EP1334361A2 - Kit et procede permettant la detection d'analytes multiples avec des mesures permettant la referenciation de la densite d'elements de reconnaissance immobilises - Google Patents

Kit et procede permettant la detection d'analytes multiples avec des mesures permettant la referenciation de la densite d'elements de reconnaissance immobilises

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Publication number
EP1334361A2
EP1334361A2 EP01994637A EP01994637A EP1334361A2 EP 1334361 A2 EP1334361 A2 EP 1334361A2 EP 01994637 A EP01994637 A EP 01994637A EP 01994637 A EP01994637 A EP 01994637A EP 1334361 A2 EP1334361 A2 EP 1334361A2
Authority
EP
European Patent Office
Prior art keywords
referencing
sensor platform
kit according
analytes
detection
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01994637A
Other languages
German (de)
English (en)
Inventor
Andreas P. Abel
Gerhard M. Kresbach
Gert L. Duveneck
Nania G. SCHÄRER-HERNANDEZ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer AG
Original Assignee
Zeptosens AG
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Filing date
Publication date
Application filed by Zeptosens AG filed Critical Zeptosens AG
Publication of EP1334361A2 publication Critical patent/EP1334361A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the invention relates to various embodiments of a kit for the simultaneous qualitative and / or quantitative detection of a large number of analytes, which in particular makes it possible to determine the density of the biological or biochemical or synthetic recognition elements immobilized for the detection of said analytes, i.e. to refer to the occupancy density of the measuring ranges identified for these detection elements.
  • the invention also relates to analytical systems based on the kit according to the invention and methods carried out therewith for the detection of one or more analytes and their use.
  • microtiter plates For the determination of a large number of analytes, above all methods are widespread in which the detection of different analytes in so-called microtiter plates is carried out in discrete sample containers or "wells" of these plates.
  • the most widespread are plates with a grid of 8 x 12 wells on a base area of typically approx. 8 cm x 12 cm, a volume of a few hundred microliters being required to fill an individual well.
  • the immobilization for the analyte-specific recognition elements in the form of small "spots" with partially significantly less than 1 mm 2 area on solid supports is proposed to by Binding of only a small part of the analyte molecules present enables the concentration of the analyte to be determined which is dependent only on the incubation time but - in the absence of a continuous flow - essentially independent of the absolute sample volume.
  • the measurement arrangements described in the associated exemplary embodiments are based on fluorescence detections in conventional microtiter plates. Arrangements are also described in which spots of up to three different fluorescence-labeled antibodies are measured in a common microtiter plate well. Following the theoretical considerations set out in these patents, it would be desirable to minimize the spot size. However, the minimum signal level, which can be distinguished from the background signal, has a limiting effect.
  • Arrays with a very high feature density ie density of discrete measurement areas on a support with detection elements immobilized in these measurement areas for the detection of different analytes
  • Arrays with a very high feature density ie density of discrete measurement areas on a support with detection elements immobilized in these measurement areas for the detection of different analytes
  • US Pat. No. 5,445,934 (Affymax Technologies) describes and claims arrays of oligonucleotides with a density of more than 1000 features per square centimeter. The excitation and layout of such arrays is based on classic optical arrangements and methods.
  • the entire array can be illuminated simultaneously with an expanded excitation light bundle, which, however, leads to a relatively low sensitivity, since the scattered light component is relatively large and scattered light or background fluorescent light is also generated from the glass substrate in the areas in which there is no binding of the analyte immobilized oligonucleotides.
  • confocal measuring arrangements are often used and the various features are read out sequentially by means of "scanning". However, this results in a greater expenditure of time for reading out a large array and a relatively complex optical structure.
  • the measured signals are not referenced for the detection of different analytes, neither with regard to the excitation light intensity available in the measuring ranges nor with respect to the distribution or (relative) number of immobilized detection elements.
  • 2 different samples labeled with different luminescence labels e.g. emitting in the green or in the red
  • a light wave is coupled into an optical waveguide that is made of optically thinner media, i.e. Media with a lower refractive index is surrounded, it is guided by total reflection at the interfaces of the waveguiding layer.
  • a fraction of the electromagnetic energy enters the optically thinner media. This proportion is known as the evanescent or cross-damped field.
  • the strength of the evanescent field is very much dependent on the thickness of the waveguiding layer itself and on the ratio of the refractive indices of the waveguiding layer and the media surrounding it.
  • thin waveguides i.e. H. Layer thicknesses of the same or lower thickness than the wavelength to be guided can be distinguished from discrete modes of the guided light.
  • the first proposed measuring arrangements of this type were based on highly multimodal, self-supporting single-layer waveguides, such as fibers or platelets made of transparent plastic or glass, with thicknesses from a few hundred micrometers to several millimeters.
  • Planar thin-film waveguides have been proposed to improve sensitivity and, at the same time, simplify mass production.
  • a planar thin-film waveguide consists of a three-layer system: carrier material, wave-guiding layer, superstrate (or sample to be examined), the wave-guiding layer has the highest refractive index. Additional intermediate layers can improve the effect of the planar waveguide.
  • luminescence denotes the spontaneous emission of photons in the ultraviolet to infrared range after optical or non-optical, such as, for example, electrical or chemical or biochemical or thermal excitation.
  • chemiluminescence, bioluminescence, electroluminescence and in particular fluorescence and phosphorescence are included under the term “luminescence”.
  • luminescence-based methods appear to be more suitable than methods based on a change in the effective refractive index (such as grating coupler sensors or methods based on surface plasmon resonance) due to the greater selectivity of signal generation.
  • the luminescence excitation is limited to the depth of penetration of the evanescent field into the optically thinner medium, i.e. to the immediate vicinity of the wave-guiding region with a depth of penetration of the order of a few hundred nanometers into the medium. This principle is called evanescent luminescence excitation.
  • WO 95/33197 describes a method described in which the excitation light is coupled into the waveguiding film as a diffractive optical element via a relief grating.
  • the surface of the sensor platform is brought into contact with a sample containing the analyte, and the isotropically illuminated luminescence in the penetration depth of the evanescent field of luminescent substances is measured by means of suitable measuring devices, such as photodiodes, photomultipliers or CCD cameras. It is also possible to decouple and measure the portion of the evanescently excited radiation fed back into the waveguide via a diffractive optical element, for example a grating. This method is described for example in WO 95/33198.
  • US 5,525,466 and US 5,738,992 describe an optical sensor based on fluorescence excitation in the evanescent field of a self-supporting multimode waveguide, preferably of a fiber-optic type.
  • the excitation light is coupled in and the fluorescent light fed back into the multimode waveguide is coupled out via coupling in and out of the end face.
  • the fluorescence signal detected in the process for the analyte detection results from the functional principle of such multimode waveguides as a single integral value for the entire surface interacting with the sample.
  • fluorescent reference materials are co-immobilized on the sensor surface in addition to the biochemical or biological detection elements for the specific detection and binding of an analyte to be detected. Due to the underlying sensor principle, however, it is not possible to have a spatially resolved, but only a normalization that affects the individual, integral measured value. Consequently, the detection of different analytes can also only be carried out by using lab in different excitation wavelengths or sequentially, after removal of previously bound analytes. For these reasons, these arrangements, together with the referencing method described, do not appear to be suitable at all, or only slightly, for the simultaneous detection of a large number of analytes.
  • WO 97/35181 describes methods for the simultaneous determination of one or more analytes in that "well" patches formed in a waveguide are formed with different detection elements, which are contacted with a sample solution containing one or more analytes.
  • solutions with defined analyte concentrations are simultaneously added to other wells with similar patches.
  • 3 wells for measurement with calibration solutions of low and high analyte concentration as well as the current sample
  • the sandwich immunoassays are carried out with sequential addition of washing solution (buffer), sample with one or more analytes, washing solution (buffer), tracer antibody (individually or as a cocktail) and washing solution (buffer).
  • the locally measured fluorescence intensities are corrected by subtracting the background signal observed next to the measuring fields.
  • this arrangement also does not make it possible to carry out an entire series of measurements for the simultaneous determination of several analytes, together with the necessary calibrations, but instead requires either the use of several different sensor platforms or repetitive, sequential measurements on one platform with interim regeneration, which is only possible to a limited extent in many cases, especially in the case of immunoassays.
  • the invention relates to a kit for the simultaneous qualitative and / or quantitative detection of a large number of analytes, comprising - a sensor platform
  • At least one array of biological or biochemical or synthetic recognition elements immobilized in discrete measurement areas (d) directly on or via an adhesion-promoting layer on the sensor platform for the specific recognition and / or binding of said analytes and / or specific interaction with said analytes, with the purpose of " Referencing the immobilization density ", ie for the spatially resolved determination of the density of the immobilized recognition elements in the measurement areas, these recognition elements are each associated with a signal-emitting component as a label and / or said biological or biochemical or synthetic recognition elements have a specific molecular sequence or a specific molecular epitope or a specific molecular recognition group , to which a detection reagent (referencing reagent), possibly with an associated signaling component as a label, binds to determine said density of immobilized recognition elements.
  • Said specific molecular sequence or said specific molecular epitope or said specific molecular recognition group is advantageous for all different, generally in different measuring ranges of a segment from several measuring ranges, particularly preferably even for all immobilized in an array of measuring ranges biological or biochemical or synthetic recognition elements.
  • an array of measurement areas can comprise discrete measurement areas with a large number of different immobilized single-stranded nucleic acids, each of which has different partial sequences, for example 10-100 or 10-1000 different partial sequences, for the detection and binding of a corresponding number of different nucleic acids complementary to these partial sequences as analytes ,
  • these different immobilized single-stranded nucleic acids can have a different partial sequence which is common to all and which can serve the purpose of “referencing the immobilization density” as described above.
  • the problem described can be solved with the kit according to the invention and detection methods based thereon.
  • a kit according to the invention it was found that it is possible, using a kit according to the invention, in multianalyt assays for Simultaneous determination of several analytes in a sample to achieve a similarly high sensitivity and reproducibility as previously in a corresponding number of individual assays for the detection of the individual analytes.
  • a referencing reagent that may be used and any signaling components associated therewith do not have an adverse effect on analyte detection.
  • spatially separate or discrete measurement areas (d) are to be defined by the closed area, which biological or biochemical or synthetic recognition elements immobilized there assume for the detection of an analyte from a liquid sample.
  • These surfaces can have any geometry, for example the shape of points, circles, rectangles, triangles, ellipses or strips.
  • optical transparency is understood below to mean that the material characterized by this property should be largely transparent and thus absorption-free at least at one or more excitation wavelengths used to excite one or more luminescence.
  • a preferred embodiment of the kit according to the invention is characterized in that the immobilized recognition elements in the measurement areas each have a general molecular sequence or a general epitope or general molecular recognition group for the purpose of referencing the immobilization density and one or more for recognizing and / or binding different analytes, have different sequence or different epitope or different molecular recognition group.
  • Said general molecular sequence or said general epitope or said general molecular recognition group can occur adjacent to one another for purposes of “referencing the immobilization density” and a different sequence or different epitope or different molecular recognition group for recognizing and / or binding different analytes or in a recognition element To improve the accessibility for an analyte to be detected, however, it is preferred that they are sufficiently far apart from one another within a recognition element so that there is no impediment to the access of an analyte to that for its recognition specific sequence or the epitope specific for its recognition or specific molecular recognition group of the immobilized recognition element comes.
  • the general and the specific recognition section (conceptually comprising recognition sequence, epitope and molecular recognition group) of an immobilized recognition element can be separated from one another by a so-called molecular spacer (for example comprising a chain molecule with hydrocarbon groups).
  • a so-called molecular spacer for example comprising a chain molecule with hydrocarbon groups.
  • sections with a general nucleic acid sequence for the purposes of “referencing the immobilization density”, for example in a hybridization step with fluorescence-labeled oligonucleotides complementary to this general sequence, and with the general nucleic acid sequence chemically linked antibodies or antibody fragments with different, in each case epitopes specific for different analytes can be present within an immobilized biological or biochemical or synthetic recognition element.
  • the said general recognition sections are preferably to be selected such that the presence of a binding partner specific for this general recognition section in a sample to be supplied with analytes to be detected can be largely ruled out, provided that this binding partner does not additionally Sample is added.
  • kits according to the invention are characterized in that, for said purposes, the “referencing rank of the immobilizing rank density” is a referencing reagent for recognition and / or binding to said general sequence or to said general epitope or to said general molecular recognition group in the same Measuring range of immobilized biological or biochemical or synthetic detection elements on the sensor platform co- is immobilized, possibly associated with said immobilized recognition elements.
  • the “referencing rank of the immobilizing rank density” is a referencing reagent for recognition and / or binding to said general sequence or to said general epitope or to said general molecular recognition group in the same Measuring range of immobilized biological or biochemical or synthetic detection elements on the sensor platform co- is immobilized, possibly associated with said immobilized recognition elements.
  • the referencing rank of the immobilizing rank density is a referencing reagent for recognition and / or binding to said general sequence or to said general epitope or to the general molecular recognition group of the immobilized biological or biochemical or synthetic recognition elements on the sensor platform after immobilization of the biological or biochemical or synthetic recognition elements is applied to the measuring areas of the sensor platform.
  • Said "referencing of the immobilization density" i.e. the spatially resolved determination of the density of the immobilized recognition elements in the measurement areas, can be part of a quality control during or after the manufacture of a sensor platform, as part of a kit according to the invention.
  • a referencing reagent for recognition and / or binding to said general sequence or to said general epitope or to said general molecular recognition group of the immobilized biological or biochemical or synthetic recognition elements on the Sensor platform is applied to the measuring areas of the sensor platform in the course of a detection method for determining one or more analytes.
  • Said general molecular sequence or said general epitope or said general molecular recognition group (such as eg biotin) of the immobilized biological or biochemical or synthetic recognition elements can for example be selected from the group consisting of polynucleotides, polynucleotides with artificial bases, PNAs (“peptide nucleic acids” ), PNA's with artificial bases, proteins, antibodies, peptides, oligosaccharides, lectins, etc. is formed.
  • a preferred embodiment is characterized in that said general sequence of the immobilized biological or biochemical or synthetic recognition elements has a length of 5 to 500, preferably 10 to 100 bases.
  • Another preferred embodiment of the kit according to the invention is characterized in that the immobilized detection elements in the measurement areas are each associated with a signal-emitting component as a label. It can also be advantageous if said signaling component changes its signaling properties as a label when an analyte is bound to the respective detection element associated with it.
  • a characteristic common to the various embodiments of a kit according to the invention is that said different sequences or different epitopes or different molecular recognition groups of the immobilized biological or biochemical or synthetic recognition elements are selected from the group consisting of nucleic acids (for example DNA, RNA, oligonucleotides) and nucleic acid analogs (eg PNA) and their derivatives with artificial bases, mono- or polyclonal antibodies, peptides, enzymes, aptamers, synthetic peptide structures, glycopeptides, glycoproteins, oligosaccharides, lectins, soluble, membrane-bound proteins isolated from a membrane, such as For example, receptors, their ligands, antigens for antibodies (z. B.
  • Biotin for streptavidin "histidine tag components” and their complexing partners, cavities generated by chemical synthesis for receiving molecular imprints, etc. born is formed. It is also provided that whole cells, cell components, cell membranes or their fragments are applied as biological or biochemical or synthetic recognition elements.
  • a referencing reagent required for certain embodiments of the kit according to the invention comprises a label which is selected from the group consisting of, for example, luminescence labels, in particular luminescent intercalators or “molecular beacons”, absorption labels, mass labels, in particular metal colloids or plastic beads, Spin labels, such as ESR or NMR labels, radioactive labels are formed.
  • a label which is selected from the group consisting of, for example, luminescence labels, in particular luminescent intercalators or “molecular beacons”, absorption labels, mass labels, in particular metal colloids or plastic beads, Spin labels, such as ESR or NMR labels, radioactive labels are formed.
  • said referencing reagent comprises a luminescence label or absorption label.
  • said referencing reagent can also comprise an intercalator or a "molecular beacon". It is preferred that said intercalator or "molecular beacon" changes its signaling properties in the presence of the referencing reagent. Said referencing reagent can be split off before or in the course of an analytical detection method or can remain associated with the recognition elements.
  • a further advantageous embodiment of the kit according to the invention is characterized in that said referencing reagent comprises a component from the group consisting, for example, of polynucleotides, polynucleotides with artificial bases, PNAs (“peptide nucleic acids”), PNAs with artificial bases, proteins, antibodies, biotin , Streptavidin, peptides, oligosaccharides, lectins, etc. is formed.
  • the quantitative and / or qualitative detection of said large number of analytes comprises the use of one or more signaling components as labels, which can be selected from the group consisting of, for example, luminescent labels, in particular luminescent ones Intercalators or "molecular beacons", absorption labels, mass labels, in particular metal colloids or plastic beads, spin labels, such as ESR or NMR labels, radioactive labels are formed.
  • labels can be selected from the group consisting of, for example, luminescent labels, in particular luminescent ones Intercalators or "molecular beacons", absorption labels, mass labels, in particular metal colloids or plastic beads, spin labels, such as ESR or NMR labels, radioactive labels are formed.
  • the label of the referencing reagent and / or an analyte detection possibly based on absorption and / or luminescence detection is based on the use of labels with the same or different absorption and / or luminescence wavelength.
  • a special embodiment based on recognition elements immobilized in the measurement areas, each with an associated signal-emitting component as a label, is characterized in that said label also serves for an analyte detection in addition to referencing the immobilization density of the recognition elements.
  • said label can be a fluorescent intercalator, which, when bound to a single-stranded nucleic acid as the immobilized recognition element, delivers a signal that is very weak but still measurable, from which the density of the recognition elements immobilized in the corresponding measuring ranges can be determined.
  • the fluorescence intensity of the sample can be greatly increased Intercalators come, based on which the analyte in question is then detected qualitatively and / or quantitatively in this measuring range.
  • the analyte detection be based on determining the change in one or more luminescences.
  • a possible embodiment is characterized in that the excitation light is irradiated by one or more light sources for generating signals of signal-generating components for the purposes of chemical referencing and / or for the detection of one or more analytes in an incident light arrangement.
  • the material of the sensor platform that is in contact with the measurement areas is transparent or absorbent within a depth of at least 200 nm from the measurement areas with at least one excitation wavelength.
  • excitation light is irradiated by one or more light sources for generating signals of signal-generating components for the purpose of referencing the immobilization density and / or for the detection of one or more analytes in a transmission light arrangement.
  • the material of the sensor platform is transparent at at least one excitation wavelength.
  • a preferred embodiment of a kit according to the invention is characterized in that the sensor platform is designed as an optical waveguide, which is preferably essentially planar.
  • the sensor platform preferably comprises a material from the group consisting of silicates, e.g. B. glass or quartz, transparent thermoplastic or sprayable plastic, for example polycarbonate, polyimide, acrylates, in particular polymethyl methacrylate, or polystyrenes is formed.
  • a particularly preferred embodiment of a kit according to the invention is characterized in that the sensor platform has an optical thin-film waveguide with a in the case of at least one excitation wavelength, comprises a transparent layer (a) on a layer (b) with a lower refractive index than layer (a), which is likewise transparent at at least this excitation wavelength.
  • the excitation light from one or more light sources is coupled into the optical waveguide by means of a method which is selected from the group consisting of end face coupling and decoupling via attached optical fibers as light guides, Prism coupling, grating coupling or evanescent coupling by overlapping the evanescent field of said optical waveguide with the evanescent field of a further waveguide that is brought into close-field contact is formed.
  • the aim should be to avoid the generation of reflections of irradiated excitation light as much as possible, since these generally lead to an increase in background signals, essentially disadvantageously.
  • the occurrence of reflections is to be expected in principle each time the excitation light passes through optical interfaces to media with different refractive indices.
  • the excitation light from one or more light sources is coupled into the optical waveguide by means of an optical coupling element in contact therewith, which is selected from the group of optical fibers as light guides, prisms, optionally via a liquid that adjusts the refractive index, and grid couplers.
  • An embodiment of the kit according to the invention is particularly preferred, which is characterized in that the excitation light from one or more light sources is coupled into the layer (a) by means of one or more grating structures (c) modulated in the layer (a).
  • Suitable geometric arrangements of such lattice structures for a sensor platform as part of a kit according to the invention are in turn, for example, in the patents US 5,822,472, US 5,959,292 and US 6,078,705 and in the patent applications WO 96/35940, WO 97/37211, WO 98/08077, WO 99/58963 , PCT / EP 00/04869 and PCT / EP 00/07529 and are also part of the present invention as an integral part of a kit according to the invention.
  • the sensor platform comprises uniform, unmodulated regions of the layer (a), which are preferably arranged in the direction of propagation of the excitation light coupled in via a lattice structure (c) and guided in the layer (a).
  • lattice structures (c) can be used to couple excitation light to the measuring areas (d) and / or to couple luminescent light fed back into layer (a).
  • the sensor platform will therefore comprise a plurality of lattice structures (c) of the same or different periods with optionally adjoining uniform, unmodulated regions of the layer (a) on a common, continuous substrate.
  • a grating structure (c) to which an unmodulated region of the layer (a) is directed in the direction of propagation of the light that is coupled in and guided in the layer (a).
  • a grating structure to which an unmodulated region of the layer (a) is directed in the direction of propagation of the light that is coupled in and guided in the layer (a).
  • this is advantageously followed by a further lattice structure with a further array of measurement areas located behind it, etc.
  • the light guided in layer (a) is in each case coupled out again.
  • each array of measurement areas following in the direction of propagation of the coupled-in excitation light is assigned a grating structure (c) specific to this array for decoupling this excitation light, wherein the grating structures can be formed specifically for individual arrays perpendicular to the direction of propagation of the coupled-in excitation light or can also be can extend over the entire sensor platform in this direction.
  • the coupling-in grating of an array following in the direction of propagation of an excitation light guided in the layer (a) of a sensor platform serves as coupling-out grating for the excitation light coupled in on the coupling-in grating of the array preceding in said direction of propagation.
  • the sensor platform comprises a superposition of 2 or more grating structures of different periodicity with parallel or non-parallel, preferably non-parallel alignment of the grating lines , which is used for coupling excitation light of different wavelengths, the grating lines in the case of 2 superimposed grating structures preferably being oriented perpendicular to one another.
  • the subdivision of the sensor platform into areas with lattice structures and subsequent unmodulated areas means that the space required for a single array of measuring areas between successive lattice structures (including at least one assigned lattice structure) cannot fall below a certain minimum, which the current technical possibilities for generating the lattice structures and for coupling in a suitable excitation light bundle are in the order of magnitude of approximately 0.1 mm to 1 mm. It is therefore particularly advantageous for arrangements in which a large number of small-area arrays is desired if a lattice structure (c) or a superposition of a plurality of lattice structures in layer (a) is modulated essentially over the entire area of the sensor platform.
  • optically or mechanically recognizable markings on the sensor platform to facilitate the adjustment in an optical one System and / or for connection to sample containers are applied as part of an analytical system.
  • a further embodiment of the arrangement according to the invention is that between the optically transparent layers (a) and (b) and in contact with layer (a) there is another optically transparent layer (b ') with a lower refractive index than that of the layer (a ) and a thickness of 5 nm - 10000 nm, preferably of 10 nm - 1000 nm.
  • the simplest form of immobilization of the biological or biochemical or synthetic recognition elements consists in physical adsorption, for example as a result of hydrophobic interactions between the recognition elements and the base plate.
  • these interactions can be greatly changed in their extent by the composition of the medium and its physicochemical properties, such as polarity and ionic strength.
  • the adhesion of the recognition elements after purely adsorptive immobilization on the surface is often inadequate.
  • the adhesiveness is improved in that an adhesion-promoting layer (f) is applied to the sensor platform for immobilizing biological or biochemical or synthetic recognition elements.
  • the adhesion-promoting layer can also serve, in particular in the case of biological or biochemical recognition elements to be immobilized, to improve the "biocompatibility" of their surroundings, ie to maintain the binding capacity in comparison to their natural biological or biochemical surroundings, and in particular to avoid denaturation. It is preferred that the adhesion promoting layer (f) has a thickness of less than 200 nm, preferably less than 20 nm. A large number of materials are suitable for producing the adhesion-promoting layer.
  • adhesion promoting layer (f) comprise one or more chemical compounds from the groups that functionalized silanes Silanes, epoxides, functionalized, charged or polar polymers and "self-organized passive or functionalized mono- or multilayers" include.
  • kits according to the invention are immobilized in spatially separated measuring areas (d). These spatially separated measuring areas (d) can be generated by spatially selective application of biological or biochemical or synthetic detection elements on the sensor platform. A large number of known processes are suitable for the application.
  • one or more methods from the group of methods used by "inkjet spotting", mechanical spotting by means of pen, pen or capillary, "micro.” are used to apply the biological or biochemical or synthetic recognition elements to the sensor platform contact printing ", fluidic contacting of the measuring areas with the biological or biochemical or synthetic recognition elements by their supply in parallel or crossed microchannels under the influence of pressure differences or electrical or electromagnetic potentials" as well as photochemical and photolithographic immobilization processes.
  • kits according to the invention are that the density of the detection elements immobilized in discrete measurement areas for the detection of different analytes on different measurement areas is selected such that the luminescence signals in the detection of different analytes in a common array are of the same order of magnitude, that is to say that if necessary the associated calibration curves for the analyte determinations to be carried out simultaneously can be recorded without changing the electronic or optoelectronic system settings.
  • kits according to the invention are characterized in that arrays of measurement areas are divided into segments of one or more measurement areas for determining analytes and areas between these measurement areas or additional measurement areas for the purposes of physical referencing, such as the excitation light intensity available in the measurement areas or the influence of changes in external parameters, such as temperature, and for the purpose of referencing the influence of additional physico-chemical parameters.
  • meters such as non-specific binding to the sensor platform of components of an applied sample.
  • two or more arrays it is advantageous for two or more arrays to have a similar geometric arrangement of measurement areas and / or segments of measurement areas for the determination of similar ones Have analytes on these arrays.
  • one or more arrays comprise segments of two or more measuring ranges with biological or biochemical or synthetic recognition elements of the same type within the segment for analyte determination or referencing.
  • the kit according to the invention with a large number of measuring ranges in discrete arrays, a large number of which in turn can be arranged on a common sensor platform offers the possibility of using relatively small amounts of sample solutions, reagents or, if appropriate, calibration solutions on one and the same platform, under largely identical conditions, many types of duplications or multiple executions of the same measurements can be carried out. In this way, for example, statistical data can be generated in a single measurement, for which a large number of individual measurements with a correspondingly longer total measurement time and higher consumption of sample and reagent quantities are conventionally required. It is preferred that 2 or more identical measurement areas are provided within a segment or array for the detection of each analyte or for referencing.
  • said identical measuring ranges can be in a continuous Row or column or diagonals of an array or segment of measurement areas can be arranged.
  • the aspects of referencing can relate to physical or physico-chemical parameters of the sensor platform, such as local differences in the excitation light intensity (see also below), as well as influences of the sample, such as its pH, ionic strength, refractive index, temperature etc.
  • said identical measuring ranges are arranged statistically within an array or segment of measuring ranges.
  • the immobilized recognition elements are generally selected so that they recognize and bind the analyte to be detected with the highest possible specificity. In general, however, it is to be expected that non-specific attachment of analyte molecules to the surface of the base plate will also take place, in particular if reactive clearances are still present between the detection elements immobilized in the measurement areas. It is therefore preferred that areas between the spatially separated measurement areas are "passivated” in order to minimize non-specific binding of analytes or their detection substances, i.e.
  • “chemically neutral” compounds are applied between the spatially separated measuring areas (d) with respect to the analyte, preferably consisting, for example, of the groups derived from albumins, in particular bovine serum albumin or human serum albumin, casein, non-specific, polyclonal or monoclonal, alien or empirically for the analyte or analytes to be detected non-specific antibodies (in particular for immunoassays), detergents - such as, for example, Tween 20 -, fragmented natural or synthetic DNA that does not hybridize with the polynucleotides to be analyzed, such as, for example, an extract of herring or salmon sperm (in particular for polynucleotide hybridization assays) , or also uncharged, but hydrophilic polymers, such as polyethylene glycols or dextrans, are formed.
  • albumins in particular bovine serum albumin or human serum albumin, casein, non-specific, polyclonal or monoclonal, alien or empirically
  • kits according to the invention are advantageous for many, if not the majority, of applications in which an adhesion promoter layer was applied to the sensor platform before the immobilization of the biological or biochemical or synthetic recognition elements.
  • Preferred embodiments are those which are characterized in that the function of passivating areas between the spatially separated measurement areas for minimizing non-specific binding of analytes or their detection substances is performed by applying said adhesion-promoting layer on the sensor platform without applying additional substances.
  • the kit according to the invention can comprise a very large number of individual measuring ranges. It is preferred that up to 100,000 measuring ranges are arranged in a 2-dimensional arrangement and that a single measuring range occupies an area of 0.001-6 mm 2 . Preferably, more than 100, more preferably more than 1000, even more preferably more than 10,000 measuring ranges are arranged on a sensor platform as part of the kit according to the invention.
  • Another object of the invention is an embodiment of the kit according to the invention, in which the upper side of the sensor platform with the measurement areas generated thereon is brought together with another body above the optically transparent layer (a) in such a way that one or more are provided between the sensor platform as the base plate and said body spatial cutouts for generating one or more sample containers fluidly sealed against one another are produced, in each of which there are one or more measuring areas or segments or arrays of measuring areas.
  • sample containers are designed as flow cells that are fluidically sealed from one another, each having at least one inlet and at least one outlet, and optionally additionally leading at least one outlet of each flow cell into a reservoir that is fluidly connected to this flow cell and that emerges from the flow cell Absorbs liquid.
  • the optionally additionally available reservoir for receiving liquid emerging from the flow cell is designed as a recess in the outer wall of the body brought together with the sensor platform as the base plate.
  • spatial structures are formed on the sensor platform as the base plate in the grid of the array of the flow cells to be generated. These structures on the base plate can form, for example, the walls or parts of the walls, such as plinths, between the flow cells arranged next to and behind one another, which are produced by bringing the base plate together with a correspondingly shaped body.
  • recesses it is also possible for recesses to be formed in the sensor platform in order to generate the spatial recesses between the sensor platform as the base plate and the body brought together with it.
  • a further embodiment consists in that recesses are formed in said body in order to produce the recesses between the base plate and the body brought together therewith.
  • the base plate is essentially planar.
  • the body to be brought together with the base plate for producing the array of flow cells can consist of a single workpiece.
  • the body brought together with the base plate is composed of several parts, the assembled components of said body preferably forming an irreversibly joined unit.
  • the body brought together with the base plate comprises auxiliary measures which facilitate the joining together of said body and the base plate.
  • the arrangement preferably comprises a multiplicity, ie 2 to 2000 sample containers, preferably 2 to 400, particularly preferably 2 to 100 sample containers.
  • a multiplicity ie 2 to 2000 sample containers, preferably 2 to 400, particularly preferably 2 to 100 sample containers.
  • the sample containers are open on the side of the body which is brought together with the sensor platform as the base plate and is opposite the measurement areas.
  • the grid (sequence in rows and / or columns) of the sample containers corresponds to the grid of the wells of a standard microtiter plate.
  • Another embodiment of the arrangement of sample containers as part of the kit according to the invention is characterized in that it is closed by an additional closure, for example a film, membrane or cover plate.
  • the receptivity of the flow cells can be varied within a wide range, so that the internal volume of each sample container is typically 0.1 ⁇ l - 1000 ⁇ l, preferably 1 ⁇ l - 20 ⁇ l.
  • the internal volumes of different flow cells of an arrangement can be the same or different.
  • the depth of the recesses between the sensor platform as the base plate and the body joined therewith is 1 to 1000 ⁇ m, particularly preferably 20 to 200 ⁇ m.
  • the size of the recesses in an array can be uniform or different, and the size surfaces can have any, preferably rectangular or polygonal or other geometry.
  • the lateral dimensions of the base areas can be varied within a wide range, typically the base areas of the recesses between the base plate and the body joined therewith each being 0.1 mm 2 - 200 mm 2 , preferably 1 mm 2 - 100 mm 2 .
  • the corners of the bases are rounded. Rounded corners have a favorable effect on the flow profile and facilitate the removal of any gas bubbles that may have formed from the flow cells or prevent their formation.
  • multi-channel pipettors can be used for manual or automatic reagent application, in which the individual pipettes are arranged in one- or two-dimensional arrays, provided the arrangement of sample containers is as Part of the kit according to the invention which has inlets in the corresponding grid.
  • the grid (sequence in rows and columns) of the arrangement therefore preferably corresponds to the grid of the wells of standard microtiter plates.
  • An arrangement of 8 x 12 wells with a (centram-to-centram) spacing of approx. 9 mm has been established as an industrial standard. Smaller arrays with 3, 6, 12, 24 and 48 wells are equally compatible with this. It is also possible to combine several arrangements of sample containers according to the invention with such smaller arrays of flow cells in such a way that the individual inlets of said flow cells are arranged in an integral multiple of the distance of approximately 9 mm.
  • plates with 384 and 1536 wells, an integral multiple of 96 wells on the same footprint with a correspondingly reduced well spacing, have also been used, which are also to be referred to as standard microtiter plates.
  • the outer basic dimensions of the arrangement of sample containers, as part of the kit according to the invention preferably correspond to the grand dimensions of these standard microtiter plates.
  • a further special embodiment of the invention is an arrangement of, for example, 2 to 8 sample containers as part of the kit according to the invention, with the properties mentioned above, in one column or, for example, 2 to 12 sample containers in a row, which on the other side is supported by a carrier ("meta carrier") are joined together with the dimensions of standard microtiter plates in such a way that the grid (sequence in rows or columns) of the inflows of the sample containers corresponds to the grid of the wells of a standard microtiter plate.
  • a carrier metal carrier
  • the grid (sequence in rows or columns) of the inflows of the flow cells corresponds to the grid of the wells of a standard microtiter plate, i.e. an integral multiple of 9 mm (corresponding to 96 - Corrugated plate) or of 4.5 mm (corresponding to 384-well plate, see above) or of 2.25 mm (corresponding to 1536-well plate, see above).
  • sample containers can of course also be designed in a different grid.
  • the materials for the body brought together with the sensor platform as the base plate and any additional cover plate that may be used must meet the requirements for the planned use of the arrangement. Depending on the specific application, these requirements relate to chemical and physical resistance, for example against acidic or basic media, salts, alcohols or detergents as components of aqueous solutions, or formamide, temperature resistance (for example between -30 ° C and 100 ° C) , possible similar thermal expansion coefficients of the base plate and the body brought together, optical properties (eg freedom from fluorescence, reflectivity), machinability etc. It is preferred that the material of the body brought together with the base plate and an optional additional closure is selected from the same category as the material of the "meta carrier".
  • the components mentioned (body combined with the sensor platform as the base plate, cover plate) can each consist of a uniform material and also comprise a mixture or layer-by-layer or lateral connection of different materials, the materials being able to replace one another.
  • An essential aspect of the present invention is the possibility of spatially resolved referencing of the available excitation light intensity.
  • the available excitation light intensities of an illuminated area are essentially determined by the excitation light density in the cross section of the excitation light bundle.
  • Local variations in the properties of the illuminated area (such as a glass plate) have only a secondary influence here.
  • there are local variations in the physical parameters of the sensor platform such as the coupling efficiency of the grating structure (c) for coupling the excitation light into the optically transparent layer (a), or local variations in the propagation losses of a guided mode in the optically transparent Layer (a), vital.
  • kits according to the invention which are characterized in that the precautions for spatially resolved referencing of the excitation light intensity available in the measuring ranges include the simultaneous or sequential generation of an image of the light emitted by the sensor platform at the excitation wavelength. It is assumed that the scattered light losses are essentially proportional to the locally guided light intensity. The scattered light losses are mainly determined by the surface roughness and homogeneity of the optically transparent layer (a) and the substrate located below (optically transparent layer (b)).
  • this type of referencing enables a reduction in the locally available excitation light intensity in its direction of propagation to be taken into account if this was done, for example, by absorption of excitation light by a high local concentration in the evanescent field of layer (a) of molecules that absorb at the excitation wavelength.
  • the precautions for spatially resolved referencing of the excitation light intensity available in the measurement areas include the simultaneous or sequential generation of an image of the light emitted by the sensor platform at the luminescence wavelength. Both methods can of course also be combined with one another. When creating a reference image, different influences of the imaging optics on the acquisition of the measurement signals should be excluded. It is therefore preferred that the image of the excitation light emitted by the sensor platform is created via the same optical path as the detection of the luminescence originating from the measurement areas.
  • the precautions for spatially resolved referencing of the excitation light intensity available in the measurement areas include the simultaneous or sequential generation of an image of the light emitted by the sensor platform at an excitation wavelength other than for excitation of a luminescence. It is further preferred that the spatial resolution of the image for referencing the excitation light emitted by the sensor platform on the sensor platform is better than 100 ⁇ m, preferably better than 20 ⁇ m. Provided that such a spatial resolution is provided, it is further preferred that the precautions for spatially resolved referencing of the excitation light intensity available in the measurement areas include determining the background signal at the respective luminescence wavelength next to or between the measurement areas.
  • kits according to the invention are characterized in that the spatially resolved referencing of the excitation light intensity available in the measurement areas by means of "luminescence marker spots", i.e.
  • the luminescence intensity is determined from measurement areas with pre-immobilized (i.e., already applied in these measurement areas before adding a sample) luminescence-labeled molecules. It is preferred that the “luminescence marker spots” are applied in a grid that spans the entire sensor platform.
  • position-resolving detectors such as CCD cameras
  • CCD cameras are preferably used for the signal detection.
  • These are characterized by the fact that their photosensitive elements (pixels) have a specific (especially thermal) background signal, which determines the lower threshold of the detection of a local light signal, and also have a maximum capacity (saturation) for the detection of high light intensities.
  • the difference between these threshold values determines the dynamic range of the signal detection for a given exposure time.
  • Both the luminescence signals to be detected for analyte detection and the reference signals should move within this dynamic range. It is advantageous if both signals are of the same order of magnitude, ie differ by no more than one or two powers of ten, for example.
  • this can be achieved, for example, by selecting the density of the luminescence-labeled molecules within a "luminescence marker spot" by means of a mixture with similar, unlabeled molecules during immobilization in such a way that the luminescence intensity from the areas of the luminescence marker spots is of a similar order of magnitude as the luminescence intensity of the from the measuring ranges provided for an analyte detection.
  • the density and concentration of the luminescence-labeled molecules within the "luminescence marker spots" within an array should preferably be uniform across the entire sensor platform.
  • its spatial resolution is essentially determined by the density of the "luminescent marker spots" within an array or on the entire sensor platform.
  • the distance and / or the size of different "luminescence marker spots” are preferably matched to the desired spatial resolution when determining the luminescence intensities from the discrete measurement areas.
  • each array on the sensor platform include at least one "luminescent marker spot”. It is advantageous if there is at least one adjacent “luminescence marker spot” for each segment of measurement areas for determining an analyte. It is particularly advantageous if each measurement area is surrounded by “luminescence marker spots”.
  • each array comprises a continuous row and / or column of “luminescence marker spots” parallel and / or perpendicular to the direction of propagation of the coupled excitation light, for determining the two-dimensional distribution of the coupled excitation light in the area of said array.
  • the precautionary measures for spatially resolved referencing of the excitation light intensity available in the measuring ranges comprise averaging over several spatially resolved reference signals.
  • excitation light intensities that do not vary statistically, but systematically, in the form of a gradient that is present over certain distances it can be advantageous if the expected value of the excitation light intensity of a measurement range lying between different areas for spatially resolved referencing range is interpolated.
  • kits according to the invention relate to precautions to calibrate luminescence signals recorded in the presence of one or more analytes.
  • said precautions for calibration of luminescences generated as a result of the binding of one or more analytes or as a result of the specific interaction with one or more analytes include the addition of calibration solutions with known concentrations of the analytes to be detected to a predetermined number of arrays. For example, it is possible that 8-12 arrays of a sensor platform are provided for calibration purposes.
  • the kit according to the invention enables a further possibility of calibration that has not been described previously. This consists in the fact that it is essentially not necessary to put a large number of calibration solutions with different, known concentrations on one or more arrays, but in the measuring ranges provided for calibration purposes, the biological or biochemical or synthetic recognition elements used for analyte detection in known, but to immobilize different local concentrations.
  • a calibration curve can be generated by adding different calibration solutions of different analyte concentrations to an array with detection elements in a single constant immobilization density
  • the invention therefore furthermore relates to a kit which is characterized in that in one or more arrays there are in each case a plurality of measurement areas with biological or biochemical or synthetic recognition elements immobilized there in a different, controlled density for the detection of an analyte common to these measurement areas. It is particularly preferred that with known concentration dependence of the binding signals between an analyte and its biological or biochemical or synthetic recognition elements and a sufficiently large "variation" of these in different controlled densities in different Measuring areas of an array of immobilized detection elements can be created by adding a single calibration solution to this array, a calibration curve for this analyte.
  • Another object of the invention is the use of a kit according to one of the abovementioned embodiments in an analytical system for determining one or more luminescences.
  • Another object of the invention is an analytical system with any embodiment of the kit according to the invention, characterized in that it additionally comprises at least one detector for detecting one or more luminescences.
  • Another object of the invention is an analytical system for determining one or more luminescence, with
  • At least one detector for detecting the light emanating from one or more measuring ranges (d) on the sensor platform.
  • a possible embodiment of the analytical system is characterized in that the excitation light is irradiated to the measurement areas in an incident light or transmission light arrangement.
  • a preferred embodiment of the analytical system according to the invention is characterized in that the excitation light emitted by the at least one excitation light source is essentially parallel and at the resonance angle for coupling into the optically transparent layer (a) onto a grating structure (c) modulated in layer (a) ) is irradiated.
  • the excitation light is expanded by at least one light source with an expansion optic to form an essentially parallel beam and at the resonance angle for coupling into the optically transparent one Layer (a) is irradiated onto a large-area lattice structure (c) modulated in layer (a).
  • the detection of the luminescent light takes place in such a way that the luminescent light coupled out from a lattice structure (c) or (c ') is also detected by the detector.
  • the analytical system according to the invention additionally comprises feed access means in order to bring the one or more samples into contact with the measurement areas on the sensor platform.
  • Another object of the invention is a method for the simultaneous qualitative and / or quantitative detection of a large number of analytes with a kit according to the invention according to one of the aforementioned embodiments and / or using an analytical system according to the invention according to one of the aforementioned embodiments, characterized in that for the purposes of "referencing the immobilization density", ie for the spatially resolved determination of the density of the immobilized biological or biochemical or synthetic recognition elements in the measurement areas, these recognition elements are each associated with a signaling component as a label and / or said recognition elements have a specific molecular sequence or a specific one have a molecular epitope or a specific molecular recognition group, followed by a detection reagent (referencing reagent), optionally with a associated signaling component as a label, for determining said density of immobilized recognition elements, and that the signals of said signaling components are recorded in a spatially resolved manner.
  • referencing the immobilization density ie for the spatially resolved determination of the density of the im
  • the determination of the immobilization density of the biological or biochemical or synthetic recognition elements on the sensor platform and the detection of said plurality of analytes can be carried out independently of one another.
  • the determination of the immobilization density of the biological or biochemical or synthetic recognition elements on the sensor platform can be part of the quality control during or after the production of said sensor platform.
  • a preferred embodiment of the method according to the invention is characterized in that the immobilized recognition elements in the measurement areas each have a general molecular sequence or a general epitope or a general molecular recognition group for the purpose of referencing the immobilization density and one for recognizing and / or binding different analytes Sequence or different epitope or different molecular recognition group.
  • Said general molecular sequence or said general epitope or said general molecular recognition group can be used for the purposes of referencing the immobilization density and a different sequence or different epitope or different molecular recognition group for the recognition and / or binding of different analytes can occur in a recognition element.
  • an analyte to be detected it is preferred that they are sufficiently far apart from one another within a recognition element so that there is no impediment to the access of an analyte to the sequence specific for its recognition or the epitope or molecular recognition group specific for its recognition of the immobilized recognition element comes.
  • the general and the specific recognition section (conceptually comprising recognition sequence and epitope) of an immobilized recognition element can be separated from one another by a so-called molecular spacer (for example comprising a chain molecule with hydrocarbon groups).
  • sections with a general nucleic acid sequence can also be used for the purposes of "referencing the immobilization density", for example in a hybridization step with fluorescence-labeled oligonucleotides complementary to this general sequence, and with the general one Nucleic acid sequence include chemically linked antibodies or antibody fragments with different recognition epitopes, each of which is specific for different analytes.
  • Nucleic acid sequence include chemically linked antibodies or antibody fragments with different recognition epitopes, each of which is specific for different analytes.
  • a possible cross-reactivity between the (specific) binding of an analyte to be detected to the specific recognition section intended for it and a possible (non-specific) binding to the general recognition section of an analyte should ideally be zero, ideally zero.
  • the said general recognition sections are preferably to be selected such that the occurrence of a binding partner specific for this general recognition section in a sample to be supplied with analytes to be detected therein can be largely ruled out, provided that this binding partner does not additionally Sample is added.
  • the referencing rank of the immobilizing density is a referencing reagent for recognition and / or binding to said general sequence or to said general epitope or to said general molecular recognition group of those immobilized in the same measuring range biological or biochemical or synthetic recognition elements is co-immobilized on the sensor platform, possibly associated with said immobilized recognition elements.
  • a referencing reagent for recognition and / or binding to said general sequence or to said general epitope or to the general molecular recognition group of the immobilized biological or biochemical or synthetic recognition elements on the sensor platform after immobilization of the biological or biochemical or synthetic recognition elements is applied to the measuring areas of the sensor platform.
  • Said "referencing of the immobilization density”, ie the spatially resolved determination of the density of the immobilized recognition elements in the measurement areas can be part of a quality control during or after the manufacture of a sensor platform, as part of a method according to the invention.
  • a referencing reagent for recognition and / or binding to said general sequence or to said general epitope of the immobilized biological or biochemical or synthetic recognition elements on the sensor platform in the course of a detection method for the determination one or more analytes is applied to the measuring areas of the sensor platform.
  • Said general molecular sequence or said general epitope or said general molecular recognition group (such as, for example, biotin) of the immobilized biological or biochemical or synthetic recognition elements can be selected, for example, from the group of polynucleotides, polynucleotides with artificial bases, PNAs (“peptide nucleic acids”) ), PNA's with artificial bases, proteins, antibodies, peptides, oligosaccharides, lectins, etc. is formed.
  • a preferred embodiment is characterized in that said general sequence of the immobilized biological or biochemical or synthetic recognition elements has a length of 5 to 500, preferably 10 to 100 bases
  • Another preferred embodiment of the method according to the invention is characterized in that the immobilized detection elements in the measurement areas are each associated with a signal-emitting component as a label. It can also be advantageous if said signaling component changes its signaling properties as a label when an analyte is bound to the respective detection element associated with it.
  • a characteristic common to the various embodiments of a method according to the invention mentioned is that said different sequences or different epitopes of the immobilized biological or biochemical or synthetic recognition elements are selected from the Grappe, which are derived from nucleic acids (for example DNA, RNA, oligonucleotides) and nucleic acid analogs (e.g. .
  • PNA PNA
  • PNA protein-binding protein
  • enzymes aptamers
  • synthetic peptide structures glycopeptides, glycoproteins, oligosaccharides, lectins, soluble, membrane-bound proteins isolated from a membrane, such as receptors, their ligands, Antigens for Antibodies, "histidine tag components" and their complex formation partners, cavities generated by chemical synthesis for the absorption of molecular imprints, etc. are formed. It is also provided that whole cells, cell components, cell membranes or their fragments are applied as biological or biochemical or synthetic recognition elements.
  • a referencing reagent required for certain embodiments of the method according to the invention comprises a label which is selected from the grappa, which is used, for example, by luminescence labels, in particular luminescent intercalators or “molecular beacons”, absorption labels, mass labels, in particular metal colloids or plastic beads, spin- Labels, such as ESR or NMR labels, radioactive labels are formed.
  • luminescence labels in particular luminescent intercalators or “molecular beacons”
  • absorption labels mass labels, in particular metal colloids or plastic beads
  • spin- Labels such as ESR or NMR labels
  • said referencing reagent comprises a luminescence label or absorption label.
  • said referencing reagent can also comprise an intercalator or a "molecular beacon". It is preferred that said intercalator or "molecular beacon" changes its signaling properties in the presence of the referencing reagent.
  • Said referencing reagent can be split off before or in the course of an analytical detection method or can remain associated with the recognition elements.
  • a further advantageous embodiment of the method according to the invention is characterized in that said referencing reagent comprises a component from the Grappe, of polynucleotides, polynucleotides with artificial bases, PNAs (“peptide nucleic acids”), PNAs with artificial bases, proteins, antibodies, biotin, streptavidin , Peptides, oligosaccharides, lectins, etc. is formed
  • the quantitative and / or qualitative detection of said multitude of analytes comprises the use of one or more signaling components as labels, which can be selected from the grapple, for example luminescent labels, in particular luminescent ones Intercalators or "molecular beacons", absorption labels, mass labels, in particular metal colloids or plastic beads, spin labels, such as ESR or NMR labels, radioactive labels are formed.
  • labels which can be selected from the grapple, for example luminescent labels, in particular luminescent ones Intercalators or "molecular beacons", absorption labels, mass labels, in particular metal colloids or plastic beads, spin labels, such as ESR or NMR labels, radioactive labels are formed.
  • the label of the referencing reagent and / or an analyte detection possibly based on absorption and / or luminescence detection is based on the use of labels with the same or different absorption and / or luminescence wavelength.
  • a special embodiment based on recognition elements immobilized in the measurement areas, each with an associated signal-emitting component as a label, is characterized in that said label also serves for an analyte detection in addition to referencing the immobilization density of the recognition elements.
  • said label can be a fluorescent intercalator, which, when bound to a single-stranded nucleic acid as the immobilized recognition element, delivers a signal that is very weak but still measurable, from which the density of the recognition elements immobilized in the corresponding measuring ranges can be determined.
  • the analyte detection be based on determining the change in one or more luminescences.
  • a possible embodiment is characterized in that the excitation light is irradiated by one or more light sources for generating signals of signal-generating components for the purpose of referencing the immobilization density and / or for detecting one or more analytes in an incident light arrangement.
  • AusNewangsformen are characterized in that the excitation light from one or more light sources for generating signals of signal-generating components for the purpose of Referenzierang the Immobilisierangs Why and / or for the detection of one or more analytes is irradiated in a transmitted light arrangement.
  • a preferred object of the invention is an embodiment of the method according to the invention, which is characterized in that the sensor platform is designed as an optical waveguide, which is preferably essentially planar, and in that the excitation light is coupled into the optical waveguide by means of one or more light sources a method which is selected from the group consisting of end face coupling, coupling via attached optical fibers as light guides, prism coupling, grating coupling or evanescent coupling by overlapping the evanescent field of said optical waveguide with the evanescent field of a further waveguide which is brought into near-field contact ,
  • the excitation light from one or more light sources is coupled into the optical waveguide by means of an optical coupling element that is in contact therewith, which is selected from the group of optical fibers as light guides, prisms, optionally via a refractive index-adjusting liquid, and grating couplers ,
  • Such an embodiment of the method according to the invention is particularly preferred, which is characterized in that the sensor platform has an optical thin-film waveguide with a layer (a) transparent at at least one excitation wavelength on a layer (b) which is also transparent at at least this excitation wavelength and has a lower refractive index than layer ( a) and that the excitation light from one or more light sources is coupled into the layer (a) by means of one or more grating structures (c) modulated in the layer (a).
  • This embodiment of the method can be carried out in such a way that one or more liquid samples to be examined for said analytes are brought into contact with the measurement areas on the sensor platform, one or more luminescences generated in the near field of layer (a) from those with said sample or said Samples brought into contact with measurement areas, as a result of the binding of one or more analytes to the biological or biochemical or synthetic recognition elements immobilized in said measurement areas or the interaction between said analytes and said immobilized recognition elements, and if necessary additionally in a spatially resolved manner those available in said measurement areas Excitation light intensity is referenced. It is preferred that (1) the isotropically emitted luminescence or (2) the luminescence or luminescence of both components (1) and (2) coupled into the optically transparent layer (a) and coupled out via lattice structures (c) are measured simultaneously.
  • a luminescent dye or luminescent nanoparticle is used as the luminescent label to generate the luminescence, which can be excited and emitted at a wavelength between 300 nm and 1100 nm.
  • the luminescence label be bound to the analyte or in a competitive assay to an analog of the analyte or in a multi-stage assay to one of the binding partners of the immobilized biological or biochemical or synthetic recognition elements or to the biological or biochemical or synthetic recognition elements.
  • Another embodiment of the method is characterized in that a second or even further luminescence label with the same or different excitation wavelength as the first luminescence label and the same or different emission wavelength is used.
  • the second or even more luminescent label can be excited at the same wavelength as the first luminescent dye, but emit at other wavelengths.
  • a variant of the method consists in that charge or optical energy transfer from a first luminescent dye serving as donor to a second luminescent dye serving as acceptor is used to detect the analyte.
  • Another possible embodiment of the method is that the extent of the quenching of one or more luminescences is determined. Another embodiment of the method is characterized in that, in addition to the determination of one or more luminescences, changes in the effective refractive index on the measurement areas are determined.
  • a further development of the method is characterized in that the one or more luminescences and / or determinations of light signals are carried out polarization-selectively at the excitation wavelength.
  • the one or more luminescences are measured with a different polarization than that of the excitation light.
  • a preferred embodiment of the method according to the invention is characterized in that the density of the detection elements immobilized in discrete measurement areas for the detection of different analytes on different measurement areas is selected such that the luminescence signals when detecting different analytes in a common array are of the same order of magnitude, that is to say that the associated calibration curves for the analyte determinations to be carried out simultaneously without changing the electronic or optoelectronic system settings.
  • a further development of the method is characterized in that arrays of measuring ranges are divided into segments of one or more measuring ranges for the determination of analytes and ranges between these measuring ranges or additional measuring ranges for purposes of physical referencing, such as the excitation light intensity or available in the measuring ranges the influence of changes in external parameters, such as temperature, and for the purpose of referencing the influence of additional physico-chemical parameters, such as non-specific binding to the sensor platform of components of an applied sample.
  • Non-specific binding components of an applied sample can be, for example, the one or more analytes themselves, detection reagents added to the sample for analyte detection, for example secondary, luminescence-labeled antibodies in a sandwich immunoassay, or components of the sample matrix, especially if the sample medium is, for example is a body fluid and the sample has not been subjected to any further cleaning steps.
  • the areas provided for this purpose can, for example, have been “passivated” on a sensor platform, ie coated with a compound “chemically neutral” with respect to the analyte, as described above as a measure to reduce non-specific binding.
  • Such an embodiment of the method according to the invention is advantageous for such applications, in which one or more measuring ranges of a segment or an array are assigned to the determination of the same analyte and whose immobilized biological or biochemical recognition elements have different affinities for said analyte.
  • the recognition elements are expediently selected such that their affinities for different, (bio) chemically similar analytes change in different, characteristic ways.
  • the identity of the analyte can then be determined from the entirety of the signals from different measurement areas with different detection elements for a single analyte, in a manner comparable to a fingerprint.
  • two or more arrays have a similar geometric arrangement of measuring ranges and / or segments of measuring ranges for the determination have similar analytes on these arrays.
  • one or more arrays comprise segments of two or more measurement areas with biological or biochemical or synthetic recognition elements of the same type within the segment for the determination of analyte or reference.
  • the method according to the invention with a kit according to the invention with a large number of measuring ranges in discrete arrays, of which in turn a large number can be arranged on a common sensor platform offers the possibility of using one and the same using relatively small amounts of sample solutions, reagents or calibration solutions Platform, under largely identical conditions, many types of duplications or multiple executions of similar measurements can be carried out. In this way, for example, statistical data can be generated in a single measurement, for which a large number of individual measurements with a correspondingly longer overall measurement time and a higher consumption of sample and reagent quantities are conventionally required. It is preferred that two or more identical measuring ranges are provided within a segment or array for the detection of each analyte or for the reference range.
  • said identical measurement areas can be arranged in a continuous row or column or diagonals of an array or segment of measurement areas.
  • the aspects of referencing can relate to physical or chemical parameters of the sensor platform, such as local differences in the excitation light intensity (see also below), as well as influences of the sample, such as its pH, ionic strength, refractive index, temperature etc.
  • said identical measuring ranges are arranged statistically within an array or segment of measuring ranges.
  • a further essential aspect of the present invention is that additional precautionary measures for the locally resolved referencing range of the excitation light intensity available in the measuring ranges are taken.
  • Excitation light intensity includes the simultaneous or sequential creation of an image of the light emitted by the sensor platform at the excitation wavelength. It is preferred that the image of the excitation light emitted by the sensor platform is created via the same optical path as the detection of the luminescence originating from the measurement areas.
  • the spatially resolved referencing range of the excitation light intensity available in the measurement areas includes the simultaneous or sequential generation of an image of the light emitted by the sensor platform at the luminescence wavelength.
  • a further embodiment is characterized in that the precautions for spatially resolved referencing of the excitation light intensity available in the measurement areas comprise the simultaneous or sequential creation of an image of the light emitted by the sensor platform at an excitation wavelength other than for excitation of a luminescence.
  • the spatial resolution of the image of the excitation light emitted by the sensor platform on the sensor platform is better than 100 ⁇ m, preferably better than 20 ⁇ m.
  • Another object of the method according to the invention is that the spatially resolved referencing range of the excitation light intensity available in the measuring ranges is achieved by means of "luminescence marker spots", i.e.
  • the luminescence intensity is determined from measurement areas with pre-immobilized (i.e., already applied in these measurement areas before adding a sample) luminescence-labeled molecules.
  • the “luminescence marker spots” are applied in a grid that spans the entire sensor platform.
  • a further development of the method according to the invention is that the density of the luminescence-labeled molecules is selected by means of a mixture with similar, unlabeled molecules during immobilization in such a way that the luminescence intensity the areas of the luminescence marker spots are of a similar order of magnitude as the luminescence intensity of the measurement areas provided for an analyte detection.
  • a preferred embodiment of the method is characterized in that the density and concentration of the luminescence-labeled molecules within the "luminescence marker spots" are uniform within an array, preferably on the entire sensor platform.
  • the spatially resolved referencing range of the excitation light intensity available in the measurement areas comprises averaging over a plurality of spatially resolved reference signals.
  • excitation light intensities that do not vary statistically, but systematically, in the form of a gradient that is present over certain distances, it can be advantageous if the expected value of the excitation light intensity of a measurement range lying between different areas for spatially resolved referencing range is interpolated.
  • the one or more samples and the detection reagents to be used in the detection method can be added sequentially in several steps. It is preferred that the one or more samples are pre-incubated with a mixture of the various detection reagents for determining the analytes to be detected in said samples and these mixtures are then fed to the arrays provided on the sensor platform in a single addition step.
  • a preferred embodiment of the method according to the invention is characterized in that the concentration of the detection reagents, such as, for example, secondary detection antibodies and / or luminescence labels and optionally additional luminescence-labeled detection reagents in a sandwich immunoassay, is selected such that the luminescence signals when detecting different analytes in a common one Arrays are of the same order of magnitude, which means that the associated calibration curves for the analyte determinations to be carried out simultaneously can be recorded without changing the optoelectronic system settings.
  • the concentration of the detection reagents such as, for example, secondary detection antibodies and / or luminescence labels and optionally additional luminescence-labeled detection reagents in a sandwich immunoassay
  • Another object of an embodiment of the method according to the invention is that the calibration of due to the binding of one or more analytes or due to the specific interaction with one or more analytes generated luminescence includes the addition of one or more calibration solutions with known concentrations of said analytes to be determined to the same or different measuring ranges or segments of measuring ranges or arrays of measuring ranges on a sensor platform, to which in the same or a separate addition step one or more samples to be examined are supplied.
  • Another preferred embodiment of the method is characterized in that the calibration of luminescence generated as a result of the binding of one or more analytes or as a result of the specific interaction with one or more analytes, the comparison of the luminescence intensities after the addition of an unknown and a control sample, such as, for example "wild type” DNA sample and a "mutant DNA” sample. It is possible that the unknown sample and the control sample are added to different arrays.
  • Another variant of this method is characterized in that the unknown sample and the control sample are added sequentially to the same array.
  • a regeneration step is generally necessary between the addition of the unknown sample and the control sample, i.e. the dissociation of recognition element-analyte complexes formed after the addition of the first sample, followed by the removal of the dissociated analyte molecules from the sample containers before the addition of the second sample can take place.
  • several samples on an array of measurement areas can be examined for their analytes in sequential form.
  • Another possible embodiment of the method is that the unknown sample and the control sample are mixed and then the mixture is fed to one or more arrays of a sensor platform.
  • a further development of the method according to the invention is characterized in that the analytes to be detected in the unknown and the control sample are detected by means of luminescence labels of different excitation and / or luminescence wavelength for the unknown and the control sample.
  • the detection is carried out using two or more luminescence labels with different excitation and / or luminescence wavelengths.
  • the kit according to the invention with the large number of measuring ranges on a sensor platform opens up the possibility of a simplified form of calibration for the qualitative and / or quantitative determination of one or more analytes on one or more arrays.
  • this new form of calibration of the signals from a sensor platform according to the invention the addition of only a single calibration solution is required.
  • Part of the invention is a method according to one of the abovementioned embodiments for the simultaneous or sequential, quantitative or qualitative determination of one or more analytes from the Grappe of antibodies or antigens, receptors or ligands, chelators or "histidine tag components", oligonucleotides, DNA or RNA strands, DNA or RNA analogs, enzymes, enzyme cofactors or inhibitors, lectins and carbohydrates.
  • Possible embodiments of the method are also characterized in that the samples to be examined naturally occurring body fluids such as blood, serum, plasma, lymph or urine or egg yolk or optically cloudy liquids or tissue fluids or surface water or soil or plant extracts or bio or Synthesis process broths or from biological tissue parts or from cell cultures or extracts.
  • body fluids such as blood, serum, plasma, lymph or urine or egg yolk or optically cloudy liquids or tissue fluids or surface water or soil or plant extracts or bio or Synthesis process broths or from biological tissue parts or from cell cultures or extracts.
  • the invention furthermore relates to the use of a kit according to the invention and / or an analytical system according to the invention and / or a method according to the invention for quantitative or qualitative analyzes for determining chemical, biochemical or biological analytes in screening processes in pharmaceutical research, combinatorial chemistry, clinical and preclinical Development, real-time binding studies and the determination of kinetic parameters in affinity screening and research, qualitative and quantitative analyte determinations, in particular for DNA and RNA analysis, for the preparation of toxicity studies and for the determination of gene or protein expression profiles as well as for the detection of antibodies, antigens, pathogens or bacteria in pharmaceutical product development and research, human and veterinary diagnostics, agrochemical product development and research, symptomatic and presymptomatic plant diagnostics, for patient stratification in pharmaceutical product development and for therapeutic drug selection, for the detection of pathogens, pollutants and pathogens, especially of salmonella, prions, viruses and bacteria, in food and environmental analysis.
  • DNA fragments (inserts) of the organism to be examined are incorporated into plasmid DNA (circular DNA sequences in bacteria or other microorganisms) with the aid of restriction endonucleases and ligases in order to produce a so-called recombinant DNA.
  • both the vectors plasmids without built-in foreign DNA
  • the DNA fragments to be amplified are “cut” in a defined and suitable manner and joined together using ligases.
  • These vehicles are used in bacterial host cells, mostly Ecoli cells, for example using From all of the cells subjected to the method, those host cells which have taken up the “vehicle” are selected by means of an antibiotic resistance method and applied to a suitable solid nutrient medium and cultivated or also cultivated in a liquid nutrient medium. The “vehicles” are multiplied by the natural growth of these bacterial cultures.
  • linear DNA contracts so-called bacteriophages, viruses that are able to infect bacterial cells can be used as vehicles.
  • 2 vectors contain a gene for tetracycline resistance: if a recombinant DNA was introduced into the bacterial cell, the cell is resistant to the antibiotic.
  • the bacterial cells which contain the desired recombinant DNA are identified by means of replica plating and labeling by means of suitable radioactively labeled - complementary - nucleic acid probes.
  • the recombinant DNA is isolated and purified using established methods: lysis of the cell wall, removal of cellular fragments via centrifugation, further purification via phenol extraction, ethanol precipitation. Alternatively, commercially available DNA isolation kits can be used.
  • a cloning ranking method can also be used using so-called “T vectors” [J. Sambrock and DW Russell, “Molecular Cloning - A Laboratory Manual”, Vol. 2 (2001), section 8.35, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York].
  • each recognition element which is applied to a carrier surface bears next to the specific recognition region one or two general DNA sequences that can be used for referencing:
  • Recombinant DNA is prepared as a plasmid or bacteriophage using the procedures listed above.
  • PCR polymerase chain reaction
  • primers oligonucleotides, which serve as starting molecules for the DNA polymerase
  • the base sequences of these primers are selected such that the amplified (amplified) DNA pieces contain both the special originally introduced DNA sequence and parts of the vector sequences at the 3 'and 5' ends.
  • additional DNA sequences are typically about 10 to 40 bases long.
  • the so-called “forward primer”, the “reverse primer” or both primers contain instead of native (unmodified) nucleotides those which are derivatized on the nucleobase with fluorescent dyes such as Cy3 or Cy5
  • the labeling step is preferably carried out during the oligonucleotide synthesis by incorporating fluorescently labeled uracil or cytosine.
  • primers each having 15 to 25 base pairs are used.
  • the primers are chosen such that an extension of preferably more than 5 nucleotides plus the length of the primer on the 5th 'and at the 3' end of the original DNA built into the vector is formed.
  • the polymerization reaction itself is carried out using commercially available Taq polymerase kits.
  • fully synthetic recognition elements with comparable properties can be used.
  • those nucleobases which are themselves derivatized with fluorescent dyes those nucleobases which are derivatized with a reactive group, for example an amino group, can also be used.
  • the fluorescent dye to be used as the fluorescent label is only covalently bound to the reactive grappa of the modified nucleotides after the amplification has been completed, in an additional step, and excess fluorescent dye is subsequently separated off.
  • sample molecules are applied to the chemically activated surface of the carrier in the same way as that of unlabelled sample molecules, by means of mechanical pin or pen spotting methods or an ink jet application.
  • the fluorescence signals measured during analyte detection can then be corrected (by division by the respective reference signal) in order to obtain the relative binding signal, based on the detection elements available for each measuring range. Since the fluorescence labels used are installed covalently, no impairment of the hybridization ability (due to steric disabilities) is to be expected. When selecting the fluorescence labels for the “reference range of the immobilization density” and for the analyte detection, it is generally preferred that the excitation and emission spectra of the different luminescence labels used overlap only slightly or not at all.
  • Recombinant DNA is prepared as a plasmid or bacteriophage using the procedures listed above.
  • PCR polymerase chain reaction
  • the base sequences of these primers are selected in such a way that the amplified DNA pieces contain both the special originally introduced DNA sequence and parts of the known vector sequences at the 3 'and 5' ends. These additional DNA sequences are typically about 10 to 40 bases long. Typically, primers with 15 to 25 bases each are used. The primers are chosen so that an extension of preferably more than 5 nucleotides plus the length of the primer at the 5 'and at the 3' end of the DNA originally incorporated into the vector is produced.
  • the polymerization reaction itself is carried out using commercially available Taq polymerase kits. Alternatively, fully synthetic recognition elements with comparable properties can be used.
  • sample molecules are applied to the chemically activated surface of the carrier in the same way as that of unlabeled sample molecules by means of mechanical pin or pen spotting methods or ink jet analog application.
  • a hybridization step fluorescence-labeled oligonucleotide sequences, the sequences of which are complementary to a general DNA part defined by the vector, are applied.
  • a fluorescence intensity can be measured from each measurement area, the height of which essentially corresponds to the immobilized sample DNA amount. If necessary, the resulting hybrid can be broken up and the fluorescence introduced by the probe can be flushed out by means of thermal or ion-induced dehybridization.
  • This method is particularly suitable for the representative quality control of production lots, since - with the exception of knowledge of the sequences of the DNA regions originating from the plasmid vector - no information about the recognition part of the DNA fragment is necessary and the measurement - if all recognition DNA about the identical one Cloning rank method was produced - only one type of nucleic acid probe was required.
  • shorter polymer sequences (or oligonucleotide sequences) ( ⁇ 100 bases) can be synthesized under inexpensive framework conditions. It is possible to construct polynucleotides from two separate building blocks - a general sequence and a specific sequence suitable for the recognition of individually expressed genes.
  • the bases of the general sequence can consist of native nucleobases or partially or completely of fluorescence-labeled bases.
  • the recognition elements can be analogous to method 2.1. and 2.2. be used.
  • Example 3 Kit according to the invention with immobilized antibodies with an associated fluorescence label as signaling component for purposes of referencing the immobilization density and method according to the invention for analyte detection
  • a sensor platform with the outer dimensions of 57 mm width (parallel to the grid lines of a grid structure (c) modulated in layer (a) of the sensor platform) x 14 mm length (perpendicular to the grid structures) x 0.7 mm thickness is used on its surface by combining with a plate made of polycarbonate with recesses open in the direction of the sensor platform with the internal dimensions 5 mm width x 7 mm length x 0.15 mm height, 6 microflow cells in the grid of a partial column of a classic microtiter plate (grid 9 mm) can be generated.
  • the polycarbonate plate can be glued to the sensor platform in such a way that the recesses are then sealed against one another.
  • This polycarbonate plate is constructed in such a way that it can be combined with a carrier ("meta carrier”) with the grand dimensions of standard microtiter plates in such a way that the grid (sequence in rows or columns) of the inflows of the flow cells corresponds to the grid of the wells of a standard microtiter plate.
  • a carrier metal carrier
  • n 1.52 at 633 nm.
  • the substrate is a pair of coupling-in and coupling-out gratings with grating lines running parallel to the width of the sensor platform (318 nm period) of 12 + / - 3 nm grating depth, the grating lines being formed over the entire width of the sensor platform.
  • the distance between the two successive grids is 9 mm, the length of the individual grating structures (parallel to the length of the sensor platform) shape) 0.5 mm.
  • the distance between the coupling-in and coupling-out gratings of a pair of gratings is selected so that the coupling of the excitation light can take place within the area of the sample containers after combining the sensor platform with the polycarbonate plate described above, while the coupling-out takes place outside the area of the sample containers.
  • the wave-guiding, optically transparent layer (a) made of Ta 2 Ü 5 on the optically transparent layer (b) has a refractive index of 2.15 at 633 nm (layer thickness 150 nm).
  • sample containers formed by the sensor platform and the combined polycarbonate plate have conically drilled openings on the boundary surfaces opposite the sensor platform, so that the sample containers can be filled or emptied by pressing in standardized, commercially available pipette tips made of polypropylene.
  • the sensor platforms are first cleaned with isopropanol, then with concentrated H 2 SO 4 , 2.5% ammonium peroxodisulfate in an ultrasound device and then incubated with 0.5 mM dodecyl monophosphate (ammonium salt) for 2 hours at room temperature, whereby the solution is constantly stirred.
  • a self-assembly forms a homogeneous, hydrophobic surface.
  • Monoclonal antibodies in the specific example against the 8 interleukins IL # 1 to EL # 8, are fluorescently labeled with Cy3 using a standard method.
  • the antibody to be labeled is dissolved at a concentration of approx. 1 mg / ml in 0.1M carbonate buffer pH 9.2, so that the primary amines of, for example, lysine side chains of the protein are in a completely deprotonated state.
  • To this solution is added a portion of a Cy3-NHS ester previously dissolved in DMSO and incubated for one hour in the dark at room temperature with gentle stirring.
  • the concentration of DMSO in the total solution must not be higher than 1% in order to denature and thus the Avoid loss of function of the antibody to be labeled.
  • the portion of the dye which has not reacted with the protein is separated off chromatographically.
  • the molar ratio of antibodies and fluorescent labels (Cy3) is selected during the reaction in such a way that not every but, for example, only about every tenth antibody molecule is covalently labeled.
  • the ratio between Cy3-labeled and unlabeled antibodies can be checked using the absorption spectrum in accordance with common methods.
  • the fluorescence-labeled 8 different (primary) antibodies against interleukins IL # 1 to IL # 8 are in a concentration of 50-150 ⁇ g / ml in phosphate-buffered saline (pH 7.4) , which additionally contains suitable additives for maintaining the functionality of the immobilized proteins, applied by means of an inkjet spotter and dried.
  • the diameter of the spots, with a distance (centram to centram) of 0.35 mm, is on average 0.15 mm.
  • Eight different antibodies for the detection of cytokines, in particular different interleukins are used in 20 rows of a single array with a total of 160 spots. In order to obtain statistical assay reproducibility data from each individual measurement for each sample to be supplied, 20 measurement areas with the same liter leukin antibodies as biological recognition elements are generated for each array.
  • the polycarbonate plate described is applied to the sensor platform prepared in such a way that the individual sample containers are fluid-tightly sealed off from one another and the protein microarrays with the associated coupling grids (c) are each located within one of the 6 sample containers.
  • the format of a sandwich assay is selected for the specific detection of the cytokines to be detected.
  • cytokines Interleukins EL # 1 to D. # 8
  • each of the 6 individual calibration solutions are filled into one of the 6 flow cells on the sensor platform and incubated for a further 2 hours at 37 ° C with the respective array on the sensor platform, so that the complexes formed in the pre-incubation step from the respective interleukins are specific secondary, biotinylated anti-interleukin antibodies and Cy-5 labeled streptavidin can bind to the primary anti-interleukin antibodies immobilized in the discrete measurement areas (spots).
  • the flow cells are washed with buffer (phosphate-buffered saline with an addition of 0.1% seramalbumin and 0.05% Tween20).
  • buffer phosphate-buffered saline with an addition of 0.1% seramalbumin and 0.05% Tween20.
  • the sensor platform with the polycarbonate plate joined to it is then inserted into a “meta carrier” as described above (Example 3.1) and, after a further 15-minute incubation period (for equilibration at room temperature) in buffer, inserted and measured in an analytical system according to the invention.
  • the "referencing rank of the immobilizing density" can be carried out in such a way that after the end of the assay the detection in an inventive according to the analytical system, for example with commercial two-color scanners or also an optical system as described in PCT / EP 01/10012, and the first color can be used for the reference, the second color for the measurement of the specific assay signal.
  • This method makes it possible to determine the relative number of immobilized antibodies as immobilized recognition elements in each measuring range. Based on this measurement, the fluorescence signals measured during analyte detection can then be corrected (by division by the respective reference signal) in order to obtain the relative binding signal, based on the detection elements available for each measuring range. Since the fluorescence labels used for the referencing of the immobilization density are incorporated covalently, there is no impairment of the functionality (as a result of steric disabilities from the fluorescence label), that is, the ability of the fluorescence-labeled immobilized antibody to specifically recognize and bind the antigen. When selecting the fluorescence labels for the “reference range of the immobilization density” and for the analyte detection, it is generally preferred that the excitation and emission spectra of the different luminescence labels used overlap only slightly or not at all.

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Abstract

La présente invention concerne différents modes de réalisation d'un kit permettant la détection qualitative et/ou quantitative simultanées d'une pluralité d'analytes qui permet notamment la référenciation de la densité des éléments de reconnaissance biologiques ou biochimiques ou synthétiques immobilisés pour permettre la détection des analytes mentionnés, c'est-à-dire la densité d'occupation des zones de mesure occupées par ces éléments de reconnaissance. Cette invention concerne également des systèmes d'analyse se basant sur le kit de l'invention, ainsi que des procédés mis en oeuvre grâce audit kit pour détecter un ou plusieurs analytes, et leur utilisation.
EP01994637A 2000-11-17 2001-11-05 Kit et procede permettant la detection d'analytes multiples avec des mesures permettant la referenciation de la densite d'elements de reconnaissance immobilises Withdrawn EP1334361A2 (fr)

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