Background of the invention

The present invention concerns a method and reagent to detect and quantify the activation of cell membrane receptors, in particular G protein-coupled receptor (GPCR) , via signal transduction at and across cell membranes. A biological array and use of an array for quantifying G protein-coupled receptor (GPCR) activation via signal transduction at cell membranes is also shown.

G protein coupled receptors (GPCRs) are structurally characterized by an amino-terminal extracellular domain, a seven transmembrane (7TM) domain and a carboxy-terminal intracellular domain. Binding of an agonist molecule at the extracellular N- terminal domain of the GPCR leads to a conformational change in the 7TM domain, which allows first association with G-proteins at the intracellular side of the cell membrane and then initiation of intracellular signalling reaction (s) eliciting a physiological cellular response (Prinster et al . , 2005; 2005 S. C. Prinster, C. Hague and R. A. Hall, Heterodimerization of G protein-coupled receptors: specificity and functional significance, Pharmacol. Rev. 57 (2005), pp. 289-298, PG Strange, Curr . Opin . Drug Discov. Devel . 11 (2008) 196-202. B Kobilka, GFX Schertler, Trends Pharmacol. Sci . 29 (2008) 79-83.

GPCRs are involved in a wide range of biological processes for instance visual sensing, olfaction, behavioral and mood regulation, regulation of immune system activity, neurotransmission and hormone regulation. GPCRs are also involved in pathologies such as pain, cancer, asthma, inflammation, and metabolic, immu- nologial, gastrointestinal or neurological disorders; in addition they are targets of a large portion of modern drugs (FiI- more D., 2004; "It's a GPCR world". Modern Drug Discovery 2004 (November) : 24-28. American Chemical Society.) . GPCRs mediate their intracellular actions through signaling pathways in which G proteins are involved (Oldham WM, Hamm HE, 2008; Hetero- trimeric G protein activation by G-protein-coupled receptors. Nat Rev MoI Cell Biol. 2008 Jan; 9 (1) : 60-71) .

In the resting state, G proteins are heterotrimers of GDP-bound G alpha, G beta and G gamma. Upon binding of an agonist, receptors undergo a conformational change that permits binding to G protein and catalyses GDP release from G alpha. Once GDP is released, a ternary complex is formed between the activated receptor and the heterotrimeric G protein. Binding of GTP to G alpha destabilizes this complex, allowing that both G beta- and G gamma-subunits interact with downstream effector proteins. Each of the already dissociated subunits can promote the regulation of different second messengers such as 5 ' -3 ' adenosine monophosphate (cAMP) or inositol triphosphate (IP3), and activate different signalling cascades, which result in a great variety of cell functions. The active state lasts until GTP is hydrolyzed to GDP by the intrinsic GTPase activity of the G subunits. Once GTP has been hydrolyzed to GDP, the G protein binds again GDP and adopts the inactive state.

G proteins regulate the activities of a structurally diverse group of effector molecules. These effectors include enzymes involved in the synthesis and degradation of intracellular signaling molecules such as cAMP, cGMP, inositol phosphates, diacyl- glycerol, arachidonic acid and ions by the opening of ion channels. The activation of second intracellular messengers and the activation of several signaling cascades, phosphorylating enzymes and promoting the regulation at gene level, which will ul- timately give rise to a specific physiological effect (Diverse- Pierluissi 2005; G Protein Effectors Sci. STKE, 281, 13) .

Summary of the Invention

One aspect of the present invention concerns a method, more particularly a method for screening, for detecting and quantifying binding of chemical compounds such as for example ligands, such as agonists, partial agonists, antagonists, etc., to and the subsequent activation or deactivation of G protein-coupled receptor (GPCR) by monitoring the receptor-mediated native signal transduction reactions at cell membranes, such as the association and dissociation between the receptor and its G protein.

A preferred embodiment comprises vesicles, comprising the receptor of interest which is inserted in the vesicle membrane such that the receptor' s binding site for G protein is located at the outer surface of the vesicular membrane and is accessible for the G protein from the bulk phase. This receptor orientation is from hereon called inside-out orientation (with respect to the receptor' s native cellular orientation) .

The vesicles can for example be produced in one of the following ways: (1) As so-called native vesicles, produced according to known procedures (Pick H, Schmid EL, Tairi AP, Ilegems E, Hovius R, Vogel H. J Am Chem Soc. 2005 9 ; 127 ( 9 ) : 2908-12., US2008139403) from living cells (such as mammalian cells) which either naturally or heterologously express the receptor of interest. The inside-out orientation can be obtained by ultra- sonifying the native vesicles. (2) Using native vesicles derived from living cells which do not express the receptor of interest. The receptors of interest will be integrated in such native vesicles by in vitro synthesis (Refs e.g. Junge, F., Schneider, B., Reckel, S., Schwarz, D., Doetsch, V., Bernhard, F., (2008) Cell MoI. Life Sci, 65, 1729-1755; Schwarz, D., Junge, F., Durst, F., Froehlich, N., Schneider, B., Reckel, S., Sobhanifar, S., Doetsch, V., Bernhard, F. (2007) Nat. Protoc. 2, 2945-2957) in the presence of said vesicles; the newly in-vitro synthesized receptor will spontaneously insert into the membrane of the native vesicle. If necessary, these vesicles might be reverted to obtain an inside-out receptor orientation. (3) Alternatively, artificial lipid vesicles can be used. The receptor of interest might be reconstituted into the artificial lipid vesicle according to standard procedures or by in-vitro synthesis as described in (2) before, now the artificial vesicles being present during the in-vitro synthesis of the receptor of interest.

A preferred embodiment comprises G proteins, which are competent to interact with the particular agonist activated GPCR. The G protein can be used either in purified form obtained either from natural sources (Zhang, J. -H., Simonds, W. F. (2000) J. Neurosci. 20, 1-5; Tanaka T. et al . , (1999) Prot. Expr. Purif. 1, 207- 212; Daulat, A. M., et al, (2008) MoI. Cell. Proteom. (2007) 6, 835-844) or recombinantly expressed in suitable mammalian cells, yeast cells or bacterial cells, or from in vitro expression. The purified G proteins then will be immobilized on a sensor surface via tags inserted in the recombinant protein during expression (such as polyhistidine sequences to be recognized by NTA (ni- trilotriacetic acid) , polypeptide sequences recognized by an antibody or antibody fragments, acylcarrier proteins (ACP) or fragments of acyl carrier proteins recognized by phospho- pantetheine transferase, SNAP tags, biotin or polypeptide sequences both recognized by avidin or streptavidin, to mention a few) which are bound to the mentioned recognition elements immobilized on the sensor surface before. Preferentially the tag for immobilizing the G protein on the sensor surface will be attached to the G alpha subunit.

As a preferred embodiment, measurement to probe the potency of a particular ligand to activate a particular GPCR will be done in the follwing way: To the sensor comprising the immobilized G protein a suspension of the GPCR comprising lipids are added. In the presence of a receptor-specific agonist and GTP the vesicles will first bind to the G proteins on the sensor surface and then dissocuiate from these G proteins in an agonist concentration- dependent manner. The specific vesicle binding to and subsequent dissociation from the sensor surface can be monitored by label- free surface-sensitive techniques such as surface plasmon resonance (SPR) or quartz crystal microbalance which detect mass changes on the sensor surface during interaction with the ana- lyte (here the ligand) .

Alternatively ultra sensitive planar waveguide techniques can be used to detect via fluorescenct probes the interactions between G proteins immobilized on the waveguide sensor surface and GPCRs integrated in inverted native or artificial vesicles: Here the GPCR containing vesicles comprise fluorescent probes (in the simplest case, fluorescent lipid probes inserted in the vesicle membrane, alternatively the fluorescent probe might be attached to the GPCR) ; the specific binding of the GPCR in the vesicle to the G protein on the sensor surface and the subsequent dissociation of the GPCR in the vesicle are measured by the evanescent field on the waveguide sensor surface as a fluorescence increase (vesicle binding to sensor surface) or fluorescence decrease (vesicle dissociation from sensor surface) , respectively.

Both the label free and the fluorescence probe based surface sensitive techniques are suited to investigate the function of GPCRs, such as odorant and taste receptors, or medically relevant receptors, and screen for compounds which activate these receptors. In the case of odorant receptors this method can be used to screen for perfumes or to detect with high sensitivity volatile (e.g. poisonous) compounds (environmental monitoring) or to use it for quality control in food industry. In the case of taste receptors the method can be used for quality control of food. In the case of medically relevant GPCRs the method can be used for drug screening. This method can further be used for investigating the basic functions of GPCRs.

The present invention can be extended to all membrane receptors which interact in a ligand dependent manner to an intracellular effector protein. These receptors are investigated in the same manner by functionally immobilizing the effector protein on a sensor surface, adding the receptor containing vesicles and observe how the interaction of the receptor in the vesicle is modulated by the presence of a receptor-specific ligand.

It is one of the aims of the preferred embodiments of the present invention to provide a rapid and controlled procedure of sample preparation from cultured cells, native tissues, in vitro expressed or purified proteins. A particular advantage of the preferred embodiments is to provide an ultrasensitive detection method for quantifying receptor activation which is due to the signal amplification of the receptor comprising vesicle (high mass for label free detection, high fluorescence for fluorescent based sensing) binding to / dissociating from the sensor surface .

The present invention can analyze small hydrophobic ligand molecules capable of passing the vesicle membrane, for example hydrophobic molecules from libraries used in drug screening in the case of pharmaceutically relevant GPCRs or odorant molecules in the context of odorant receptors.

If the ligand molecules to be analyzed are polar and thus not spontaneously traversing the vesicle membrane, it is necessary to make the vesicle membrane permeable for these ligands before performing the measurement (e.g. by adding small amounts of detergents sufficient to make membrane permeable without dissolving it) .

In a further particular embodiment the present invention can analyze peptides directly expressed in the vesicles.

In a preferred embodiment, the present invention can be used for analyzing odorant receptors.

Odorant compounds, which influence the behavior of consumers in the choice of food, personal hygiene, household products and fashion, represent a market of large economic impact. Odorants are generally small, hydrophobic organic molecules with highly variable chemical structures. Olfactory GPCRs which are capable of discriminating thousands of different odors constitute a large, diverse family encoded by 1000 different genes in rodents and about 350 full length genes in humans. Each neuron in the olfactory epithelium expresses just one type of the olfactory receptors (OR) .

Olfactory receptors act as signal transducers through the cytoplasmic membrane. The binding of the odorant molecule induces an increase of the second messengers cyclic adenosine 3', 5'- monophosphate (cAMP) inside the cell which opens a cyclic nucleo- tide-gated channel and finally results in depolarization and a neuronal response that triggers the perception of a smell in the brain .

Each OR subtype is broadly tuned to bind only odorant molecules with specific structures and chemical properties, and each OR subtype is expressed in only a subset of olfactory neurons (Buck 2004) . These neurons are directly connected to specific target structures in the brain, and binding of an odorant thus results in a characteristic pattern of activation in the olfactory bulb that is interpreted by the olfactory cortex as perception of a certain smell and a physiological behaviour. Applications of the present invention are not only restricted to the development of new fragrances and their quality control but also to the detection of toxic volatile compounds which can adversely affect human health.

The development of recombinant DNA techniques has allowed to obtain cell preparations expressing a particular GPCR. They can be prepared in a cell culture laboratory by means of transfecting cell lines. Native vesicles are prepared from these cell lines expressing a particular GPCR in their membrane using cyto- chalasin treatment and the native vesicles are subsequently inverted by ultra-sonication to present the naturally intracellular receptor carboxyl termini and its intracellular interaction sites for its G proteins on the outer vesicle surface.

A particular aspect of the present invention relates to an array of native vesicles with inverted membrane orientation containing transmembrane G protein-coupled receptors (GPCRs) for analyzing the interaction of compounds (for example drugs, odorants etc.) with cell membrane receptor proteins, as well as for analyzing the intracellular signaling mechanisms triggered by this interaction mechanism mediated by such compounds (ligands) . The patent application WO0246766 describes a method for production of native vesicles representing a bioanalytical reagent comprising a vesicle generated from a living cell, wherein the vesicle comprises at least one receptor, and membrane and lumen cell products and/or cell proteins, besides said at least one receptor, which are involved in a mechanism of signal transduction triggered by the receptor in the cell used for vesicle generation from said living cell is effected after application of cytochalasin B and/or cytochalasin D. These fungal toxins act on the actin cytoskeleton . The cytoskeleton is a dynamic network responsible for various essential biological functions in the cell, such as cell division, regulation of cell volume, change of cell form and cell movement. It has been shown that cytochalasin B and D bind to the polymerization end of actin filaments and prevent their extension by inhibiting the attachment of further globular actin monomers. As a result, budding of the cell membrane occurs. These buds are either bullous or pedunculate in form. In some cases without further external influences, the buds are pinched off as vesicles or can be detached from the cell surface through the application of shear forces.

Membrane fragments containing transmembrane receptors can alternatively be prepared by cell homogenization in presence of protease inhibitors. In general, the protease inhibitors block the enzymatic digestion of the membrane receptors by released cell proteases. The method for the production of native vesicles as a bioanalytical reagent as described in patent application WO0246766 according, avoids the external addition of protease inhibitors. This can be advantageous, because additional components (in this case most often mixtures of protease inhibitors) in a test solution may affect the receptor-ligand interaction. According to the patent application WO0246766, it is characteristic that a vesicle produced by said method has a diameter of 50 nm - 5000 nm, especially preferably of 100 nm - 2000 nm.

The term "inverted native vesicle" relates to a cell-derived vesicle containing the receptor protein in a functional state but with inverted membrane topology presenting the ^λ naturally' cytoplasmic located carboxyl terminus of the receptor on the vesicle surface which interacts and binds exogenously added purified heterotrimeric G proteins or functionally immobilized G proteins on a sensory surface.

The native vesicle of the present invention are derived either from primary cells prepared from any tissue, organ or cell type of any member of an animal species, including human species, or they are derived form cultured mammalian cell over-expressing a specific receptor in their cell membrane using recombinant DNA techniques and subsequent transfection of these cells by the receptor encoding DNA construct.

Inverted native vesicles represent a bioanalytical reagent wherein receptors are provided in such a form and in such a biocompatible environment that signal transduction processes associated with receptor activation, can be tested in the presence of different biological or biochemical or synthetic components supplied in a sample or resulting from other changes of other external parameters influencing the transduction mechanism. This is of major importance here to avoid disadvantages of assays based on whole living cells, i.e. the high variation of the test results due to the continuous change in living organisms and the frequent difficulties of assigning cause-and-effect relationships. This is due to the complex nature of whole cells which contain many additional biochemical components that might not be directly involved in the mechanism of signal transduction by a receptor, but which can also be effected in their function during a test procedure, which may lead to further changes in the observed test results.

The term "G protein-coupled receptor (GPCR) " relates to cell membrane receptors with seven transmembrane domains and which are associated to their coeffector, the heterotrimeric G protein. These receptors transduce the extracellular signal (com- pound/ligand binding) into an intracellular signal (G protein activation) . The GPCR protein superfamily is the largest known protein family, the members of which participate in virtually all intracellular biological processes and bind a very diverse molecules as ligands, like odorants, tastants, peptides, hormones, neurotransmitter etc.

After a compound binds to the membrane GPCR, a conformational change of the GPCR occurs such that the protein binding sites of the G protein, which were previously covered are exposed. This interaction catalyzes a guanine nucleotide change, resulting in the binding of a GTP to the subunit of the G protein. This binding makes the G alpha-GTP dissociate from the G beta/gamma sub- units. As a result of the intrinsic GTPase activity of the G alpha subunit, the bound GTP is hydrolyzed to GDP, the system thus returning to its initial heterotrimer state.

One aspect of the present events is quantifying GPCR activation using an array comprising (i) inverted, cell-derived native vesicles expressing a GPCR in their vesicle membrane attached to (ii) purified and functionally immobilized G proteins on a sensory surface. In the presence of (iii) GTP and a receptor- specific agonist the vesicles will dissociate form the G protein in an agonist concentration-dependent manner by label-free or fluorescent based surface-sensitive techniques.

Following this approach, numerous measurement arrangements are conveivable, wherein the detection of an analyte is based on its interaction with the evanescent field that is associated with light guidance in an optical waveguide, wherein biochemical or biological or synthetic recognition elements for specific recognition and binding of analyte molecules are immobilized on the surface of the waveguide, as outlined in detail in WO0246766 and are all applicable for the present invention. When a light wave is coupled into an optical waveguide surrounded by optically rarer media, i.e. media of lower refractive index, the light wave is guided by total reflection at the interfaces of the waveguiding layer. In this arrangement, a fraction of the electromagnetic energy penetrates the media of lower refractive index. This portion is termed the evanescent (=decaying) field. The strength of the evanescent field depends to a very great extent on the thickness of the waveguiding layer itself and on the ratio of the refractive indices of the waveguiding layer and of the media surrounding it. In the case of thin waveguides, i.e. layer thicknesses that are the same as or smaller than the wavelength of the light to be guided, discrete modes of the guided light can be distinguished. As an advantage of such methods, the interaction with the analyte is limited to the penetration depth of the evanescent field into the adjacent medium, being of the order of some hundred nanometers, and interfering signals from the depth of the (bulk) medium can be largely avoided. The first proposed measurement arrangements of this type were based on highly multimodal, self-supporting single-layer waveguides, such as fibers or plates of transparent plastics or glass, with thicknesses from some hundred micrometers up to several millimeters . For improved sensitivity and at the same time easier manufacturing in mass production, planar thin-film waveguides can be used. In the simplest case, a planar thin-film waveguide consists of a three-layer system: support material (substrate) , waveguiding layer, superstrate (i.e. the sample to be analyzed), wherein the waveguiding layer has the highest refractive index. Additional intermediate layers can further improve the action of the planar waveguide .

Several methods for the incoupling of excitation light into a planar waveguide are known. The earliest methods used were based on butt coupling or prism coupling, wherein generally a liquid is introduced between the prism and the waveguide in order to reduce reflections resulting from air gaps. These two methods are particularly suitable with respect to waveguides of relatively large layer thickness, i.e. especially self-supporting waveguides, and with respect to waveguides with a refractive index significantly below 2.

For incoupling of excitation light into very thin waveguiding layers of high refractive index, however, the use of coupling gratings is a significantly more elegant method. Various methods of analyte determination in the evanescent field of lightwaves guided in optical film waveguides can be distinguished. Based on the measurement principle applied, for example, a distinction can be drawn between fluorescence, or more general luminescence methods on the one hand and refractive methods on the other.

In this context, methods for the generation of surface plasmon renonance in a thin metal layer on a dielectric layer of lower refractive index can be included in the group of refractive methods, provided the resonance angle of the launched excitation light for generation of the surface plasmon resonance is taken as the quantity to be measured. Surface plasmon resonance can also be used for the amplification of a luminescence or the improvement of the signal-to-background ratios in a luminescence measurement. The conditions for generation of a surface plasmon resonance and the combination with luminescence measurements, as well as with waveguiding structures, are described in the literature, for example in U.S. Pat. No. 5,478,755, No. 5,841,143, No. 5,006,716, and No . 4,649,280.

In the case of the refractive measurement methods, the change in the effective refractive index resulting from molecular adsorption to or desorption from the waveguide is used for analyte detection. This change in the effective refractive index is determined, in the case of grating coupler sensors, from changes in the coupling angle for the in- or out-coupling of light into or out of the grating coupler sensor and, in the case of interfer- ometric sensors, from changes in the phase difference between measurement light guided in a sensing branch and a referencing branch of the interferometer.

The aforesaid refractive methods have the advantage that they can be applied without using additional marker molecules, so- called molecular labels.

Alternatively, for achieving lower detection limits, luminescence-based methods are suitable in view of the higher selectivity of signal generation. In this arrangement, luminescence excitation is confined to the penetration depth of the evanescent field into the medium of lower refractive index, i.e to the immediate proximity of the waveguiding area, with a penetration depth of the order of some hundred nanometers into the medium. This principle is called evanescent luminescence excitation. By means of highly refractive thin-film waveguides, based on a waveguiding film measuring only a few hundred nanometers in thickness on a transparent support material, it has been possible to increase sensitivity considerably in recent years. In WO 95/33197, for example, a method is described, wherein the excitation light is coupled into the waveguiding film via a relief grating as diffractive optical element. The isotropically emitted luminescence from substances capable of luminescence, which are located within the penetration depth of the evanescent field, is measured by suitable measurement devices, such as pho- todiodes, photomultipliers or CCD cameras. The portion of eva- nescently excited radiation that has back-coupled into the waveguide can also be out-coupled via a diffractive optical element, such as a grating, and be measured. This method is described, for example, in WO 95/33198. The in-coupling and out- coupling grating in this method may also be identical, because each in-coupling grating can be used as an out-coupling grating under the same conditions as for in-coupling, in view of the reversibility of the light path.

For the simultaneous or sequential performance of exclusively luminescence-based, multiple measurements with essentially mono- modal, planar inorganic waveguides, for example in the specification WO 96/35940, arrangements (arrays) have been proposed wherein at least two discrete waveguiding areas are provided on one sensor platform, such that the excitation light guided in one waveguiding area is separated from other waveguiding areas. By means of such an arrangement it is possible, in particular, to determine different analytes simultaneously in an applied sample, using different recognition elements immobilized in discrete measurement areas . According to the present invention, spatially separated measurement areas (d) should be defined by the area that is occupied by biological or biochemical or synthetic recognition elements immobilized thereon for recognition of an analyte in a liquid sample. These areas may have any geometry, for example the form of dots, circles, rectangles, triangles, ellipses or lines.

For the investigation of receptor-ligand interactions, especially the functionality of a transduction mechanism controlled by a receptor, there is a need for a solid carrier, in particular for a sensor platform with high detection sensitivity, designed in such a way that the mechanism of signal transduction, is not impaired, in particular by the immobilization of the receptor on a solid surface. Various embodiments of solid carriers and/or sensor platforms are provided within the scope of this invention .

A particular aspect of the invention is a bioanalytical reagent with at least one vesicle, generated from a living cell, comprising at least one receptor, characterized in that a mechanism of signal transduction triggered by said receptor in the cell used for vesicle generation is preserved in said vesicle as a component of the bioanalytical reagent.

The membrane topology of the receptor associated with said vesicle will be inverted by sonication that the ligand-binding site faces the inside of the vesicle and the carboxyl terminus face the exterior of the vesicle and thereby can interact with purified G proteins which are functionally immobilized on a sensory surface .

Said mechanism of signal transduction may be triggered in this case by the effect both of external signals or of signals inside the vesicle or of signal-generating biological or biochemical or synthetic components possibly added externally. "External or vesicle-internal signals" are here understood, for example, to be changes in macroscopic properties, such as changes in ion concentrations in the medium or in the vesicle. By contrast, "signal-generating biological or biochemical or synthetic components" are understood, for example, to be ligands binding specifically to a receptor.

An important characteristic of the inverted native vesicles according to the invention is that a binding capability of said one or more receptors to a specific ligand is preserved, this binding capacity being present in said vesicle-generating cell and the receptor being associated with the vesicle as a component of the bioanalytical reagent. Small lipophilic molecules directly pass the vesicle membrane to interact with the ligand- binding site of the receptor inside the vesicle. Bigger or hy- drophilic molecules will pass by increasing the membrane permeability using mild detergents or pore forming proteins.

Additional components for generation of an experimentally detectable signal may be incorporated in the vesicle lumen and may be selected from the group of components formed by absorptive indicators and luminescent indicators, luminescence labels, luminescent nanoparticles, absorptive indicator proteins and luminescent indicator proteins, such as BFP ("blue fluorescent protein"), GFP ("green fluorescent protein") or RFP ("red fluorescent protein"), artificial luminescent (i.e. in particular fluorescent) amino acids, radioactive labels, spin labels, such as NMR labels or ESR labels. Said additional components for generation of an experimentally detectable signal may also be inserted into the vesicle after its formation. A preferred characteristic for the application of the inverted native vesicles as a bioanalytical reagent according to the invention in practice is that the functionality of a receptor associated with a vesicle as a component of said bioanalytical reagent is preserved upon storage under deep-frozen conditions for at least one week, preferably for at least one month, especially preferably for at least one year.

Preservation of the functionality of said receptor is intended to mean that a mechanism of signal transduction to be triggered by said receptor in the vesicle is also when the vesicle is thawed out again intact after storage of the vesicle under the conditions described.

Alternatively, the inverted native vesicles are immobilized on a sensory surface to investigate the interaction of the receptor with G proteins. To allow the arrangement of the inverted native vesicles in predetermined patterns and to reduce nonspecific binding of a vesicle, as part of a bioanalytical reagent according to the invention, to a surface that is to be brought into contact with the vesicle, it is advantageous if, after production of said vesicle from a living cell, lipids comprising for example hydrophilic polymers (such as polyethylene glycols) are additionally integrated into the vesicle membrane. Vesicles with surface-associated polymers are described, for example, in PCT/EP/00/04491.

Further embodiments of the method according to the invention are one or more vesicles produced by this method that additionally comprise components for generation of an experimentally detectable signal. These additional components for generation of an experimentally detectable signal may be comprised in the further biological compounds (components) associated with the inverted native vesicles as part of said bioanalytical reagent. In this case, said further biological compounds (components) may be comprised in the group that is formed e.g. by G proteins and G-protein regulators (e.g. rasGAP) , enzymes such as adenylate cyclases, phos- pholipases which form intracellular secondary messenger compounds (e.g. cAMP (cyclic adenosine monophosphate), cGMP (cyclic guanosine monophosphate) , diacyl glycerol (DAG) or inositol triphosphate (IP3)), enzymes such as serine, threonine and tyrosine kinases, and tyrosine phosphatases that activate or inhibit proteins by phosphorylation or de-phosphorylation . These components are purified and fluorescently labeled either using recombinant fusions to different variants of the green fluorescent protein or fusions to the 06-alkylguanine-DNA alkyltransferase (Keppler et al, 2004) allowing the free choice of the time of fluorescence pulse labeling with a variety of different fluoro- phores .

A further subject of the invention is a bioanalytical detection method with a bioanalytical reagent according to any of the aforementioned embodiments, wherein said detection method is selected from the group that is formed, for example, by optical detection methods, such as refractrometric methods, surface plasmon resonance, optical absorption measurements (e.g. internal reflection methods using a highly refractive material, in combination with infrared spectroscopic measurements) or luminescence detection (e.g. fluorescence correlation spectroscopy (FCS) or fluorescence cross-correlation spectroscopy (FCCS) ) , detection of fluorescence energy transfer (FRET) or charge transfer, mass spectroscopy, impedance measurements, electronic resonance measurements, such as electron spin resonance or nu- clear spin resonance, gravimetric methods (e.g. electrical crystal balance measurements) , radioactive methods, or by electro- phoretic measurements.

It has surprisingly been found that it is advantageous if the inverted native vesicles as a bioanalytical reagent according to the invention, can be positioned in a patterned array format by means of a hydrodynamic or electrokinetic flow or by other mechanical manipulation (e.g. by means of optical tweezers, force microscope or by a micro manipulator) .

The inverted native vesicles generated from a living cell are immobilized on the surface of said solid support, for example, by means of covalent binding or physical adsorption (electrostatic or van der Waals interaction or hydrophilic or hydrophobic interaction or a combination of these interactions) .

In a further preferred embodiment, an adhesion-promoting layer is deposited between the surface of said solid support and the one or more vesicles immobilized thereon. In this case, according to the invention, the adhesion-promoting layer is designed in such a way that a mechanism of signal transduction triggered by the one or more receptors in said living cell is preserved also after immobilization of the vesicles generated from a living cell as part of a bioanalytical reagent comprising at least one receptor, according to the invention, on said adhesion- promoting layer.

In a further preferred embodiment, the adhesion-promoting layer has a thickness of preferably less than 200 nm, most preferably of less than 20 nm. The adhesion-promoting layer may comprise a chemical compound of the group of silanes, epoxides, functional- ized, charged or polar polymers and "self-organized functional- ized mono or multiple layers".

Characteristic of a preferred embodiment is that the adhesion- promoting layer comprises a monomolecular layer of mainly one kind of protein, such as serum albumins or streptavidin, or of modified proteins, such as biotinylated serum albumin. Characteristic of another preferred embodiment is that the adhesion- promoting layer comprises self-organized alkane-terminated monolayers of mainly one kind of chemical or biochemical molecules .

Especially preferred is an embodiment wherein the adhesion- promoting layer is provided as a double layer (bilayer) , comprising an initial self-organized alkane-terminated anchoring layer and a second layer formed by self-organization (self- assembly) of synthetic or natural lipids. The immobilization of the one or more vesicles generated from a living cell on the adhesion-promoting layer may be performed, for example, upon cova- lent binding or upon physical adsorption (electrostatic or van der Waals interaction or hydrophilic or hydrophobic interaction or a combination of these interactions) .

A further embodiment of the bioanalytical detection method according to the invention, using a particularly specific variant of vesicle immobilization, comprises association with the adhesion-promoting layer of biological or biochemical or synthetic recognition elements which recognize and bind a vesicle generated from a living cell with surface-associated biological or biochemical or synthetic components for specific recognition and binding, as part of the corresponding above-described specific embodiment of a bioanalytical reagent according to the invention. These specific interactions for the recognition and bind- ing of the vesicles to their recognition elements on the adhesion-promoting layer may for example be based on interactions with biotin/streptavidin, so-called "histidine tags" (references: Schmid E. L., Keller T. A., Dienes Z., Vogel H., "Reversible oriented surface immobilization of functional proteins on oxide surfaces", Anal Chem 69 (1997) 1979-1985; Sigal G. B., Bamdad C, Barberis A., Strominger J., Whitesides G. M., "A self-assembled monolayer for the binding and study of histidine- tagged proteins by surface plasmon resonance", Anal Chem 68 (1996) 490-497), sugars or peptide affinity interactions, wherein any one of the two binding partners in each case may be associated with the vesicle surface and the other anchored on the surface of said adhesion-promoting layer.

In a further particularly preferred embodiment of a solid carrier (support) according to the invention regions between the laterally separated measurement areas, with vesicles generated from living cells (from a bioanalytical reagent according to any of the described embodiments) immobilized in these measurement areas, or with ligands for receptors that are bound to vesicles generated from living cells (from a bioanalytical reagent according to any of the described embodiments) , and/or regions within these measurement areas, between the compounds immobilized therein, are "passivated" in order to minimize nonspecific binding of analytes or of their detection reagents, i.e. that compounds which are "chemically neutral" towards the analyte are deposited between the laterally separated measurement areas (d) and/or within these measurement areas (d) between said immobilized compounds, the "chemically neutral" compounds preferably being composed of the groups that are formed by albumins, casein, detergents, such as Tween 20, detergent I lipid mixtures (of synthetic and/or natural lipids) , synthetic and natural lip- ids or also hydrophilic polymers, such as polyethylene glycols or dextrans .

It is also possible to passivate an activated surface (activated for immobilization of the biological, biochemical or synthetic recognition elements), this surface comprising e.g. poly-L-lysin or functionalized silanes (e.g. comprising aldehyde or epoxy groups) , for example by the addition of reducing reagents such as sodium borate (in the case of aldehyde or epoxy groups) . The material of the surface of the solid support (carrier) with immobilized vesicles generated from living cells (from a bioana- lytical reagent according to the invention and any of the described embodiments), or with immobilized ligands for receptors that are bound to vesicles generated from living cells (from a bioanalytical reagent according to the invention and any of the described embodiments) , may comprise a material of the group which is formed e.g. by moldable, sprayable or millable plastics, carbon compounds, metals, such as gold, silver, copper, metal oxides or silicates, such as glass, quartz or ceramics, or silicon or germanium or ZnSe or a mixture of these materials. In this case, said solid carrier (support) may be provided in a variety of different embodiments. It may be provided e.g. as a glass or microscope plate. It may also be a microtiter plate of the type that is, for example in widespread use for screening assays (for testing numerous compounds, e.g. using classical fluorescence methods or fluorescence correlation spectroscopy) . It is preferred if the surface of said solid support (carrier) is essentially planar.

For numerous applications such an embodiment of a sensor platform according to the invention, used as a solid carrier (support) , is preferred when the first optically transparent layer (a) comprises a material of the group of Tiθ2, ZnO, Nb ₂ Os, Ta2θ ₅ , HfO ₂ , or ZrO ₂ , preferably of TiO ₂ or Ta ₂ O5 or Nb ₂ O ₅ . The first optically transparent layer (a) preferably has a thickness of 40 to 300 nm, most preferably of 100 to 200 nm.

Characteristic of a further embodiment of the sensor platform according to the invention, used as solid carrier (support) is that an additional optically transparent layer (b ¹ ) with lower refractive index than layer (a) and with a thickness of 5 nm-10 000 nm, preferably of 10 nm-1000 nm, is located between the optically transparent layers (a) and (b) and in contact with layer (a) .

It is preferred if the in-coupling of excitation light into the optically transparent layer (a) to the measurement areas (d) is performed using one or more optical in-coupling elements from the group formed by prism couplers, evanescent couplers comprising joined optical waveguides with overlapping evanescent fields, butt-couplers with focusing lenses, preferably cylindrical lenses, arranged in front of a front face (distal end) of the waveguiding layer, and grating couplers. In this case, it is especially preferred if the in-coupling of excitation light into the optically transparent layer (a) to the measurement areas (d) is performed using one or more grating structures (c) that are formed in the optically transparent layer (a) .

Characteristic of an improvement of a sensor platform according to the invention and any of the aforementioned embodiments is that they additionally comprise one or more sample compartments with said sensor platform as the base plate, said sample compartments being open towards the sensor platform at least in the region of the one or more measurement areas, wherein said sample compartments may be open or closed except for inlet and/or outlet openings at the side facing away from the sensor platform. A variety of further embodiments of sensor platforms, which are suitable in combination with a bioanalytical reagent according to the invention and may be applied in a bioanalytical detection method according to the invention, are described in detail, for example, in patents U.S. Pat. No. 5,822,472, U.S. Pat. No. 5,959,292, and U.S. Pat. No. 6,078,705, and in patent applications WO 96/35940, WO 97/37211, WO 98/08077, WO 99/58963, PCT/EP 00/04869, and PCT/EP 00/07529.

The embodiments of sensor platforms and methods for the detection of one or more analytes, as well as the optical and analytical systems described therein, are also a subject of the present invention, as part of sensor platforms according to the invention, as solid carriers (supports) comprising a bioanalytical reagent according to the invention, and as parts of bioanalytical detection methods according to the invention which are performed therewith.

A further subject of the present invention is the use of a vesicle as a component of a bioanalytical reagent according to the invention and any of the aforementioned embodiments and/or of a solid carrier (support) according to the invention, comprising one or more vesicles immobilized thereon, as described in any of the aforementioned embodiments for the enrichment of membrane receptors or for the enrichment of proteins (such as antigens) triggering an immunological response in a two- or three- dimensional phase, which may then e.g. be administered to living organisms (e.g. to stimulate immune defense processes) .

A further subject of the invention is the use of a vesicle as a component of a bioanalytical reagent according to the invention, as described in any of the aforementioned embodiments, as a com- partment for therapeutic, diagnostic, photosensitive or other biologically active compounds for administration to a living organism.

The present invention also comprises the use of a bioanalytical reagent according to the invention as described in any of the aforementioned embodiments and/or of a solid carrier (support) according to the invention as described in any of the aforementioned embodiments, comprising one or more immobilized vesicles, and/or of a bioanalytical detection method according to the invention as described in any of the aforementioned embodiments for investigating receptor-ligand interactions, especially for determining the binding strength and kinetic parameters of these interactions between a receptor and its ligand, or for determining the channel activity of an ion channel receptor after ligand binding or other inducing influences on said receptor, or for determining the enzymatic activity of enzymes associated with a vesicle, as a component of a bioanalytical reagent according to the invention, or for determining secondary messenger compounds after ligand binding to a receptor resulting in a signal transduction, or for determining protein-protein interactions, or for determining protein kinases.

The present invention additionally comprises the use of a bioanalytical reagent according to the invention as described in any of the aforementioned embodiments and/or of a solid carrier (support) according to the invention as described in any of the aforementioned embodiments, comprising one or more immobilized vesicles, and/or of a bioanalytical detection method according to the invention as described in any of the aforementioned embodiments for quantitative and/or qualitative analyses for determining chemical, biochemical or biological analytes in screening methods in pharmaceutical research, combinatorial chemistry, clinical and pre-clinical development, for real-time binding studies and for determining kinetic parameters in affinity screening and in research, for qualitative and quantitative analyte determinations, especially for DNA and RNA analytics, for generation of toxicity studies and for the determination of expression profiles, and for determining antibodies, antigens, pathogens or bacteria in pharmaceutical product development and research, human and veterinary diagnostics, agrochemical product development and research, for symptomatic and pre-symptomatic plant diagnostics, for patient stratification in pharmaceutical product development and for therapeutic drug selection, for determining pathogens, nocuous agents and germs, especially of salmonella, prions and bacteria, in food and environmental analytics, and for analysis and quality control of odorous and flavoring substances.

Said additional components for generation of an experimentally detectable signal my also be associated with a receptor or may be parts of fusion proteins associated with the vesicle.

Using the present invention it is possible to establish a sensing device based on multi-arrays of different ORs, which directly couple to the native cellular signal amplification cascade. This approach copies from a conceptional point of view the native olfactory system enabling the highest possible sensitivity to detect and distinguish different volatile compounds (Fig. D •

Detailed Description of the Invention

Principle of the sensor device: 1. Production of native vesicles as central sensing and ampl ification units. Cytochalasin, an active agent for destabilizing cytoskeleton-membrane interactions, induces mammalian cells to extrude from their plasma membrane sub-micrometer-sized native vesicles, which allows monitoring cellular signalling reactions. They comprise functional cell surface receptors correctly exposing their extracellular ligand binding sites on the outer vesicle surface and retaining cytosolic signalling proteins (e.g. G proteins) in the vesicle interior. They are capable of responding to the exogenously added receptor-specific agonists by transient second messenger signalling with a similar time course and amplitude like observed in intact cells, indicating that receptor-mediated signalling is preserved in native vesicles. Thus, native vesicles are the smallest autonomous containers capable of performing cellular signalling reactions under physiological conditions .

2. The light-activation of rhodopsin is monitored with optical techniques (surface plasmon microscopy) . The subsequent interaction of the activated rhodopsin with its heterotrimeric G protein (transducin) forming a ternary protein complex is detected, and finally the dissociation of this ternary complex (Bieri C, Ernst OP, Heyse S, Hofmann KP, Vogel H. (1999) Nat Biotechnol. 17,1105-1108) . This central finding will be used in our present sensing device. From mamalian cells, heterologously producing a particular OR, functionally active native vesicles are produced. By simple sonification, they are turned inside out, thus having their ligand binding site now in the inside of the vesicle. On the vesicle outside there are the receptor binding sites for the receptors G proteins (e.g. Golf) . The Golf can be either co- expressed heterologously with its OR in the same cell line, or can be added in purified form after formation of the inside-out native vesicles. The particular receptor can be activated by simply adding a suitable odorant (or other agonistic ligand) from the bulk solution to the vesicle. Because all presently known odorant molecules are highly hydrophobic and membrane permeable, they will diffuse into the vesicle and subsequently bind and activate the particular OR. As a consequence, the Golf will then bind to the activated OR and finally dissociate from the receptor and the vesicle as shown in the case of rhodopsin By using fully functional cellular sensing units (the native vesicles) , advantage is taken of the intrinsic receptor mediated signaling process: Each single OR can activate many G proteins thus amplifying the detection of odorant binding to the receptors .

(i) Detection is not restricted to surface-bound G proteins, but can also be carried out in solution. E.g., both the OR and the Golf can be orthogonally labelled with two different, distinguishable fluorescent probes suitable to measure fluorescence resonance energy transfer (FRET) between these probes if they are located within a molecular complex like in the ternary complex OR-GoIf. FRET monitors first activation of the receptor

(formation of ternary OR-GoIf complex) and then activation of Golf by detecting the dissociation of the OR-GoIf complex.

In general terms, both the GPCR and the G protein can be labelled with two different optical probes. Both these optical probes may form a suitable FRET pair. However, formation of a FRET pair is not necessary; dissociation of the GPCR / G protein complex can also be detected e.g. by FCS or FCCS.

In yet a further embodiment, both optical probes can be placed at the G protein, since it is a homotrimer. Again, detection can then be achieved by FRET or e.g. FCS or FCCS, as outlined above. (ii) Activation of the OR and then of the Golf can also be monitored by surface-sensitive techniques such as total internal reflection fluorescence (TIRF) or by label-free techniques such as surface plasmon resonance (SPR) microscopy or surface accoustic wave techniques. Both label-free techniques monitor mass changes due to the dissociation and subsequent release of the beta-gamma subunits of Golf from the sensing.

3. Arrays of native inside-out vesicles are established, where each vesicle will carry a different odorant receptor. The location of each particular OR receptor is defined by either using multiple color codes inside of a particular vesicle (in this case the native vesicles can be randomly immobilized on the sensing TIRF surface) , or by using microcontact printed arrays of oligonucleotides on the sensor surface which serve as defined addressable locations for native vesicles comprising a defined complementary oligonucleotide attached to the vesicle surface.

The invention is hereafter illustrated by means of schematic figures; the invention is however not to be limited to these embodiments :

Fig. IA: shows an illustration of the human olfactory nervous system.

Fig. IB: shows a high-magnification view of the olfactory epithelium.

Fig. 1C: shows a sensing surface of cell-derived, sub-micrometer sized native vesicles comprising a library of different ORs. Fig. ID: shows surface-sensitive measurements of odorant responses .

Fig. 2: shows a classical GPCR signal transduction mechanism.

Fig. 3: shows the dissociation of inverted native vesicles from the immobilized G protein.

Fig. 4: shows the dissociation of inverted native vesicles from the immobilized G alpha protein.

Fig. 5: shows an inverted native vesicle with high magnification of the inverted cell layer with the inverted membrane topology such as to present the carboxyl termini of the receptor on its outer surface. Artificial nose based on OR multi-arrays coupled to native cellular signal amplification (A) Illustration of the human olfactory nervous system. (B) High-magnification view of the olfactory epithelium. Schematic of individual sensory neurons, each expressing a particular type of OR (C) Sensing surface of cell-derived, sub-micrometer sized native vesicles comprising a library of different ORs. (D) Surface-sensitive measurements of odorant responses. I: Interactions of heterotrimeric G protein complexes with ORs presented by native vesicles. II. Vesicle membrane passage of odorant molecules and specific binding to OR. Ill: Measurements of odorant- mediated receptor activation by detecting the dissociation and release of the heterotrimeric G protein complex .