescence or fluorescence produced in the near field of the sensor. The bioanalytical detection method according to any one of 1. 18. The bioanalytical detection method according to claim 16, wherein a planar or fiber type waveguide is used as the sensor platform. 19. A planar thin film waveguide comprising a first optically transparent layer (a) on a second optically transparent layer (b) is used as a sensor platform.
The bioanalytical detection method according to claim 18. 20. The excitation light is coupled to the planar thin film waveguide acting as a sensor platform by one or several gratings (c).
8. The bioanalytical detection method according to 8. 21. Lower refractive index than layer (a) and 5 nm to 1000 μm
A further optically transparent layer (b '), preferably 10 nm to 1000 nm thick,
21. Located between layers (a) and (b) in contact with layer (a).
The bioanalytical detection method according to any one of 1. 22. The adhesion promoting layer (f) is deposited on the optically transparent layer (a) for immobilization of biological or biochemical recognition elements. The bioanalytical detection method according to any one of 1. 23. A thin film waveguide acting as a sensor platform comprises:
Bioanalysis with a sensor platform according to any one of claims 19 to 22, characterized in that it comprises several measurement areas for the simultaneous or sequential measurement of one or more analytes in one or more samples. Detection method. 24. The laterally separated measuring regions (d) are produced by laterally selective deposition of biological or biochemical or synthetic recognition elements on the sensor platform. 23. A bioanalytical detection method using the sensor platform according to 23. 25. The lattice structure (c) has various periodicity in the lateral direction in a direction orthogonal or parallel to the direction of propagation of the incoupled excitation light in the layer (a). A bioanalytical detection method using the sensor platform according to claim 1. 26. The material of the second optically transparent layer (b) is glass, quartz,
Or a bioanalytical detection method with a sensor platform according to any one of claims 19 to 25, characterized in that it comprises a transparent thermoplastic of the group comprising polycarbonate, polyimide or polymethylmethacrylate. 27. A bioanalytical detection method with a sensor platform according to any one of claims 19 to 26, wherein the refractive index of the first optically transparent layer is greater than 2. 28. The thickness of the first optically transparent layer (a) is 40 nm to 300.
28. A bioanalytical detection method with a sensor platform according to any one of claims 19 to 27, which is nm. 29. The grating (c) has a period of 200 nm to 1000 nm and 2 nm to 1
30 to 28, having a grating modulation depth of 00 nm, preferably 10 nm to 30 nm.
A bioanalytical detection method using the sensor platform according to claim 1. 30. The modulation depth to thickness ratio of the first optically transparent layer (a) is
30. The bioanalytical detection method with a sensor platform according to claim 29, which is 0.2 or less. 31. A bioanalytical detection method with an optical system for the measurement of one or more luminescence, comprising at least one excitation light source, a sensor platform according to any one of claims 19 to 30, a sensor A method comprising at least one detector for measuring light from at least one or more measurement areas (d) on the platform. 32. Excitation light from at least one light source is emitted towards one or more measurement areas at a resonance angle for coupling to a coherent and optically transparent layer (a). 32. A bioanalytical detection method using the sensor platform according to any one of claims 20 to 31. 33. The bioanalytical detection method with a sensor platform according to claim 17, wherein at least one laterally dividing detector is used for signal detection. 34. A lens or lens system for shaping the transmitted light flux, a flat or curved mirror for deflection of the light flux and optionally additional shaping, a prism for deflection of the light flux and optionally spectral separation, one of the light fluxes. Dichroic mirror for spectrally selective deflection of a part, neutral density filter for adjusting the transmitted light intensity, optical filter or monochromator for spectrally selective transmission of a part of the luminous flux or excitation or luminescence light A group of optical components including polarization-selective elements for the selection of discrete polarization directions of, between the one or more excitation light sources and the sensor platform, and / or between the sensor platform and one or more detectors. 17. Located,
34. A bioanalytical detection method using the sensor platform according to any one of 33 to 33. 35. The sensor platform according to claim 17, wherein the emission of the excitation light and the detection of the luminescence from the one or more measurement areas are carried out sequentially for a single or a plurality of measurement areas. Bioanalytical detection method. 36. The bioanalytical detection method according to claim 35, wherein the sequential excitation and detection is performed by using a group of movable optics comprising mirrors, deflecting prisms and dichroic mirrors. 37. The bioanalytical detection method according to any one of claims 35 to 36, wherein the sensor platform is moved during the sequential steps of excitation and detection. 38. One or more luminescent or fluorescent labels for detecting an analyte are attached to the analyte or to an analyte analog in a competitive assay or an analyte binding partner in a multi-step assay. 38. A bioanalytical detection method according to any one of claims 17 to 37, which is also bound to one of the applied biological, biochemical or synthetic recognition elements. 39. Luminescence which is (1) isotropically radiated luminescence or (2) incoupled to the optically transparent layer (a) and outcoupled by a lattice structure (c). Alternatively, the luminescence of both (1) and (2) moieties is measured simultaneously, the bioanalytical detection method according to any one of claims 17 to 38. 40. An antibody or an antigen, a receptor or a ligand, a chelating agent or "
A histidine-tag component ", an oligonucleotide, a DNA or RNA chain, a DNA or RNA analog, an enzyme, an enzyme cofactor or an inhibitor, a lectin and a carbohydrate, a simultaneous or sequential quantification of one or more analytes. 40. The bioanalytical detection method according to any one of claims 14 to 39 for qualitative determination. 41. The sample to be tested is a natural body fluid such as blood, serum, plasma, lymph or urine or egg yolk, or an optically cloudy liquid or surface water or soil or plant extract or bio- or synthetic. 41. The bioanalytical detection method according to any one of claims 14 to 40, which is a process broth or is taken from the tissue of a living or dead organism.