Bioanalytical reagent, method for production thereof, sensor platforms and detection methods based on use of said bioanalytical reagent

• US 20050019836A1
• Filed: 12/04/2001
• Published: 01/27/2005
• Est. Priority Date: 12/06/2000
• Status: Abandoned Application

• Current Assignee: Bayer Technology Services GmbH (Bayer AG), Ecole polytechnique fãdãrale de lausanne epfl (Schweizerische Eidgenossenschaft)
• Original Assignee: Bayer Technology Services GmbH (Bayer AG)
• Inventors: Pawlak, Michael, Vogel, Horst, Tairi, Ana-Paula, Schmid Osborne, Evelyne, Pick, Horst Matthias, Preuss, Axel Kurt
• Application Number:
  US10/433,823
• Publication Number:
  US 20050019836A1
• Time in Patent Office: Days
• US Class Current: 435/7.200
• CPC Class Codes: G01N 33/554 the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells

Abstract

The invention relates to various embodiments of 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 invention further relates to methods for production of the bioanalytical reagent according to the invention, to bioanalytical detection methods based on the application of said reagent, and to the use of said detection method and of the bioanalytical reagent.

Claims

1-112. -112. (canceled).

113. 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.

114. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein the vesicle as a component of said reagent comprises further cell products and/or cell proteins, besides said one or more receptors, which are involved in said mechanism of signal transduction, besides said one or more receptors.

115. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein said one or more vesicles are generated from a eukaryotic cell.

116. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein said one or more vesicles are generated from a cell of a native tissue.

117. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein the interior of said one or more vesicles is free from cell nucleus material, so that replication processes do not occur within said one or more vesicles.

118. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein said one or more vesicles have a diameter of 50 nm-5000 nm, preferably of 100 nm-2000 nm.

119. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein the one or more receptors are present in natural form in said one or more vesicles as a component of the bioanalytical reagent.

120. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein the one or more receptors are present in a modified form in said one or more vesicles as a component of the bioanalytical reagent.

121. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein the one or more receptors are present in a form resulting from a recombinant fabrication process in said one or more vesicles as a component of the bioanalytical reagent.

122. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, comprising the preservation of a binding capability of said one or more receptors to a specific ligand, 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.

123. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein the one or more receptors are selected from the group of signal-transducing receptors that is formed by plasma membrane receptors, such as ion channel receptors, G protein-coupled receptors (GPCR), orphan receptors, enzyme-coupled receptors, such as receptors with an intrinsic tyrosine kinase activity, receptors with an intrinsic serine/threonine kinase activity, furtheron by receptors for growth factors (peptide hormone receptors), receptors for chemotactic substances, such as the class of chemokine receptors, and by intracellular hormone receptors, such as steroid hormone receptors.

124. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein said one or more vesicles comprise, besides said one or more receptors, further biological compounds (components) from the group that is formed, e.g., by G-proteins and G-protein regulators (e.g. rasGAP), enzymes, such as adenylate cyclases, phospholipases forming 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.

125. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein biological, biochemical or synthetic compounds like cell surface proteins or cell surface sugars are associated with the outer membrane of the one or more vesicles, which associated compounds are used for the transport of said vesicle to pre-determined destinations, such as cells and/or organs and/or pre-determined tissue in a living organism, and/or for the binding to a biological or biochemical or synthetic recognition element, which specifically recognizes and binds said biological or biochemical or synthetic recognition element.

126. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein the mechanism of signal transduction comprises a mechanism from among the group of mechanisms that is formed e.g. from ion conducting, G-protein coupling, activation or inhibition of intra-vesicular ion channels, intra-vesicular release of calcium, protein activation or inhibition by enzymatic phosphorylation or de-phosphorylation (kinase cascades;

phosphatases), and release or enzymatic formation of secondary messenger compounds, such as cAMP, cGMP or diacyl glycerol (DAG), inositol triphosphate (IP3).

127. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein said mechanism of signal transduction comprises a (secondary) functional response of the one or more vesicle-associated receptors after a primary specific interaction of said one or more receptors with one or more natural and/or synthetic ligands contained in a sample that is brought into contact with said vesicle.

128. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein said mechanism of signal transduction comprises the activation of an ion channel of a receptor associated with a vesicle, as a component of said bioanalytical reagent.

129. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein said mechanism of signal transduction comprises the binding of a G-protein to a receptor associated with a vesicle, as a component of said bioanalytical reagent.

130. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein said mechanism of signal transduction comprises the internal release of ions, such as Ca²⁺, or of other messenger compounds, such as cAMP or cGMP.

131. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein said mechanism of signal transduction comprises the enzymatic decomposition of a substrate to a product by a vesicle-associated enzyme.

132. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 127, wherein a (secondary) functional response as part of said mechanism of signal transduction occurs after interaction between one or more natural and/or synthetic ligands or co-factors contained in a sample brought into contact with said vesicle on the one hand and naturally or recombinantly generated proteins associated with said vesicle on the other.

133. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein said one or more vesicles additionally comprise components for generation of an experimentally detectable signal.

134. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 133, wherein said additional components for generation of an experimentally detectable signal are associated with the further biological compounds (components) according to claim 128, these further biological compounds (components) being associated with the one or more vesicles as a component of said bioanalytical reagent.

135. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 133, wherein said additional components for generation of an experimentally detectable signal are associated with a receptor.

136. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 133, wherein said additional components for generation of an experimentally detectable signal are parts of fusion proteins that are associated with said one or more vesicles.

137. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 133, wherein said additional components for generation of an experimentally detectable signal are 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 amino acids, radioactive labels, spin labels, such as NMR labels or ESR labels, ion indicators, especially pH and calcium indicators, or potential-dependent indicators, such as potential-dependent luminescence labels, or redox complexes.

138. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 133, wherein said additional components for generation of an experimentally detectable signal are generated from the same cell from which the vesicle was generated.

139. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 133, wherein said additional components for generation of an experimentally detectable signal are inserted into the vesicle after its formation.

140. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein the functionality of a receptor associated with a vesicle being a component of said bioanalytical reagent is preserved upon storage under deep-frozen conditions for a period of at least one week, preferably of at least one month, especially preferred for at least one year.

141. A bioanalytical reagent with at least one vesicle, generated from a living cell, according to claim 113, wherein it is characterized by a shelf life of at least one week under sterile conditions in cooled buffer solution, i.e. at a temperature below ambient temperature, e.g. at 4°

C.

142. A method for production of a bioanalytical reagent with a vesicle generated from a living cell, according to claim 113, wherein said vesicle was produced from a living cell comprising at least one receptor, and wherein a mechanism of signal transduction triggered by said receptor in said living cell is preserved in said vesicle as a component of the bioanalytical reagent.

143. A method for production of a bioanalytical reagent with a vesicle generated from a living cell, according to claim 142, wherein the constriction of said vesicle from said living cell is effected after application of cytochalasin B and/or cytochalasin D.

144. A method according to claim 142, wherein said method comprises the application of shear forces and/or of centrifugation steps, for example upon exposure to a gradient of sucrose, and/or the application of chromatographic steps, for example by separation into fractions of different size distributions, and/or the application of filtration steps and/or the application of electrophoretic methods.

145. A method according to claim 142, wherein the interior of a vesicle produced by said method is free from cell nucleus material, so that replicative processes do not occur.

146. A method according to claim 142, comprising the preservation of a binding capability of said one or more receptors to a specific ligand, this binding capability being present in said vesicle-generating cell and the receptor being associated with the vesicle as a component of the bioanalytical reagent.

147. A method according to claim 142, wherein the one or more receptors are selected from the group of signal-transducing receptors that is formed by plasma membrane receptors, such as ion channel receptors, G protein-coupled receptors (GPCR), orphan receptors, enzyme-coupled receptors, such as receptors with an intrinsic tyrosine kinase activity, receptors with an intrinsic serine/threonine kinase activity, furtheron by receptors for growth factors (peptide hormone receptors), receptors for chemotactic substances, such as the class of chemokine receptors, and by intracellular hormone receptors, such as steroid hormone receptors.

148. A method according to claim 142, wherein said one or more vesicles produced by this method comprise, besides said one or more receptors, further biological compounds (components) from the group that is formed e.g. by G-proteins and G-protein regulators (e.g. rasGAP), enzymes, such as adenylate cyclases, phospholipases forming 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.

149. A method according to claim 142, wherein biological, biochemical or synthetic compounds, such as cell surface proteins or cell surface sugars, are associated with the outer membrane of the one or more vesicles produced by this method, these compounds being used for the transport of said vesicle to pre-determined destinations, such as cells and/or organs and/or pre-determined tissue in a living organism, and/or for the binding to a biological or biochemical or synthetic recognition element, which specifically recognizes and binds said biological or biochemical or synthetic recognition element.

150. A method according to claim 142, wherein said one or more vesicles produced by this method additionally comprise components for generation of an experimentally detectable signal.

151. A method for production of a bioanalytical reagent with a vesicle generated from a living cell, according to claim 142, wherein said vesicle is merged with an artificial lipid vesicle to form a mixed vesicle.

152. A method according to claim 151, wherein said mixed vesicle is substantially enlarged in comparison to the vesicle generated from a living cell, for example to a diameter of 5 μ

m-50 μ

m.

153. A method according to claim 151, wherein said mixed vesicle comprises additional natural and/or artificial lipids and/or also additional proteins with additional functionalities, in comparison to the vesicle generated from a living cell.

154. A bioanalytical detection method with a bioanalytical reagent according to claim 113, 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), detection of energy or charge transfer, mass spectroscopy, electrical or electrochemical detection methods, such as electrophysiology, patch clamp techniques, impedance measurements, electronic resonance measurements, such as electron spin resonance or nuclear spin resonance, gravimetric methods (e.g. electrical crystal balance measurements), radioactive methods, or by electrophoretic measurements.

155. A bioanalytical detection method according to claim 154, wherein said method is performed in homogeneous solution.

156. A bioanalytical detection method according to claim 154, wherein said method is performed using a measurement arrangement with at least 2 electrodes and separate compartments adequate for receiving liquids, wherein a solid carrier (preferably as an electrically isolating separation wall), comprising at least one aperture and separating at least 2 compartments, is located between two electrodes facing each other, the electrodes being of any geometrical form and each extending into at least one compartment or being in contact with at least one compartment.

157. A bioanalytical detection method according to claim 156, wherein said measurement arrangement is provided with means on one side or on both sides of the carrier which enable a supply of liquid and/or a storage of liquid and/or an exchange of liquid and/or the addition of vesicles generated from a living cell, from a bioanalytical reagent according to the invention, between carrier and electrode.

158. A bioanalytical detection method according to claim 156, wherein the one or more apertures of said measurement arrangement have such a diameter that, in the presence of a potential difference over the measurement arrangement and mediated by the two or more electrodes, an inhomogeneous electrical field is generated around the aperture, said field having an increasing value with decreasing distance from the aperture and said field being capable of moving vesicles electrophoretically towards the aperture, said vesicles being located close to said aperture and generated from a living cell, from a bioanalytical reagent according to the invention.

159. A bioanalytical detection method according to claim 156, wherein the one or more apertures of said measurement arrangement have such a diameter that vesicles generated from a living cell, from a bioanalytical reagent according to the invention, can be positioned over or within the aperture 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).

160. A bioanalytical detection method according to claim 156, wherein the carrier of said measurement arrangement is provided with an electrically charged surface which exerts attractive force on vesicles generated from a living cell, from a bioanalytical reagent according to the invention, or is provided with an adhesion promoting layer for binding said vesicles on its surface.

161. A bioanalytical detection method according to claim 156, wherein vesicles generated from a living cell, from a bioanalytical reagent according to the invention, are inserted between separation wall or carrier and electrode into a compartment filled or not filled with buffer beforehand, and wherein said vesicles are moved towards the aperture by means of an electrical potential difference applied to the electrodes, or are positioned on the aperture by hydrodynamic or electrokinetic flow and/or are positioned on the aperture mechanically (e.g. by means of optical tweezers, force microscope or by a micro manipulator).

162. A bioanalytical detection method according to claim 156, wherein vesicles generated from a living cell, from a bioanalytical reagent according to the invention, are positioned on said aperture, the vesicle membranes form an electrically close contact with the carrier over the aperture, and a measurement of the (electrical) membrane resistance is enabled.

163. A bioanalytical detection method according to claim 156, wherein artificial lipid vesicles with a diameter larger than the diameter of said aperture are added to at least one compartment, in order to generate a planar lipid bilayer on the surface of the carrier and extending over the aperture, and wherein then vesicles generated from a living cell, from a bioanalytical reagent according to the invention are added to said compartment, in order to merge said vesicles with the generated lipid membrane and to make receptors that are associated with said vesicles generated from living cells accessible for electrical or optical measurements.

164. A bioanalytical detection method according to claim 156, wherein membrane proteins are inserted into a vesicle generated from a living cell, after positioning said vesicle on an aperture.

165. A bioanalytical detection method according to claim 156, wherein a vesicle generated from a living cell located over an aperture or a planar membrane generated from said vesicle and spanning an aperture is accessible for optical measurements, especially for fluorescence measurements, or for simultaneous optical and electrical measurements, to which it is subjected.

166. A bioanalytical detection method according to claim 156, wherein a measurement arrangement or a measurement system with several apertures on one carrier is used, and wherein measurements on at least two apertures are performed sequentially and/or in parallel.

167. A bioanalytical detection method according to claim 156, wherein a multitude of vesicles generated from living cells, from a bioanalytical reagent according to the invention, is arranged in an array on a solid, electrically isolating carrier, wherein said array of vesicles is brought into electrically isolating contact with an array of patch-clamp pipets in a geometrical arrangement similar to that of the vesicle array, in order to enable a simultaneous performance of electrical measurements independently of each other or simultaneous electrical and optical measurements on a multitude of individual vesicles.

168. A bioanalytical detection method according to claim 154, wherein the at least one vesicle generated from a living cell, comprising at least one receptor, from a bioanalytical reagent according to the invention, is immobilized on the surface of a solid support.

169. A bioanalytical detection method according to claim 168, wherein a mechanism of a signal transduction triggered by said receptor in said living cell is retained in a vesicle generated from the cell after immobilization of the vesicle.

170. A bioanalytical detection method with at least one vesicle immobilized on the surface of a solid support, the vesicle being generated from a living cell, from a bioanalytical reagent according to claim 113, comprising at least one receptor, wherein a mechanism of a signal transduction triggered by said receptor in said living cell is preserved in a vesicle generated from the cell after immobilization of the vesicle.

171. A bioanalytical detection method according to claim 170, wherein vesicles, each comprising at least one receptor, are immobilized in discrete measurement areas (d) with one or more vesicles each on the surface of said solid support.

172. A bioanalytical detection method according to claim 170, wherein vesicles with at least two different kinds of receptor are immobilized in a multitude of measurement areas (d), wherein preferably within an individual measurement area always vesicles with a uniform kind of receptor are immobilized.

173. A bioanalytical detection method according to claim 168, wherein the immobilization of the one or more vesicles generated from a living cell, on the surface of said solid support, is performed upon covalent binding or upon physical adsorption (electrostatic or van-der-Waals interaction or hydrophilic or hydrophobic interaction or a combination of these interactions).

174. A bioanalytical detection method according to claim 167, wherein an adhesion-promoting layer is deposited between the surface of said solid support and the one or more vesicles immobilized thereon.

175. A bioanalytical detection method according to claim 174, wherein the adhesion-promoting layer comprises a chemical compound of the group of silanes, epoxides, functionalized, charged or polar polymers and “

self-organized functionalized mono or multiple layers”

.

176. A bioanalytical detection method according to claim 174, wherein 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.

177. A bioanalytical detection method according to claim 174, wherein the adhesion-promoting layer comprises self-organized alkane-terminated monolayers of mainly one kind of chemical or biochemical molecules.

178. A bioanalytical detection method according to claim 174, comprising 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 from the bioanalytical reagent, wherein biological, biochemical or synthetic compounds like cell surface proteins or cell surface sugars are associated with the outer membrane of the one or more vesicles, which associated compounds are used for the transport of said vesicle to pre-determined destinations, such as cells and/or organs and/or pre-determined tissue in a living organism, and/or for the binding to a biological or biochemical or synthetic recognition element, which specifically recognizes and binds said biological or biochemical or synthetic recognition element.

179. A bioanalytical detection method according to claim 154, wherein at least one ligand for a receptor, which is bound to a vesicle generated from a living cell, from a bioanalytical reagent according to the invention, is immobilized, optionally by means of a spacer molecule, on the surface of the solid support.

180. A bioanalytical detection method according to claim 179, wherein at least two different ligands for receptors, which are bound to a vesicle generated from a living cell, from a bioanalytical reagent according to the invention, are immobilized in a multitude of measurement areas (d), wherein preferably a uniform kind of ligand is immobilized within an individual measurement area.

181. A bioanalytical detection method according to claim 179, wherein said ligands are immobilized on the surface of the solid support upon covalent binding or upon physical adsorption (e.g. electrostatic or van-der-Waals interaction or hydrophilic or hydrophobic interaction or a combination of these interactions).

182. A bioanalytical detection method according to claim 179, wherein an adhesion-promoting layer is applied between the surface of the solid support and said ligands immobilized thereon.

183. A bioanalytical detection method according to claim 171, wherein regions between the laterally separated measurement areas, with vesicles, generated from living cells (from a bioanalytical reagent according to the invention) 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 the invention), and/or regions within these measurement areas, between the compounds immobilized therein, are “

passivated”

in order to minimize non-specific 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/lipid mixtures (of synthetic and/or natural lipids), synthetic and natural lipids or also hydrophilic polymers, such as polyethylene glycols or dextrans.

184. A bioanalytical detection method according to claim 156, wherein the material of the solid support (carrier) with immobilized vesicles, generated from living cells (from a bioanalytical reagent according to the invention), or with immobilized ligands for receptors that are bound to vesicles generated from living cells (from a bioanalytical reagent according to the invention), comprises 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.

185. A bioanalytical detection method according to claim 156, wherein the surface of said solid support (carrier) is essentially planar.

186. A bioanalytical detection method according to claim 156, wherein said solid support (carrier) is an optical or electronic sensor platform.

187. A bioanalytical detection method according to claim 156, wherein said solid support (carrier) is transparent at least in a region of wavelengths in the ultraviolet to infrared spectrum and comprises preferably a material from the group that is formed e.g. by moldable, sprayable or millable plastics, carbon compounds, metals, metal oxides or silicates, such as glass, quartz or ceramics, or silicon or germanium or ZnSe or a mixture of these materials.

188. A bioanalytical detection method according to claim 186, wherein said solid support is an optical waveguide used as a sensor platform.

189. A bioanalytical detection method according to claim 186, wherein said solid support is an optical thin-film waveguide used as a sensor platform, with an initial optically transparent layer (a) with refractive index n1 on a second optically transparent layer (b) with refractive index n₂, wherein n₂, wherein n, >

n₂.

190. A bioanalytical detection method according to claim 188, wherein the sensor platform as a solid support is divided into two or more discrete waveguiding regions.

191. A bioanalytical detection method according to claim 189, wherein the material of the second optically transparent layer (b) of the sensor platform as a solid support is selected from the group that is formed by silicates, such as glass or quartz, or transparent moldable, sprayable or millable, especially thermoplastic plastics, such as polycarbonates, polyimides, polymethyl methacrylates, or polystyrenes.

192. A bioanalytical detection method according to claim 189, wherein the refractive index of the first optically transparent layer (a) of the sensor platform as a solid support is greater than 1.8.

193. A bioanalytical detection method according to claim 189, wherein the first optically transparent layer (a) comprises a material of the group of TiO₂, ZnO, Nb₂O₅, Ta₂O₅, HfO₂, or ZrO₂, preferably of TiO₂ or Ta₂O₅ or Nb₂O₅.

194. A bioanalytical detection method according to claim 189, wherein an additional optically transparent layer (b′

) 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).

195. A bioanalytical detection method according to claim 189, wherein the in-coupling of excitation light into the optically transparent layer (a) to the measurement areas (d) on the sensor platform as a solid support 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.

196. A bioanalytical detection method according to claim 189, wherein 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).

197. A bioanalytical detection method according to claim 156, wherein one or more liquid samples, comprising vesicles generated from living cells (from a bioanalytical reagent according the invention), with associated receptors, are brought into contact with the ligands for these receptors, immobilized in one or more measurement areas, and wherein a signal change caused by a binding of the receptors associated with said vesicles to their immobilized ligands is measured.

198. A bioanalytical detection method according to claim 197, wherein the signal transduction of receptors associated with vesicles generated from living cells (from a bioanalytical reagent according to the invention) after binding of these receptors to their immobilized ligands, is measured, wherein this signal transduction can be triggered, for example, by binding of further ligands to the receptors associated with said vesicles, or by other inducing influences.

199. A bioanalytical detection method according to claim 197, wherein the binding of receptors that are associated with vesicles generated from living cells (from a bioanalytical reagent according to the invention) to said immobilized ligands occurs in competition with the binding of these receptors associated with said vesicles to ligands in free solution.

200. A bioanalytical detection method according to claim 156, wherein one or more liquid samples are brought into contact with the vesicles, which are generated from living cells (from a bioanalytical reagent according to the invention) and immobilized in one or more measurement areas, along with their associated receptors, and wherein a signal change resulting from the binding of ligands to said receptors or from other inducing influences on said receptors is measured.

201. A bioanalytical detection method according to claim 156, wherein one or more liquid samples are brought into contact with the vesicles, which are generated from living cells (from a bioanalytical reagent according to the invention) and immobilized in one or more measurement areas, along with their associated receptors, and wherein the signal transduction of those receptors resulting from the binding of ligands to said receptors or from other inducing influences on said receptors is measured.

202. A bioanalytical detection method according to claim 200, wherein the binding of ligands from a supplied sample to receptors that are associated with the immobilized vesicles generated from living cells (from a bioanalytical reagent according to the invention) occurs in competition with the binding of these ligands to receptors in free solution which are optionally associated with vesicles.

203. A bioanalytical detection method according to claim 197, wherein one or more liquid samples, comprising vesicles generated from living cells (from a bioanalytical reagent according to the invention) with associated receptors, are brought into contact with the ligands for these receptors, the ligands being immobilized in one or more measurement areas, excitation light from one or more light sources of similar or different wavelengths is in-coupled to the measurement areas (d) by one or more grating structures (c), and the change of optical signals emanating from one or more measurement areas (d), caused by a binding of the receptors associated with said vesicles to their immobilized ligands, is measured.

204. A bioanalytical detection method according to claim 200, wherein one or more liquid samples are brought into contact with the vesicles immobilized in one or more measurement areas (d), along with their associated receptors, excitation light from one or more light sources of similar or different wavelengths is in-coupled to the measurement areas (d) by one or more grating structures (c), and the change of optical signals emanating from one or more measurement areas (d), caused by the binding of the ligands to said receptors or by other inducing influences on said receptors, is measured.

205. A bioanalytical detection method according to claim 204, wherein said changes of optical signals from the measurement areas (d) are caused by changes of the effective refractive index in the near-field of the optically transparent layer (a) in these measurement areas and are measured at the actual excitation wavelength.

206. A bioanalytical detection method according to claim 204, wherein said changes of optical signals from the measurement areas (d) are changes of one or more luminescences of similar or different wavelength, which have been excited in said measurement areas in the near-field of the optically transparent layer (a), and which are measured each at a wavelength different from the corresponding excitation wavelength.

207. A bioanalytical detection method according to claim 204, wherein the one or more luminescences and/or measurements of light signals at the excitation wavelength are determined polarization-selectively, wherein preferably the one or more luminescences are measured at a polarization that is different from the polarization of the excitation light.

208. A bioanalytical detection method according to claim 154, for the simultaneous or sequential, quantitative and/or qualitative determination of one or more analytes from the group of receptors or ligands, chelators or “

histidine tag components”

, enzymes, enzyme co-factors or inhibitors.

209. A bioanalytical detection method according to claim 154, wherein the samples to be examined are, for example, aqueous solutions or surface water or soil or plant extracts or bio- or process broths, or are taken from biological tissue fractions or from food, or odorous or flavoring substances or cosmetic compounds.

210. The use of a vesicle as a component of a bioanalytical reagent according to claim 113 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 can then e.g. be administered to living organisms (e.g. to stimulate immune defense processes).

211. The use of a vesicle, as a component of a bioanalytical reagent according to claim 113 as a compartment for therapeutic, diagnostic, photosensitive or other biologically active compounds for administration to a living organism.

212. The use of a bioanalytical reagent according to claim 113 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.

213. The use of a bioanalytical reagent according to claim 113 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.

214. The use of a bioanalytical detection method according to claim 154 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.

215. The use of a bioanalytical detection method according to claim 154 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.