EP1190236A1

EP1190236A1 - Sensor platform and method for analysing multiple analytes

Info

Publication number: EP1190236A1
Application number: EP00940279A
Authority: EP
Inventors: Andreas P. Abel; Wolfgang Budach; Gert L. Duveneck; Markus Ehrat; Gerhard M. Kresbach; Dieter NEUSCHÄFER
Original assignee: Zeptosens AG
Current assignee: Novartis AG
Priority date: 1999-06-05
Filing date: 2000-05-29
Publication date: 2002-03-27
Also published as: WO2000075644A1; WO2000075644A9; US20020074513A1; US20040046128A1; JP2003501654A; AU5526500A

Abstract

The invention relates to a variable configuration of a sensor platform, based on a planar thin-film waveguide, for determining one or more luminescences of one or more measuring areas on said sensor platform. The configuration comprises an optical film waveguide with a first optically transparent layer (a) on a second optically transparent layer (b) with a lower index of refraction than layer (a) and at least one grating structure for coupling excitation light into the measuring areas or coupling luminescent light out of said measuring areas. The invention also relates to an optical system for determining luminescence and to an analytical system comprising a sensor platform, an inventive optical system and supply means for bringing one or more samples into contact with the measuring areas on the sensor platform. The invention also relates to methods for determining luminescence and to the use of these methods.

Description

Sensor platform and method for multi-analyte determination

The invention relates to a variable embodiment of a sensor platform based on a planar thin-film waveguide for determining one or more luminescences from one or more measurement areas on this sensor platform, comprising an optical layer waveguide with a first optically transparent layer (a) on a second optically transparent layer (b ) with a lower refractive index than layer (a) and at least one grating structure for coupling excitation light to the measuring ranges. The invention also relates to an optical system for determining luminescence with an excitation light source, an embodiment of a sensor platform according to the invention and at least one detector for detecting the light emanating from the measuring areas on the sensor platform, and an analytical system which comprises a sensor platform according to the invention, an optical system according to the invention and supply means, to bring one or more samples into contact with the measurement areas on the sensor platform. Further objects of the invention are luminescence detection methods with sensor platforms according to the invention, optical systems and analytical systems and the use of these methods for quantitative affinity sensors and for various other applications.

The objectives of the present invention are to provide sensor platforms and optical and analytical measurement arrangements for highly sensitive detection of one or more analytes.

If a light wave is coupled into a planar thin-film waveguide, which is surrounded by optically thinner media, it is guided through total reflection at the interfaces of the wave-guiding layer. In the simplest case, a planar thin-film waveguide consists of a three-layer system: carrier material, waveguiding layer, superstrate (or sample to be examined), the waveguiding layer having the highest refractive index. Additional intermediate layers can improve the effect of the planar waveguide.

A fraction of the electromagnetic energy enters the optically thinner media. This portion is known as an evanescent or cross-damped field. The strength of the evanescent field is very much dependent on the thickness of the waveguiding layer itself, and the ratio of the refractive indices of the waveguiding layer and the media surrounding it. In the case of thin waveguides, that is to say layer thicknesses of the same or a lower thickness than the wavelength to be guided, discrete modes of the guided light can be distinguished.

Various methods for coupling excitation light into a planar waveguide are known. The earliest methods used were based on face coupling or prism coupling, with a liquid generally being applied between the prism and the waveguide to reduce reflections due to air gaps. These two methods are especially in connection with waveguides of relatively large layer thickness, i. H. Particularly suitable for self-supporting waveguides, as well as with a refractive index of the waveguide of significantly less than 2. In contrast, the use of coupling gratings is a much more elegant method for coupling excitation light into very thin, highly refractive waveguiding layers.

Different methods for the detection of analytes in the evanescent field of guided light waves in optical layer waveguides can be differentiated. On the basis of the measuring principle used, a distinction can be made, for example, between fluorescence or general luminescence methods on the one hand and refractive methods on the other. In this case, methods for generating a surface plasmon resonance in a thin metal layer on a dielectric layer with a lower refractive index can be included in the group of refractive methods, provided that the resonance angle of the irradiated excitation light for generating the surface plasmon resonance serves as the basis for determining the measurement variable. The surface plasmon resonance can also be used to enhance luminescence or to improve the signal-to-background ratio in a luminescence measurement. The conditions for generating a surface plasmon resonance, as well as for combination with luminescence measurements, as well as with waveguiding structures, have been described many times in the literature, for example in US Patents US 5478755, US 5841143, US 5006716 and US 4649280.

In this application, the term “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. For example, chemiluminescence, bioluminescence, electroluminescence and in particular fluorescence and phosphorescence are included under the term "luminescence".

In the refractive measurement methods, the change in the so-called effective refractive index due to molecular adsorption or desorption on the waveguide is used to detect the analyte. This change in the effective refractive index, in the case of grating coupler sensors, is determined from the change in the coupling angle for the coupling or decoupling of light into or out of the grating coupler sensor, and in the case of interf erometric sensors from the change in the phase difference between the measurement light guided in a sensor arm and a reference arm of the interface.

The prior art for using one or more coupling gratings for coupling and / or coupling out guided waves by means of one or more coupling gratings is described, for example, in K. Tiefenthaler, W. Lukosz, "Sensitivity of grating couplers as integrated-optical chemical sensors", J Opt. Soc. At the. B6, 209 (1989); W. Lukosz, Ph.M. Nellen, Ch. Stamm, P. Weiss, "Output Grating Couplers on Planar Waveguides as Integrated, Optical Chemical Sensors", Sensors and Actuators Bl, 585 (1990), and in T. Tamir, S.T. Peng, "Analysis and Design of Grating Couplers", Appl. Phys. 14, 235-254 (1977).

The refractive methods mentioned have the advantage that they can be used without the use of additional labeling molecules, so-called molecular labels. The disadvantage of these label-free methods is, however, that the detection limits that can be achieved with them are limited to pico- to nanomolar concentration ranges due to the low selectivity of the measuring principle, depending on the molecular weight of the analyte, which is not the case for many applications of modern trace analysis, for example for diagnostic applications is sufficient.

To achieve lower detection limits, luminescence-based methods appear more suitable due to the greater selectivity of the signal generation. The luminescence excitation is based on the penetration depth of the evanescent field into the optically thinner medium, i.e. on the immediate vicinity of the wave-guiding region with a penetration depth of the order of a few hundred nanometers ins Medium limited. This principle is called evanescent luminescence excitation.

The evanescent luminescence excitation is of great interest for analysis, since the excitation is limited to the immediate vicinity of the wave-guiding layer and disruptive influences from the depth of the medium can be minimized.

Photometric instruments for determining the luminescence of biosensors under evanescent excitation conditions with planar optical waveguides are also known and are described, for example, in WO 90/06503. The waveguiding layers used there are 160 nm to 1000 nm thick, the excitation wave is coupled in without a grating coupler.

Various attempts have been made to increase the sensitivity of evanescently excited luminescence and to produce integrated optical sensors. For example, Biosensors & Bioelectronics 6 (1991), 595-607 reports on planar mono- or low-modal waveguides which are produced in a two-stage ion exchange process and in which the excitation wave is coupled in with prisms. Human immunoglobulin G / fluorescein-labeled protein A is used as the affinity system, the antibody being immobilized on the waveguide and the fluorescein-labeled protein A to be detected in phosphate buffer is added to a film of polyvinyl alcohol, with which the measurement region of the waveguide is coated.

A major disadvantage of this method is that only small differences in refractive index can be achieved between the waveguiding layer and the substrate layer, which results in a relatively low sensitivity.

The sensitivity is given as 20 nM fluorescein-labeled protein A. In order to be able to determine the slightest trace, this is still unsatisfactory and a further increase in sensitivity is therefore necessary. In addition, the reproducibility and practical feasibility of coupling light through prisms appears difficult due to the large dependence of the coupling efficiency on the quality and size of the contact surface between the prism and the waveguide. Another principle is proposed in US-A-5 081 012. The planar wave-guiding layer is 200 nm to 1000 nm thick and contains two gratings, one of which is designed as a reflection grating, so that the injected light wave has to pass through the sensor region lying between the gratings at least twice. In this way, increased sensitivity is to be achieved. A disadvantage is that the reflected radiation can lead to an undesirable increase in the background radiation intensity.

WO 91/10122 describes a thin-layer spectroscopic sensor, which is characterized in that it has a coupling-in grating and a spatially distant coupling-out grating. It is particularly suitable for absorption measurement if a highly refractive inorganic metal oxide is used as the wave-guiding layer. Various embodiments are described which are suitable for coupling in and out multichromatic light sources. The preferred thickness of the waveguiding layer is greater than 200 nm and the grating depth should be approximately 100 nm. These conditions are not suitable for luminescence measurements in affinity sensors because only a low sensitivity is obtained. This is described in Appl. Optics Vol. 29, No. 31, (1990), 4583-4589 confirmed by the data of the total efficiency at 633 nm of 0.3% and at 514 nm of 0.01% for these systems.

In another embodiment of the same sensor, a plurality of polymeric planar waveguiding layers are applied to a substrate, which can be used as a gas mixture analyzer. You make yourself the change in the effective refractive index or the change in layer thickness of the polymeric waveguide when in contact with z. B. Use solvent vapors. The wave-guiding structure is physically changed. However, such changes are completely unsuitable for luminescence measurements in affinity sensor technology, since this changes the coupling, increases the scatter and the sensitivity can decrease significantly.

Other arrangements have become known in which luminescence amplification is to take place without direct coupling of excitation light, but mediated via near-field effects by excitation of luminescent molecules at or near (ie at a distance of up to a few hundred nanometers) the surface of a waveguide. In US 4649280 a multi-layer system made of a conductive and reflective material (for Example silver) on a substrate, a dielectric optical waveguide (for example made of lithium fluoride with a refractive index of only 1.39) and a film of fluorescent molecules deposited thereon. In a further development it is additionally proposed in US 5006716 to design the conductive film in the form of a surface relief grid, the shape of which is depicted in the course of the deposition processes for producing the overall structure down to the surface. As an advantage of this modification, it is described that luminescent light coupled into the waveguiding layer can be coupled out in discrete spatial directions, corresponding to the decoupled diffraction orders and the modes guided in the waveguide, so that a larger proportion of the luminescence can be detected by a detector, if it is positioned in the direction of the coupled out luminescent light. However, an essential component of these arrangements with a wave-guiding layer of relatively low refractive index is the existence of the reflective metal layer underneath.

However, a simpler two-layer system, such as a thin-layer waveguide, seems more suitable for reproducible production. It is also desirable to use a wave-guiding film with the highest possible refractive index in order to increase the intensity of the evanescent field.

Using high-index thin-film waveguides based on a waveguiding film that is only a few hundred nanometers thin on a transparent substrate, the sensitivity has been increased significantly in recent years. For example, WO 95/33197 describes a method 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 emitted luminescence in the penetration depth of the evanescent field of luminescent substances is measured by means of suitable measuring devices such as, for example, 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. A disadvantage of all of the methods described above in the prior art, in particular in WO 95/33197 and WO 95/33198, for detecting evanescently excited luminescence is that only one sample can be analyzed in each case on the wave-guiding layer of the sensor platform formed as a homogeneous film. In order to be able to carry out further measurements on the same sensor platform, complex washing and cleaning steps are continuously necessary. This applies in particular if an analyte different from the first measurement is to be detected. In the case of an immunoassay, this generally means that the entire immobilized layer on the sensor platform must be replaced or a new sensor platform as a whole must be used.

There is therefore a need to develop a method which allows several samples to be analyzed in parallel, that is to say simultaneously or in succession without additional cleaning steps.

In WO 95/03538, for example, it is proposed to mount a plurality of sample cells over a continuous wave-guiding layer, which are formed in the form of depressions in a sample plate above the wave-guiding layer. There is a grating under each sample cell, which couples out part of the light guided in the wave-guiding layer. The detection of the analyte is based on the change in the coupling-out angle depending on the analyte concentration. These methods, which are based on the change in the refractive index, are generally significantly less sensitive than luminescence methods.

WO 94/27137, for example, proposes a device and a method for carrying out immunoassays by means of evanescently excited fluorescence. The device consists of a continuous optical waveguide with two plane-parallel surfaces and a side edge, which acts as a coupling element in connection with a lens. A large number of specific binding partners are immobilized on at least one surface. In a preferred embodiment, these specific binding partners are arranged spatially separated on the continuous waveguide. In the exemplary embodiment, they are distributed in spots over the waveguide surface.

On the basis of the disclosed embodiments, it must be assumed that the coupling-in efficiency over the side edge is lower than in the case of a grid coupling-in, because of the large layer thickness (self-supporting waveguide) the evanescent field strength and thus also the excitation efficiency is significantly lower than with monomodal waveguides of lower layer thickness. Overall, this limits the sensitivity of the arrangement.

These arrangements, in which various specific binding partners are applied to a continuous wave-guiding layer, also have the disadvantage that the excitation light excites all of the fluorophore-labeled molecules. A spatial selection of measuring points is not possible. In addition, evanescently fed-back fluorescence photons can contribute to the signal of the neighboring spot and thus lead to measurement errors.

In the integrated optics for applications in telecommunications, planar glass-based optical components are known which contain waveguides in channel form, the waveguiding channels of which are produced by exchanging individual ions on the surface with the aid of masks (Glastechnischeberichte Vol. 62, page 285, 1989 ). The result is a physically continuous layer which has a slightly increased refractive index in the channels doped with ions. The increase is usually less than 5%. The manufacture of such components is complex and expensive.

R. E. Kunz in SPIE Vol 1587 Chemical, Biochemical and Environmental Fiber Sensors III (1991), pages 98-113 describes branching and re-merging optical waveguides which are particularly suitable for integrated optical devices such as interferometers. Structures of this type are unsuitable for the evanescently excited luminescence measurement, since the elements cannot be addressed individually, and the arrangement of several branches quickly results in high intensity losses of the light wave coupled into the first branch. Since the opening angle of such branches is small, typically 3 °, the distances between the two branches of the branch are small in the case of small components or the components must be dimensioned correspondingly larger, which is generally undesirable. In addition, the fixed phase relationship of the branched waves to one another is unnecessary for luminescence measurements.

WO 99/13320 claims an optical sensor for the detection of at least two different light components. This The application mainly relates to refractive measurement methods, however fluorescence or phosphorescence methods are also used to generate the measurement signal. In WO 99/13320, which also refers to multi-analyte determinations, a number of different definitions for the generation of a plurality of “sensing pads” are used, also in the same physical area (waveguide grating structure according to the nomenclature used in WO 99/13320) of the claimed sensor, given. However, there is no indication of an arrangement of several measuring ranges, according to the definition below in our application, on a continuously modulated lattice structure in accordance with the definition of our application, which is also below. Furthermore, there is no indication of how a disturbing crosstalk of measurement light to adjacent measurement areas, in particular of luminescent light fed back into the wave-guiding layer, can be prevented with a high density of measurement areas on the sensor platform.

The solution to this problem is of central importance in order to achieve the greatest possible miniaturization of the sensor platform in order to provide the greatest possible number of different measuring ranges on a common platform.

For the simultaneous or successive execution of exclusively luminescence-based multiple measurements with essentially monomodal, planar inorganic waveguides, e.g. B. WO 96/35940, devices (arrays) are known in which at least two separate waveguiding areas are arranged on a Sensoφlatform, which are acted upon separately with excitation light. The division of the sensor platform into separate wave-guiding areas has the disadvantage, however, that the space required for discrete measurement areas, in discrete wave-guiding areas on the common sensor platform, is relatively large and therefore only achieves a relatively low density of different measurement fields (or so-called "features") can be.

There is therefore a need to increase the feature density or to reduce the required area per measuring range.

Arrays with a very high feature density are known based on simple glass or microscope plates, without additional waveguiding layers. For example, in US 5445934 (Affymax Technologies) Arrays of oligonucleotides with a density of more than 1000 features per square centimeter are described and claimed. 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. In order to limit the excitation and detection to the areas of the immobilized features and to suppress light generation in the neighboring areas, 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.

There is therefore a need for an embodiment of the sensor platform and for an optical measuring arrangement, with which a sensitivity that is as high as that achieved with sensor platforms based on thin-film waveguides can be achieved, and with which the measuring area per feature can be minimized at the same time.

It has now surprisingly been found that with a suitable choice of parameters, in particular the grating depth, of a grating structure (c ') adjoining a measurement area on a sensor platform with a waveguiding layer (a), into which excitation light was coupled via a grating structure (c), the Luminescent light fed back into the layer (a) over the shortest distances, ie completely coupled out a few hundred micrometers and thus prevented from spreading further in the waveguiding layer (a). For the purposes of the present invention, spatially separated measuring areas (d) are to be defined by the areas which biological or biochemical or synthetic recognition elements immobilized there for the recognition of one or more analytes from a liquid sample. These surfaces can have any geometry, for example the shape of points, circles, rectangles, triangles, ellipses or lines. Different measurement areas can be separated from one another by lattice structures (c) and (c ') if a disturbing crosstalk of luminescent light generated in adjacent measurement areas and fed back into layer (a) is to be prevented. she can also be located on a common, continuous lattice structure, which, depending on the coupling efficiency of the lattice, leads to a partial or complete prevention of interfering crosstalk from luminescence.

In order to detect several analytes in a sample at the same time, it can be advantageous to combine two or more spatially separated measuring areas into segments on the sensor platform. Different segments can be separated from one another by lattice structures (c) and (c ') if crosstalk of luminescent light generated in adjacent segments and layer (a) is to be prevented. In addition, different segments can be delimited from one another by an applied border, which contributes to the fluidic sealing against adjacent areas and / or to a further reduction in optical crosstalk between adjacent segments.

The present invention relates to a sensor platform for determining one or more luminescence from an array of at least two or more spatially separated measuring areas (d) or at least two or more spatially separated segments (d '), into which several measuring areas are combined, on this Platform comprising an optical layer waveguide with a first optically transparent layer (a) on a second optically transparent layer (b) with a lower refractive index than layer (a), with one or more grating structures (c) for coupling excitation light to the measurement areas (d ) at least two or more spatially separated measuring areas (d) or at least two or more spatially separated segments (d '), in which several measuring areas are combined, and the same or different biological or biochemical or synthetic recognition elements (e) immobilized on these measuring areas qualitative or quantitative detection of one or more analytes in a sample brought into contact with the measuring ranges, characterized in that crosstalk of luminescence generated in the measuring ranges or in the segments comprising several measuring ranges and fed back into the optically transparent layer (a) of the layer waveguide into adjacent measuring ranges or adjacent segments by means of those adjacent to the measuring ranges or to the segments Lattice structures (c ') with the same or different period as the lattice structures (c) for coupling out the luminescent light is prevented.

Since the luminescent light fed back into the optically transparent, wave-guiding layer (a) spreads isotropically therein, it is possible to couple both excitation light into the wave-guiding layer and to couple out the fed back luminescent light from the same via the same lattice structure. A lattice structure (c) or (c ') can therefore be used both as a coupling-in and a coupling-out grating. Furthermore, the function of the lattice structures (c) and (c ') can also be interchanged, i.e. that is, the lattice structures (c) and (c ') can be used alternately as coupling-in and / or coupling-out gratings.

In order to prevent crosstalk that is fed back and propagates isotropically in the optically transparent layer (a) between adjacent measurement areas or adjacent segments not only in the direction of propagation of the guided excitation light, but also perpendicularly to it, it is advantageous if the grating structures (c) and (c ') enclose spatially separated measuring ranges (d)) or spatially separated segments (d'), in which several measuring ranges are combined.

A circular, rectangular or polygonal arrangement of the lattice structures (c) and (c ') around the measurement areas or segments is preferred.

In the simplest embodiment, the grating structures (c) and (c ') limit a measuring range or a segment only in the direction of propagation of the coupled excitation light. Then they are advantageously aligned parallel to each other. In particular, the lattice structures (c) and (c ') can also form a common continuous lattice structure on which there are several measurement areas or segments.

Since both the excitation light and the feedback luminescent light with a suitable grating structure (c), the coupling and decoupling efficiency of which is essentially determined by the suitable choice of the grating depth, are decoupled again at the point of coupling, it is possible to have a very high density of To create measurement areas on a common grid structure. The density that can be achieved is essentially determined by the minimum size of the spots, which are associated with the immobilization of the biological or biochemical or synthetic Detection elements can be achieved. The Sensoφlattformen used can have clearances of several centimeters side length. It is therefore possible that up to 100,000 measuring ranges are arranged on a sensor platform in a 2-dimensional arrangement. A single measuring range can have an area of 0.001 - 6 mm.

Another object of the invention is therefore a sensor platform for the simultaneous determination of one or more luminescences of at least two or more spatially separated measuring areas (d) or at least two or more spatially separated segments (d '), into which several measuring areas are combined, on this platform comprising an optical layer waveguide with a first optically transparent layer (a) on a second optically transparent layer (b) with a lower refractive index than layer (a), with one in the region of the at least two or more measuring ranges or at least two or more spatially separated segments (d '), in which several measuring ranges are combined, continuously modulated grating structure (c) for coupling excitation light to the measuring ranges (d)

- at least two or more spatially separated measuring ranges (d) or at least two or more spatially separated segments (d '), in which several measuring ranges are combined, and

- Identical or different biological or biochemical or synthetic recognition elements (e) immobilized on these measurement areas for the qualitative or quantitative detection of one or more analytes in a sample brought into contact with the measurement areas, characterized in that the density of the measurement areas on the sensor platform is at least 16

Measuring ranges per square centimeter and crosstalk of luminescence generated in the measuring ranges or within a segment and fed back into the optically transparent layer (a) of the layer waveguide into adjacent measuring ranges or segments by means of coupling this luminescent light through the grating structure that is continuously modulated in the area of the relevant measuring ranges (c ) is prevented. This arrangement of the sensor platform according to the invention is additionally distinguished by the advantage that the intensity of the interfering transmission light has a minimum when the coupling angle is reached, ie it disappears almost completely, which occurs when the excitation light is irradiated from the rear of the sensor platform, that is to say through the optically transparent layer (b) in the direction of the grating structure, which minimizes excitation light not serving for luminescence excitation in an optical system. The physical conditions for the disappearance of the transmission light and the simultaneous occurrence of an extraordinary "reflection" (as the sum of the regular portion of the reflection, in accordance with the radiation laws, and the light coupled out via the lattice structure) are described, for example, in D. Rosenblatt et al., " Resonant Grating Waveguide Structures ", IEEE Journal of Quantum Electronics, Vol. 33 (1997) 2038-2059.

For applications in which the requirements for sensitivity are reduced, it can be advantageous if the excitation light is irradiated to the measurement areas not under coupling conditions, but in a simple incident light or transmission light arrangement. Even then there will be an intensification of the luminescence in the near field of the optical layer waveguide, and a high feature density without optical crosstalk of signals from neighboring measurement areas can in turn be achieved by decoupling the signals by means of the grating structure.

The invention therefore also relates to a sensor platform on this platform for the simultaneous determination of one or more luminescences of at least two or more spatially separated measurement areas (d) or at least two or more spatially separated segments (d '), into which several measurement areas are combined comprising an optical layer waveguide with a first optically transparent layer (a) on a second optically transparent layer (b) with a lower refractive index than layer (a), with one in the region of the at least two or more measuring ranges or at least two or more spatially separated segments (d '), in which several measuring ranges are combined, continuously modulated lattice structure (c) - at least two or more spatially separated measuring ranges (d) or at least two or more spatially separated segments (d '), in which several measuring ranges are combined, and

Measuring ranges per square centimeter and crosstalk of luminescence generated in the measuring ranges or within a segment and fed back into the optically transparent layer (a) of the waveguide layer into neighboring measuring ranges or segments by means of coupling this luminescent light through the grating structure that is continuously modulated in the area of the relevant measuring ranges ( c) is prevented.

In many applications, particularly in the field of biology, it is desirable to use excitation light of different wavelength and luminophores of different excitation wavelength and the same or different emission wavelength, or excitation light of the same wavelength and luminophore of different emission wavelength, for the purposes of referencing with a control substance or for calibration.

For this purpose, it is advantageous if the grating structure continuously modulated in the area of the one or more measuring areas or segments is a superimposition of 2 or more grating structures of different periodicity with parallel or non-parallel, preferably non-parallel, alignment of the grating lines, which differentiates the coupling of excitation light Wavelength is used, and in the case of two superimposed grating structures, their grating lines are preferably aligned perpendicular to one another.

The extent of the propagation losses of a mode guided in an optically wave-guiding layer (a) is determined to a large extent by the surface roughness of an underlying carrier layer and by absorption due to possibly present in this carrier layer Chromophores determine what additionally harbors the risk of excitation of undesired luminescence in this carrier layer by penetration of the evanescent field of the mode carried out in layer (a). Furthermore, thermal stresses may occur as a result of different coefficients of thermal expansion of the optically transparent layers (a) and (b). In the case of a chemically sensitive, optically transparent layer (b), provided that it consists, for example, of a transparent thermoplastic, it is desirable to prevent solvents, which could attack the layer (b), from penetrating through the optically transparent layer (a). to prevent existing micropores.

It is therefore advantageous if there is another optically transparent layer (b ') with a lower refractive index than that of layer (a) and a thickness between the optically transparent layers (a) and (b) and in contact with layer (a) from 5 nm to 10,000 nm, preferably from 10 nm to 1000 nm. The function of the intermediate layer is to reduce the surface roughness under layer (a) or to reduce the penetration of the evanescent field of light guided in layer (a) into the one or more layers below or to improve the adhesion of layer (a) the one or more layers below or the reduction of thermally induced voltages within the optical sensor platform or the chemical isolation of the optically transparent layer (a) from layers below by sealing micropores in layer (a) against layers below.

There are a variety of methods for applying the biological or biochemical or synthetic recognition elements to the optically transparent layer (a). For example, this can be done by physical adsorption or by electrostatic interaction. The orientation of the recognition elements is then generally statistical. In addition, there is a risk that if the composition of the sample containing the analyte or the reagents used in the detection method differ, some of the immobilized recognition elements will be washed away. It can therefore be advantageous if an adhesion-promoting layer (f) is applied to the optically transparent layer (a) for the immobilization of biological or biochemical or synthetic recognition elements (e). This adhesive layer should also be optically transparent. In particular, the adhesive layer should not be over the penetration depth of the evanescent field protrudes from the wave-guiding layer (a) into the medium above. Therefore, the adhesion promoting layer (f) should have a thickness of less than 200 nm, preferably less than 20 nm. For example, it can include chemical compounds from the group consisting of silanes, epoxides, "self-organized functionalized monolayers".

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

To apply the biological or biochemical or synthetic recognition elements, one or more methods from the group of methods can be used, from inkjet spotting, mechanical spotting, micro contact printing, fluidic contacting of the measurement 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 ".

Components from the group consisting of nucleic acids (DNA, RNA, ...), nucleic acid analogs (PNA ..), antibodies, aptamers, membrane-bound and isolated receptors, their ligands, antigens can be applied as biological or biochemical or synthetic recognition elements for antibodies, "histidine tag components", cavities generated by chemical synthesis to accommodate molecular imprints, etc. is formed.

The latter type of recognition elements are understood to mean cavities which are produced in a process which has been described in the literature as "molecular imprinting". To do this, mostly encapsulated in organic solution, the analyte or an analogue of the analyte, in a polymer structure. It is then called the "imprint". Then the analyte or its analogue is removed from the polymer structure with the addition of suitable reagents, so that it leaves an empty cavity there. This empty cavity can then be used as a binding site with high steric selectivity in a later detection method.

It is also possible for whole cells or cell fragments to be applied as biochemical or biological recognition elements. In many cases the detection limit of an analytical method is limited by signals of so-called non-specific binding, i.e. H. by signals which are generated by binding the analyte or other compounds used to detect the analyte, which are bound not only in the area of the immobilized biological or biochemical or synthetic recognition elements used, but also in areas of a sensor platform which are not covered by them, for example by hydrophobic adsorption or through electrostatic interactions. It is therefore advantageous if "chemically neutral" compounds to reduce non-specific binding or adsorption are applied between the spatially separated measuring areas (d) with respect to the analyte. "Chemically neutral" compounds are substances which do not themselves have any specific binding sites for the detection and binding of the analyte or an analogue of the analyte or another binding partner in a multi-stage assay and which, due to their presence, give access to the analyte or its analogue or block another binding partner to the surface of the sensor platform.

For example, substances from the groups formed by bovine serum albumin or polyethylene glycol can be used as "chemically neutral" compounds.

For many applications it is advantageous if the lattice structure (c) is a diffractive lattice with a uniform period. The resonance angle for coupling the excitation light via the grating structure (c) to the measuring areas is then uniform in the entire area of the grating structure. However, if, for example, you want to couple the excitation light of different light sources with a clearly different wavelength, the corresponding resonance angles for the coupling can be used make a clear distinction as to what the use of additional adjusting elements in an optical system for receiving the sensor platform may require or which may lead to spatially very unfavorable coupling angles. For example, to reduce widely differing coupling angles, it can therefore be advantageous if the lattice structure (c) is a multidiffractive lattice.

To relax the requirements on the parallelism of the excitation light bundle and the exact setting of the resonance angle, it can be advantageous if the grating structure (c) has a periodicity which varies spatially perpendicular or parallel to the direction of propagation of the excitation light coupled into the optically transparent layer (a). Then, from a large-area irradiated, convergent or divergent beam, coupling will take place at the location on the lattice structure where the resonance condition is fulfilled.

In particular, such a grating structure with a periodicity varying spatially or perpendicular to the direction of propagation of the excitation light coupled into the optically transparent layer (a) enables a method in which, in addition to the determination of one or more luminescences, changes in the effective refractive index on the measurement areas are determined. It can be advantageous if the one or more luminescences and / or determinations of light signals are carried out polarization-selectively at the excitation wavelength.

To improve the signal-to-background ratio, it can furthermore be advantageous if the one or more luminescences are measured with a different polarization than that of the excitation light.

The material of the second optically transparent layer (b) can consist of glass, quartz or a transparent thermoplastic, for example from the group formed by polycarbonate, polyimide or polymethyl methacrylate.

To generate the strongest possible evanescent field on the surface of the optically transparent view (a), it is desirable that the refractive index of the wave-guiding, optically transparent layer (a) is significantly larger than the refractive index of the adjacent layers. It is particularly advantageous if the refractive index of the first optically transparent layer (a) is greater than 2. For example, the first optically transparent layer (a) can consist of Ti0 ₂ , ZnO, Nb ₂ θ ₅ , Ta ₂ 0 ₅ , Hf0 ₂ , or Zr0 ₂ . It is particularly preferred if the first transparent optical layer (a) consists of Ti0 ₂ or Ta ₂ Os.

In addition to the refractive index of the wave-guiding optically transparent layer (a), its thickness is the second relevant parameter for generating the strongest possible evanescent field at its interfaces with neighboring layers with a lower refractive index. The strength of the evanescent field increases with decreasing thickness of the waveguiding layer (a), as long as the layer thickness is sufficient to lead at least one mode of the excitation wavelength. The minimum "cut-off" layer thickness for guiding a mode depends on the wavelength of this mode. It is larger for longer-wave light than for short-wave light. However, as the "cut-off" layer thickness is approached, undesired propagation losses also increase sharply to what further limits the choice of preferred layer thickness. Preferred are layer thicknesses of the optically transparent layer (a) which only allow the guidance of 1 to 3 modes of a predetermined excitation wavelength; layer thicknesses which lead in monomodal waveguides for this excitation wavelength are very particularly preferred. It is clear that the discrete mode character of the guided light only refers to the transverse modes.

These requirements mean that the thickness of the first optically transparent layer (a) is advantageously 40 to 300 nm. The thickness of the first optically transparent layer (a) is very particularly advantageously 70 to 160 nm.

Given predetermined refractive indices of the wave-guiding, optically transparent layer (a) and the adjacent layers, the resonance angle for the coupling of the excitation light in accordance with the above-mentioned resonance condition depends on the diffraction order to be coupled in, the excitation wavelength and the Gitteφeriode. To increase the coupling efficiency, coupling the first diffraction order is advantageous. In addition to the height of the diffraction order, the grating depth is decisive for the level of the coupling efficiency. In principle, the coupling efficiency increases with increasing grid depth. Since the The process of decoupling is completely reciprocal for coupling, but at the same time the coupling efficiency increases, so that it is used to excite luminescence in a measuring area (d) arranged on or adjacent to the lattice structure (c), depending on the geometry of the measuring areas and the irradiated excitation light beam gives an optimum. Because of these boundary conditions, it is advantageous if the grating (c) has a period of 200 nm - 1000 nm and the modulation depth of the grating (c) is 3 to 100 nm, preferably 10 to 30 nm.

As shown in the exemplary embodiment of this invention shown below, it is possible, on a continuous grating structure by incomplete coupling and decoupling of excitation and / or feedback luminescent light parallel to the direction of propagation of the coupled excitation light, a positive gradient of the intensity of the guided that can be controlled via the grating depth To generate excitation and / or excited luminescent light within a single and / or over several measuring ranges. This gradient arises from the fact that a smaller proportion of excitation light is coupled out over the relevant region of the grating structure that is simultaneously irradiated under coupling conditions with an expanded, largely parallel excitation bundle than is additionally coupled in in the direction of propagation of the coupled excitation light. As a result, the available total excitation intensity increases under these conditions up to the end of the illuminated area on the continuous lattice structure, in the direction of propagation of the guided light. This gradient of the intensity of available excitation light has the advantage that it can be used to expand the dynamic range.

The coupling-in and coupling-out efficiency of a diffractive grating is essentially determined by the grating depth under other specified parameters. Therefore, said gradient of the intensity of the guided excitation and / or excited luminescent light can be additionally influenced and controlled if the grating (c) has a spatially varying grating depth in the direction of propagation of the coupled excitation light.

In contrast, propagation losses of the coupled excitation light in an optically transparent, wave-guiding layer lead to a negative gradient of the guided excitation light in its direction of propagation. Accordingly, through a targeted definition of the Amount of this propagation loss, for example through a targeted doping of the waveguiding layer with absorbing molecules but not interfering with the luminescence to be generated or by applying such absorbing molecules on the waveguiding layer, a controllable negative gradient of the intensity of the excitation and / or excited luminescent light within of a single and / or over several measuring ranges.

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

The grating structure (c) can be a relief grating with a rectangular, triangular or semicircular profile or a phase or volume grating with a periodic modulation of the refractive index in the essentially planar optically transparent layer (a).

To enhance luminescence or to improve the signal-to-background ratio, it can furthermore be advantageous if a thin metal layer, preferably made of gold or silver, is optionally present between the optically transparent layer (a) and the immobilized biological or biochemical or synthetic recognition elements an additional dielectric layer with a lower refractive index than layer (a), for example made of silica or magnesium fluoride, is applied, the thickness of the metal layer and the possible further intermediate layer being selected such that a surface plasmon is excited at the excitation wavelength and / or the luminescence wavelength can.

Furthermore, it can be advantageous if optically or mechanically recognizable markings are applied to the sensor platform to facilitate adjustment in an optical system and / or for connection to sample containers as part of an analytical system.

The present invention also relates to an optical system for determining one or more luminescences, with at least one excitation light source of a sensor platform according to at least one of the named

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

For applications in which the highest demands are not placed on the sensitivity, it can be advantageous if the excitation light is irradiated to the measurement areas in a simple incident light or transmission light arrangement. This results in significantly reduced requirements for the positioning of the sensor platform according to the invention in an optical system. In particular, this also enables the use of the sensor platform in a large number of luminescence excitation and detection systems already on the market, such as, for example, scanner systems. It is preferred that the detection of the luminescent light takes place in such a way that the luminescent light coupled out from a grating structure (c) or (c ') is also detected by the detector.

To achieve the lowest detection limits, however, it is advantageous if the excitation light is irradiated onto the grating structure (c) or (c ') under coupling conditions.

It is advantageous if the excitation light emitted by the at least one light source is coherent and is irradiated onto the one or more measurement areas at the resonance angle for coupling into the optically transparent layer (a).

In order to excite a large number of measurement areas or all measurement areas simultaneously and to determine their luminescence at the same time, it is preferred that the excitation light is expanded by at least one light source with an expansion lens to form a substantially parallel beam and is irradiated onto the one or more measurement areas, wherein this preferably takes place at the resonance angle for coupling into the optically transparent layer (a).

To reduce luminescence signals outside the measuring ranges, however, it can also be advantageous if the excitation light from the at least one light source through one or, in the case of several light sources, optionally a plurality of diffractive optical elements, preferably Dammann gratings, or refractive optical elements, preferably microlens Arrays, in a variety of individual beams intensity of the partial beams originating from a common light source, which are radiated essentially parallel to one another onto spatially separated measuring areas, is broken down as far as possible.

In the case of insufficient intensity of a single light source or the need for light sources of different emission wavelengths, for example for biological applications, it is advantageous if two or more coherent light sources with the same or different emission wavelength are used as excitation light sources. In the case of light sources of different emission wavelengths, it is advantageous if the excitation light from 2 or more coherent light sources is irradiated simultaneously or sequentially from different directions onto a grating structure (c) which comprises a superposition of grating structures with different periodicity.

In order to record the signals separately from a large number of measuring ranges, it is preferred that at least one spatially resolving detector is used for the detection.

At least one detector from the group formed by CCD cameras, CCD chips, photodiode arrays, avalanche diode arrays, multichannel plates and multichannel photomultipliers can be used as the at least one spatially resolving detector.

In the optical system according to the invention according to one of the embodiments mentioned, optical components from the group that are used by lenses or lens systems can be used between the one or more excitation light sources and the sensor platform according to one of the embodiments mentioned and / or between said sensor platform and the one or more detectors for shaping the transmitted light bundles, planar or curved mirrors for deflection and, if necessary, additionally for shaping light bundles, prisms for deflecting and possibly for spectrally dividing light bundles, dichroic mirrors for spectrally selective deflection of parts of light bundles, neutral filters for regulating the transmitted light intensity, optical Filters or monochromators for spectrally selective transmission of parts of light beams or polarization-selective elements for the selection of discrete polarization directions of the excitation or L be formed by fluorescent light. For a number of applications, it is advantageous if the excitation light is irradiated in pulses with a duration of between 1 fsec and 10 minutes.

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

It is further preferred that the optical system according to the invention comprises components with which light signals from the group are measured for referencing, which are from excitation light at the location of the light sources or after their expansion or after their subdivision into partial beams, scattered light at the excitation wavelength from the range of one or more spatially separated measuring areas, and via the grating structure (c) in addition to the measuring areas, coupled light of the excitation wavelength is formed. It is particularly advantageous if the measuring ranges for determining the emission light and the reference signal are identical.

It is also possible for the excitation light to be radiated onto and detection of the emission light from one or more measurement areas sequentially for individual or more measurement areas, in which case the sequential excitation and detection can be carried out using movable optical components, which consist of the group of mirrors, deflection prisms and dichroic mirrors is formed. Commercially available so-called scanners are usually used for sequential excitation and detection in bioanalytical array systems, with which an excitation light beam is guided sequentially over the areas to be examined, usually by means of movable mirrors. Most scanner systems change the angle between the illuminated area and the excitation light beam. In order to meet the resonance condition for the coupling of the excitation light into the wave-guiding layer of the sensor platform according to the invention, however, this angle must remain essentially constant, ie a scanner to be used in the optical system according to the invention must work at the correct angle. This requirement is met by some commercially available scanners. At the same time, however, the size of the stimulated areas on the sensor platform must not change. Therefore is a Another object of this invention is an optical system, which is characterized in that sequential excitation and detection is carried out using a scanner that is essentially true to the angle and focus.

Another possible embodiment is that the sensor platform is moved between steps of sequential excitation and detection. In this case, the one or more excitation light sources and the components used for detection can be spatially fixed.

The invention relates to a derived complete analytical system for luminescence detection of one or more analytes in at least one sample on one or more measurement areas on a sensor platform, comprising an optical layer waveguide, with a sensor platform according to one of the aforementioned

Embodiments, an optical system according to one of the aforementioned

Embodiments, as well

Feeding means to mix the one or more samples with the

Bring measuring ranges on the Sensoφlatform in contact.

Advantageously, the analytical system additionally comprises one or more sample containers which are opened towards the sensor platform at least in the area of the one or more measurement areas or the measurement areas combined into segments. The sample containers can each define a volume of 0.1 nl - 100 μl.

The Sensoφlattform can be operated in a closed giant system as well as in an open system. In the first case, the analytical system is designed in such a way that the sample containers on the side facing away from the optically transparent layer (a) are closed, with the exception of inlet and / or outlet openings for the supply or outlet of the samples and, if appropriate, additional reagents and the supply or discharge of samples and, if appropriate, additional reagents take place in a closed flow-through system, wherein in the case of liquid supply to several measurement areas or segments with common inlet and outlet openings, these are preferably addressed in columns or rows. In the case of an open system, the analytical system according to the invention is designed in such a way that the sample containers have openings on the side facing away from the optically transparent layer (a) for the locally addressed addition or removal of the samples or other reagents. In addition, containers for reagents can be provided, which are wetted during the method for the detection of the one or more analytes and brought into contact with the measuring areas

Another object of the invention is a method for the luminescence detection of one or more analytes in one or more samples on at least two or more, spatially separated measurement areas on a sensor platform for determining one or more luminescence from an array of at least two or more, spatially separated measurement areas (i.e. ) or at least two or more spatially separated segments (d '), in which several measuring ranges are combined, on this platform, comprising an optical layer waveguide

with a first optically transparent layer (a) on a second optically transparent layer (b) with a lower refractive index than layer (a),

- With one or more lattice structures (c) for coupling excitation light to the measuring ranges (d)

- Identical or different biological or biochemical or synthetic recognition elements (s) immobilized on these measuring areas for the qualitative or quantitative detection of one or more analytes in a sample brought into contact with the measuring areas, characterized in that crosstalk in the measuring areas or in the segments of several measurement areas generated and fed back into the optically transparent layer (a) of the layer waveguide into adjacent measurement areas or adjacent segments by means of lattice structures (c ') adjacent to the measurement areas or to the segments with the same or different period as the lattice structures (c) Coupling of the luminescent light is prevented.

The present invention in particular relates to a method for the simultaneous detection of luminescence of one or more analytes in one or several samples on at least two or more spatially separated measuring areas on a sensor platform for the simultaneous determination of one or more luminescence from at least two or more spatially separated measuring areas (d) or at least two or more spatially separated segments (d ') into which Several measuring ranges are combined on this platform, including an optical layer waveguide

- With a grid structure (c) which is continuously modulated in the area of the at least two or more measuring areas or at least two or more spatially separated segments (d '), in which several measuring areas are combined.

- The same or different biological or biochemical or synthetic recognition elements (e) immobilized on these measurement areas for the qualitative or quantitative detection of one or more analytes in a sample brought into contact with the measurement areas, characterized in that

- The density of the measuring areas on the sensor platform is at least 16 measuring areas per square centimeter and

- Crosstalk of luminescence generated in the measurement areas or within a segment and fed back into the optically transparent layer (a) of the layer waveguide into adjacent measurement areas or segments by means of coupling this luminescence light through the grating structure (c) continuously modulated in the area of the measurement areas or segments concerned becomes.

It is preferred that the excitation light is coupled to the measurement areas via the grating structure (c) in the optically transparent layer (a).

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

This invention also relates to a method for luminescence detection of one or more analytes according to the above statements, using an analytical system according to one of the aforementioned embodiments, which comprises an optical system according to at least one of the aforementioned embodiments with a sensor platform according to at least one of the aforementioned embodiments, characterized in that one or more liquid samples, which are to be examined for the one or more analytes, are brought into contact with one or more measuring areas on the sensor platform, excitation light is directed into the measuring areas, luminescent substances in the samples or on the measuring areas are stimulated to luminescence and the emitted luminescence is measured.

As a further development, a method is claimed, which is characterized in that, by means of a controllable gradient of the guided excitation and / or excited luminescent light within a single and / or over several measuring ranges, parallel to the direction of propagation of the coupled excitation light, the dynamic range for the signal detection and / or quantitative analyte determination can be expanded or limited.

In the method according to the invention, a luminescence or ruorescence label can be used to generate the luminescence or ruorescence, which label can be excited and emitted at a wavelength between 300 nm and 1100 nm. The luminescence or ruorescence labels can be conventional luminescence or ruorescence dyes or so-called luminescent or fluorescent nanoparticles based on semiconductors (WCW Chan and S. Nie, "Quantum dot bioconjugates for ultrasensitive nonisotopic detection", Science 281 (1998) 2016 - 2018) act.

The luminescence label can be on the analyte or in a competitive assay on an analog of the analyte or in a multistage assay on one of the binding partners of the immobilized biological or biochemical or synthetic recognition elements or on the biological or biochemical or synthetic recognition elements.

In addition, a second or even more luminescence label with the same or different excitation wavelength as the first luminescence label and the same or different emission wavelength can be used. It can be advantageous here if the second or even more luminescence label can be excited at the same wavelength as the first luminescence label, but can emit at other wavelengths.

For other applications, it is advantageous if the excitation spectra and emission spectra of the luminescence labels used overlap only slightly or not at all.

In the method according to the invention it can furthermore be advantageous if charge or optical energy transfer from a first luminescence label serving as donor to a second luminescence label serving as acceptor is used to detect the analyte.

In addition, it can be advantageous if, in addition to the determination of one or more luminescences, changes in the effective refractive index are determined on the measurement areas. It can be of further advantage if the one or more luminescences and / or determinations of light signals at the excitation wavelength are carried out in a polarization-selective manner. Furthermore, the method allows the possibility that the one or more luminescences are measured at a different polarization than that of the excitation light.

The inventive method according to one of the preceding embodiments enables simultaneous or sequential, quantitative or qualitative determination of one or more analytes from the group of antibodies or antigens, receptors or ligands, chelators or "histidine tag components", oligonucleotides, DNA or RNA Strands, DNA or RNA analogs, enzymes, enzyme factors or inhibitors, lectins and carbohydrates.

The samples to be examined can be naturally occurring body fluids such as blood, serum, plasma, lymph or urine or egg yolk. However, a sample to be examined can also be an optically cloudy liquid, surface water, a soil or plant extract, a bio- or synthesis process broth.

The samples to be examined can also be taken from biological tissue parts.

The present invention furthermore relates to the use of a method according to at least one of the preceding embodiments for determining chemical, biochemical or biological analytes in screening methods in pharmaceutical research, combinatorial chemistry, clinical and preclinical development, for real-time binding studies and for determining kinetic parameters in affinity screen mg and in research, for qualitative and quantitative analyte determinations, in particular for DNA and RNA analysis, for the preparation of toxicity studies as well as for the determination of expression profiles and 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 nd for the therapeutic selection of medication, for the detection of pathogens, pollutants and pathogens, in particular salmonella, prions and bacteria, in food and environmental analysis.

The following exemplary embodiments are intended to explain and demonstrate the invention in more detail.

Example 1;

a)

Sensor platform with 2 separate lattice structures and several

Measuring ranges and a segment of measuring ranges

A sensor platform with the outer dimensions 16 mm wide x 48 mm long x 0.5 mm thick was used. The substrate material (optically transparent layer (b)) consisted of Corning glass 7059 (Refractive index n = 1,538 at 488 nm). In the substrate, by means of holographic exposure of the waveguiding layer (a) covered with spun-on photoresist and subsequent wet chemical etching while covering the areas of the sensor platform which are not to be structured, 2 structures of surface relief gratings with a period of 320 nm and a depth of 12 +/- 3 nm were produced . The grids were 5 mm long x 12 mm wide (grid structure I) or 1 mm long x 12 mm wide (grid structure II), with the grid lines oriented parallel to the specified width. The lattice structures were arranged centrally symmetrically with respect to their inner sides for the excitation light to be coupled in and to be guided in the wave-guiding layer (a), at an inner distance of 20 mm on the sensor platform. The wave-guiding, optically transparent layer (a) on the optically transparent layer (b) was produced by "ion plating" and subsequent annealing at 300 degrees (see RE Kunz, J. Edlinger et al., "Grating Couplers in tapered waveguides for integrated optical sensing ", in Proc. SPIE vol. 2068 (1994), p. 321), and had a refractive index of 2,317 at 488 nm (layer thickness 150 nm). The lattice depths on the wave-guiding layer, into which the lattice structure is transferred almost to scale as a result of the deposition process, were later checked using AFM. In the later exemplary embodiment (4.b) measurement method), the grating structures (I) serve as continuous grating structures for coupling the excitation light to the measuring areas located thereon or to the measuring areas located between the grating structures (I) and (II), which, summarized as Segment, by coupling out, fed back luminescent light and guided excitation light through grating structure (II), from crosstalk into potential further measuring areas or segments located in this direction of propagation, according to which the grating structure (II) serving as coupling-out grating is separated.

To prepare for the immobilization of the biochemical or biological or synthetic recognition elements, the sensor platforms were cleaned and with epoxy silane in liquid phase (10 ml (2% v / v) 3-glycidyloxypropyltrimethoxysilane and 1 ml (0.2% v / v) N-ethyldiisopropylamine in 500 ml Ortho-xylene (d = 0.881 g / cm ³ ; m = 440.5 g)) silanized. Then with a commercial spotter (GESIM) solutions of 16-mer oligonucleotides (NH2-3'CAACACACCTTAACAC-5 ', concentration of the deposited solution: 0.34 mM, 3 nl per spot) were applied and thus approximately circular measuring areas with a diameter of 140 - 150 μm at a distance of 600 μm (center-to-center) in a 6 x 6 array both on the lattice structure (I) and in the area between the lattice structures (I) and (II) generated.

b)

Sensor platform with several measuring ranges on one continuous

Lattice structure

A sensor platform with the outer dimensions of 16 mm wide x 48 mm long x 0.7 mm thick was used. The substrate material (optically transparent layer (b) consisted of AF 45 glass (refractive index n = 1.52 at 633 nm). A continuous structure of a surface relief grating was formed in the substrate by means of holographic exposure of the waveguiding layer (a) covered with spun-on photoresist and subsequent wet chemical etching The wave-guiding, optically transparent layer (a) on the optically transparent layer (b) made of Ta ₂ 0 ₅ with a period of 364 nm and a depth of 25 +/- 5 nm, with orientation of the grating lines parallel to the specified width of the sensor platform was generated by reactive, magnetic field-assisted DC sputtering (see DE 4410258) and had a refractive index of 2.15 at 633 nm (layer thickness 150 nm). The grating depths on the waveguiding layer, into which the grating structure is due to the deposition process almost to scale 1: 1 transmissions were later checked using AFM.

In preparation for the immobilization of the biochemical or biological or synthetic recognition elements, the sensor platforms were cleaned and silanized with epoxy silane in the liquid phase, as described above. Afterwards, solutions of 16-mer oligonucleotides (concentration of the deposited solution: 0.34 mM, 3 nl per spot) were applied with a commercial spotter and thus approximately circular measuring areas with a diameter of 140-150 μm at a distance (center to center) of 600 μm created in a 6 x 6 array on the continuous grid structure. Example 2

Optical system a) Excitation units

The Sensoφlattform is mounted on a computer-controlled adjustment unit, which allows the translation parallel and perpendicular to the grid lines as well as a rotation with a pivot point in the axis of the excitation light spot hitting the grid structure (I) for coupling into the Sensoφlattform mentioned in example la. Immediately after the laser used as the excitation light source, there is a shutter in the light path to block the light path if no measurement data are to be recorded. In addition, neutral filters or polarizers can be placed at this point or at other positions in the further path of the excitation light to the sensor platform in the light path in order to vary the excitation intensity step by step or continuously.

Excitation unit a.i) / sensor platform l.a)

The excitation light beam of a helium-neon laser (2 mW) is directed onto the right edge of the lattice structure I without the use of further beam-shaping components. The size of the excitation light spot corresponds to the diameter of the exciting laser beam. The sensor platform is adjusted for maximum coupling, which can be recognized from the maximum intensity of the scattered light which is emitted by scattering along the coupled-in excitation light. This maximum can be determined both visually and by imaging the scattered light detected with an imaging system along the excitation mode onto an optoelectronic detector, e.g. B. on the pixels of a CCD camera as an example of a spatially resolving detector or a photodiode as an example of a non-spatially resolving detector. Under the same coupling conditions, a maximum signal is also measured with a second optoelectronic detector at the coupling angle for the guided excitation light on the second grating structure II. An angle of -3.8 ° is determined as the resonance angle for the coupling.

Excitation unit a.ii) / sensor platform la) The excitation light beam of a helium-neon laser (2 mW) is expanded with a lens combination, using a cylindrical lens, to form a slit-shaped light beam (parallel to the grating lines of the sensor platform). The upper and lower marginal areas of the excitation light, which is slightly divergent parallel to the grid lines, but parallel in the perpendicular projection, are hidden by a slit. The resulting light beam with a slit-shaped cross section on the lattice structure is directed onto the right edge of the lattice structure I. The excitation light spot has a size of 1 mm length x 12 mm width. The sensor platform is adjusted for maximum coupling, which can be recognized from the maximum intensity of the scattered light which is emitted by scattering along the coupled-in excitation light. This maximum can be determined both visually and by imaging the scattered light detected with an imaging system along the excitation mode onto an optoelectronic detector, e.g. B. on the pixels of a CCD camera as an example of a spatially resolving detector or a photodiode as an example of a non-spatially resolving detector. Under the same coupling conditions, a maximum signal is also measured with a second optoelectronic detector at the coupling angle for the guided excitation light on the second grating structure II. An angle of -3.9 ° is determined as the resonance angle for the coupling.

Excitation unit a.üi) / sensor platform l.a)

The excitation light of a helium-neon laser is broken down with a Dammann grating into 16 partial beams arranged linearly parallel to the grating lines. The manufacturer optimized the non-uniform sequence of grooves and ridges of the Dammann grating in such a way that all even-numbered diffraction orders, in particular the zero order, were suppressed and the same intensity (with a variation of less than 5%) was achieved in the odd-numbered diffraction orders . An aspherical lens after the Dammann grating in the direction of the sensor platform, in the focal point of which is the Dammann grating, is used to generate a bundle of parallel partial beams from the partial beams divergent after the Dammann grating. The divergence of the partial beams emerging after the Dammann grating and the focal length of the lens located behind them can be coordinated with one another in such a way that a desired beam distance on the Sensoφlattform is generated. In the present example, 16 partial beams were generated with the Dammann grating used, of which, after passing through a slit-shaped diaphragm, 8 partial beams were directed via a deflection prism onto the right edge of the grating structure I serving as a coupling grating. The coupling condition could be fulfilled for all 8 partial beams at the same time, which was evident from the simultaneous maximum intensity of the scattered light generated along the coupled partial beams and guided in the wave-guiding layer (a). The coupling angle was -3.8 °.

Excitation unit a.iv) / sensor platform l.a)

The excitation light beam of a helium-neon laser at 632.8 nm is widened to a parallel beam with a circular cross section of 2 cm in diameter using a 25-fold expansion optics. An area of 1 mm length x 9 mm width (corresponding to the designations for the lattice structures) is selected from the central part of the excitation light bundle and guided via a deflection prism to the right edge of the lattice structure I (in the direction of the excitation light to be coupled in and guided). The sensor platform is adjusted for maximum coupling, which can be recognized from the maximum intensity of the scattered light which is emitted by scattering along the coupled-in excitation light. This maximum can be determined both visually and by imaging the scattered light detected with an imaging system along the excitation mode onto an optoelectronic detector, e.g. B. on the pixels of a CCD camera as an example of a spatially resolving detector or a photodiode as an example of a non-spatially resolving detector. Under the same coupling conditions, a maximum signal is also measured with a second optoelectronic detector at the coupling angle for the guided excitation light on the second grating structure II.

An angle of -3.8 ° is determined as the resonance angle for the coupling. The amount of light that passes through is measured with a laser power meter behind the position of the sensor platform. A value of 88 μW is determined as the available excitation intensity (without the sensor platform in the beam path). With the sensor platform in between, but without coupling into the wave-guiding layer, the transmission is 79 μW. When the coupling is fulfilled this value decreases to 21 μW, ie 24% of the total available excitation light.

Excitation unit a.v) / sensor platform l.a)

The excitation light beam of a helium-neon laser at 632.8 nm is widened to a parallel beam with a circular cross section of 2 cm in diameter using a 25-fold expansion optics. An area of 4 mm length x 9 mm width (corresponding to the designations for the lattice structures) is selected from the central part of the excitation light bundle and then directed via a deflection prism to the right edge of the lattice structure I (in the direction of the excitation light to be coupled in and guided) . The Sensoφlattform is adjusted to maximum coupling, which is recognizable by the excitation light guided along the coupled mode by scattered light. An angle of -4 ° is determined as the resonance angle for the coupling. Then the sensor platform is laterally shifted without changing the angle so that the 4 mm long excitation light surface is in the middle of the 5 mm long grid structure. The amount of light that passes through is measured with a laser power meter behind the position of the sensor platform. A value of 250 μW is determined as the available excitation intensity (without a sensor platform in the beam path). With the sensor platform in between, but without meeting the coupling condition, the transmission is 240 μW. If the coupling is fulfilled, this value decreases to 51 μ W, i.e. H. 20% of the total available excitation light.

b) detection units

(i) Detection systems for the simultaneous measurement of several

Measuring ranges

(I) A CCD camera (TE3 / A Astrocam, Cambridge, UK) with Peltier cooling (operating temperature - 30 ° C) was used as the spatially resolving detector. The signal was captured and focused on the CCD chip using a 35 mm Nikon lens (Nikkor 35 mm). There were 2 interference filters (Omega Optical, Brattleborough, Vermont) between the lens and the CCD chip, in a beam path with only a small convergence, which did not significantly affect the mode of operation of the interference filter. with a central length of 679 nm and 25 nm bandwidth. The spatially resolved signals recorded with the addition of the hybridization buffer without a luminescent tracer sample, with a time lag to the luminescence signal during hybridization with complementary, luminescence-labeled tracer molecules, were used both for determining the background signal and for referencing.

(II) A CCD camera (TE3 / A Astrocam, Cambridge, UK) with Peltier cooling (operating temperature

-30 ° C). The signal was captured and focused on the CCD chip using a Heligon tandem lens (Rodenstock, two XR Heligon 1.1 / 50 mm). Mounted on a filter wheel, between the two halves of the Heligon tandem objective were 2 interference filters (Omega, Brattleborough, Vermont) with a central wavelength of 679 nm and 25 nm bandwidth, as well as either a neutral filter (for transmission of the attenuated, scattered excitation and much weaker luminescent light from the measuring ranges) or a neutral filter in combination with an interference filter (for the transmission of the attenuated excitation light scattered from the measuring ranges). The signals at the excitation and the luminescence wavelength were measured alternately.

(III) A CCD camera (TE3 / A Astrocam, Cambridge, UK) with Peltier cooling (operating temperature

- 30 ° C). The signal acquisition and focusing on the CCD chip was carried out with a Heligon tandem lens as in the previous example. Between the two halves of the Heligon tandem lens, in the direction of propagation of the emission beam path in the direction of the detector, there was initially a beam part plate at 45 ° for right-angled reflection of the light component reflected by Fresnel reflection (predominantly consisting of light at the excitation wavelength) and then 2 interference filters (Omega, Brattleborough, Vermont) with a central wavelength of 679 nm and 25 nm bandwidth for the selective transmission of luminescent light. The light reflected from the emission beam path via the beam part plate was passed either directly or after passing through an interference filter for the excitation wavelength to a spatially resolving detector or a non-spatially resolving detector. The reference signals, which, as in the above examples for the detection unit, each capture the same areas on the sensor platform, were recorded simultaneously with the luminescence signals from the measurement areas. (ii). Detection system for sequential measurement of measuring ranges

The measuring range to be imaged on the Sensoφlatform is in the focus of a lens system, which depicts the measuring range on a diaphragm in a 1: 1 image, with which areas outside the relevant measuring range can be masked out. The diaphragm is in turn in the focal point of the first of a system consisting of at least 2 lenses, so that a parallel beam path is then generated again in the direction of the detector. In the parallel beam path there is initially a beam partial plate at 45 ° to the parallel beam path, with which part of the detected light, which mainly comprises scattered light at the excitation wavelength, is used in the direction of the reference detector, for example by means of Fresnel reflections. B. a phodiode mt downstream amplifier, reflected, possibly after passing through an interference filter at the excitation wavelength. The luminescent light that spreads further after the beam part plate is selected by means of two interference filters (Omega Optical, 679 DF25) and focused on the detector, which serves as a selected photomultiplier in conjunction with a photon counting unit (Hamamatsu H6240-02 select).

For the sequential recording of signals from different measuring ranges, the sensor platform is shifted in the x and y directions by means of the adjusting elements described under example 2.a).

A combination of simultaneous excitation of several measuring ranges and signal detection by means of spatially resolving detectors and translation steps for detecting larger parts of the sensor platform than can be excited and detected in a single step can also be carried out.

Example 3

Analytical system

All of the examples listed below are designed in such a way that the sensor platforms with the assigned sample containers can be thermostatted in whole or in part, and the fluid supply system can be thermostatted in whole or in part. (a) A single continuous, closed flow cell + fluidic system

A sensor platform according to example la) is used with an excitation unit according to example 2 a.iv). The detection unit is designed according to Example 2.b.i.I). For the sequential addition of various reagents and the samples in a closed pouring system, a closed sample container with a sample chamber open to the sensor platform is used, which encloses the entire area including the lattice structures I and II with a width of 8 mm. The material of the sample container advantageously consists of a self-adhesive, flexible and fluid-sealing, fluorescence-free and low-reflection plastic, in the exemplary embodiment of blackened polydimethylsiloxane. The depth of the sample chamber is 0.1 mm, so that the total volume of the sample chamber is approximately 25 μL. The continuous sample chamber is used for the simultaneous application of one and the same sample or reagents to all measuring ranges. On the left and right edge of the sample chamber, on the opposite side of the sensor platform, there are 2 openings that can be used alternately as inlets or outlets. The sample and reagents are supplied with syringe pumps (Cavro XL 3000, Cavro, Sunnyvale, CA, USA) with a dosing accuracy of 1 μl - 10 μl, depending on the size of the syringe. The syringe pumps are part of a Ruidik system, which also includes a commercial autosampler (Gilson 231 XL), one or more multi-way valves and a sample loop. By switching one or more valves and transporting them through the pumps, different reagents or samples can be directed to the measuring ranges.

(b) Flow cell with 5 parallel, closed flow channels + fluidic system

A sensor platform according to example la) is used with an excitation unit according to example 2 a.ii). The detection unit is designed according to Example 2.biI). For the sequential addition of various reagents and the samples in a closed pouring system, a closed flow cell with 5 parallel sample chambers open to the sensor platform, each 1 mm wide, at a distance of 1 mm, is used, which extends up to the lattice structures I and II extend out. The depth of the sample chamber is 0.1 mm, so that the total volume of the sample chambers comprises approximately 2.5 μL each. The 5th Sample chambers are used to apply the same or different samples or reagents to the measuring ranges addressed from above. On the left and right edge of the sample chambers, on the opposite side of the sensor platform, there are 2 openings, which can be used alternately as an inlet or outlet. The sample and reagents are supplied with syringe pumps (Cavro XL 3000, Cavro, Sunnyvale, CA, USA) with small syringe sizes 50 μl - 250 μl, with which a dosing accuracy of approximately 0.5 μl can be achieved. The syringe pumps are part of a fluidic system that also includes a commercial autosampler (Gilson 231 XL), one or more multi-way valves, and one or more sample loops. By switching one or more valves and transporting them through the pumps, various reagents or samples can be directed to the measuring ranges.

c) Sample vessels open to the outside for the individually addressable addition of reagents

A sensor platform according to example lb) is used with a monodiffractive lattice structure formed over the entire sensor platform, and an excitation unit according to example 2 a.v). The detection unit is designed according to Example 2.b.i.I).

In order to enable the addition or removal of samples and reagents in individually addressable, open sample containers, the sensor platform is arranged horizontally. The structure for the sample containers is formed from a 1 to 3 mm thick, self-adhesive and fluid-sealing, flexible plate made of blackened polydimethylsiloxane, into which a multitude of continuous recesses (with typical diameters of 1 mm - 3 mm) have been made, which geometrically correspond to the correspond to measurement areas that are individually fluidically addressed or measurement areas combined into segments. The PDMS plate structured in this way, which can be molded in high piece steel from a corresponding master (just like the sample containers mentioned above as an example), is brought into contact with the surface of the sensor platform and adheres to one another on this sub-fluidic seal of the cutouts. The same or different samples and reagents, individually addressed, are added to or drawn from the sample containers with a single or multiple parallel dispenser. To avoid evaporation, especially in the case of volatile samples or reagents, the Ruidik application steps are carried out under a saturated water vapor atmosphere.

The dispenser is part of a Ruidik system, which also includes a commercial autosampler (Gilson 231 XL), one or more multi-way valves and a sample loop. By switching one or more valves and transporting them through the pumps, different reagents or samples can be directed to the measuring ranges.

d) Sample and reagent addition using a dispenser, without additional sample containers

A sensor platform according to example lb) is used with a monodiffractive lattice structure formed over the entire sensor platform, and an excitation unit according to example 2.a.v). The detection unit is designed according to Example 2.b.i.I).

In order to allow the addition or removal of samples and reagents to the individually addressable measuring areas or segments, the sensor platform is arranged horizontally. The same or different samples and reagents, individually addressed, are applied to the measuring areas or segments with a single or multiple parallel dispenser or are extracted from them. To avoid evaporation, especially in the case of volatile samples or reagents, the Ruidik application steps are carried out under a saturated water vapor atmosphere.

The dispenser is part of a Ruidik system, which also includes a commercial autosampler (Gilson 231 XL), one or more multi-way valves and a sample loop. By switching one or more valves and transporting them through the pumps, different reagents or samples can be directed to the measuring ranges. Example 4

Luminescence detection method

4.a Solutions used:

1) Hybridization buffer (pH 7.7), consisting of 326 mL 0.070 M phosphate buffer (pH 7), 29.5 g KCL, 0.09 g EDTA x 2 H ₂ 0, 2.25 g poly (acrylic acid) 5100 sodium salt, 2.25 g Tween 20, 1.13 g of sodium azide, made up to 4.5 l with distilled water and adjusted to pH 7.7 with 1 molar sodium hydroxide solution.

2) Sample solution (16 * c-Cy5): Cy 5 -labeled oligomer which is complementary to the immobilized in the measurement areas and consists of 16 base pairs (Cy5-5'-GTTGTGTGGAATTGTG-3 '(10 ^" 9 M) in hybridization buffer 1)

3) Regeneration solution: 0.22 g sodium chloride, 0.11 g sodium citrate, 2.5 g Tween 20, 142 g formamide and 0.13 g sodium azide, dissolved in 250 mL deionized water.

4.b) Measuring method:

(i) A sensor platform according to example la) is used with a

Excitation unit according to example 2.a.v) and a detection unit according to example 2.b.i.I) and a closed soot cell according to the example

3.a).

The measuring procedure consists of the following individual steps:

-5 minutes washing with hybridization buffer 1) (0.5 ml / min), recording of the background signal;

-5 minutes supply of the sample solution (1 nM 16 * c-Cy5; 0.5 ml / min);

-5 minutes of rinsing with hybridization buffer

-5 minutes supply of the regeneration solution (0.5ml / min)

-5 minutes rinsing with hybridization buffer (re-equilibration)

During the process, camera images of the sensor platform with the measurement areas located thereon are recorded at the luminescence wavelength at intervals of one minute each. (ii) A sensor platform according to example lb) is used with a

3.a).

The measuring procedure consists of the following individual steps:

-5 minutes supply of the sample solution (1 nM (16 * c-Cy5; 0.5 ml / min);

-5 minutes rinsing with hybridization buffer (1 nM

-5 minutes supply of the regeneration solution (0.5ml / min)

-5 minutes rinsing with hybridization buffer (re-equilibration)

During the process, camera images of the sensor platform with the measurement areas located thereon are recorded at the luminescence wavelength at intervals of one minute each.

4.c) Results

(i) In the following, results according to the measuring method 4.bi) are discussed as examples. Initially, the widened excitation light beam was directed onto the center of the lattice structure I under coupling conditions, and there it produced a directly illuminated area 4 mm long x 9 mm wide. Of the 6 x 6 measurement areas (columns x rows), the bottom row was not taken into account in the evaluation because it was on the edge of the soot cell and therefore the analyte supply was not carried out under the same conditions as for the other measurement areas. After the hybridization phase, the following average net luminescence signals resulted, as the difference between the absolute signals and the background signals in these measurement ranges (Table 1, unit: "counts per second, cps"):

Table 1:

In the case of the sensor platform la) used in this example with a grating depth of 12 +/- 3 nm, the efficiency of coupling and decoupling of the excitation light was incomplete, which led to a positive gradient of the intensity of the available excitation light, in the direction of the guided mode . This leads to an increase in the luminescence signals observed with an increasing number of columns in Table 1. For graphical visualization, the course of the gross luminescence signals, that is to say before the background signals are subtracted, is shown along line 5 of the measurement ranges, according to the table above, in FIG. 1 .

In the further course of the measuring procedure 4.b.i. the sensor platform was shifted so far in the longitudinal direction without changing the angle that part of the excitation light, which was coupled in near the right edge of the lattice structure 1, could spread further in the optically transparent layer a) in the direction of the lattice structure II, where it was then was decoupled. When passing through the area between the two lattice structures I and II, the second 6 x 6 array of measurement areas lying between these lattice structures was excited, as an example for a segment of measurement areas. The top two rows of the array were at the top of the sample container. The signals from the relevant measuring ranges were not taken into account in the evaluation.

After the hybridization phase, the following average net luminescence signals resulted, as the difference between the absolute signals and the background signals in these measuring ranges (Table 2, unit: "counts per second, cps"):

Table 2:

The propagation losses in the optically transparent layer (a) between the grating structures (I) and (II), corresponding to a negative gradient of the intensity of the guided excitation light available, were quite large in this example and led to a significant reduction in the net luminescence signals with increasing propagation length of the guided excitation light or increasing number of columns in the measuring ranges (see Table 2). For graphic visualization, the course of the gross luminescence signals, i.e. before the background signals are subtracted, is shown along line 5 of the measuring ranges, according to the table above, as an example.

In the further course of this measuring process, the setting angle of the sensor platform with respect to the normal of -4 °, which leads to a mode of excitation light in the optically transparent layer (a) which spreads to the right in relation to the above images, was changed to + 4 °. The coupling condition for generating a mode that propagates to the left is thus fulfilled.

Under the test conditions, 4 columns of the 6 x 6 array of measurement areas on the lattice structure I could be excited under coupling conditions. Due to the incomplete efficiency of the coupling and decoupling, a gradient of the left increases Intensity introduced available excitation light, as can be seen, for example, from the course of the gross luminescence signals, that is to say before the background signals are subtracted, along line 5 of the measurement ranges in FIG. 3.

(ii) In the following, results according to measurement method 4.b.ii) are discussed as examples. The widened excitation light beam was directed, under coupling conditions, onto an array of measurement areas which had been applied to the sensor platform which had been completely modulated with a uniform lattice structure.

Due to the greater grating depth of 25 +/- 5 nm, the efficiency of coupling and decoupling the excitation light was significantly greater, which led to only a very slight positive gradient of the intensity of the available excitation light in the direction of the guided mode, the effect of which was hardly that statistical fluctuation of the measurement results exceeds. For graphic visualization, the course of the gross luminescence signals, i.e. before the background signals are subtracted, is shown along line 5 of the measuring ranges in Figure 4.

Example 5

a) Sensor platforms

(i) A sensor platform with the outer dimensions 16 mm wide x 48 mm long x 0.7 mm thick was used. The substrate material (optically transparent layer (b)) consisted of AF 45 glass (refractive index n = 1.52 at 633 nm). The optically transparent layer (a) on the optically transparent layer (b) made of Ta ₂ 0 ₅ was produced by reactive, magnetic field-assisted DC sputtering and had a refractive index of 2.15 at 633 nm (layer thickness 150 nm). In addition, the sensor platform contained 2 discrete lattice structures, each with a period of 360 nm, with the same arrangement as in example la) (dimensions of 5 mm length x 12 mm width or 1 mm length x 12 mm width, with depths of 12 + / - 3 nm). However, the lattice structures were not used in the measuring method described below, neither for luminescence excitation nor for luminescence detection. (ii) A sensor platform with the outer dimensions 16 mm wide x 48 mm long x 0.7 mm thick was used, with similar physical parameters as example b). The substrate material (optically transparent layer (b)) consisted of AF 45 glass (refractive index n = 1.52 at 633 nm). A continuous structure of a surface relief grating with a period of 360 nm and a depth of again 25 +/- 5 nm was produced in the substrate by means of holographic exposure of the waveguiding layer (a) covered with spun-on photoresist and subsequent wet chemical etching, with the grating lines being oriented in parallel to the declared width of the sensor platform. The wave-guiding, optically transparent layer (a) on the optically transparent layer (b) made of Ta ₂ θ ₅ was generated by reactive, magnetic field-assisted DC sputtering (see DE 4410258) and had a refractive index of 2.15 at 633 nm (layer thickness 150 nm ). Under coupling conditions, excitation light of 633 nm can be coupled into the structure at an angle of approximately + 3 °; Coupling or coupling out of light with a wavelength of 670 nm (corresponding to the maximum of the ruorescence of Cy5) takes place at an angle of approximately -6 °.

To prepare for the immobilization of the biochemical or biological or synthetic recognition elements, the sensor platforms 5.a) (i) and (ii) were cleaned and treated with epoxy silane in the liquid phase (10 ml (2% v / v) 3-glycidyloxypropyltrimethoxysilane and 1 ml (0.2 % v / v) N-ethyldiisopropylamine silanized in 500 ml ortho-xylene (7 hours at 70 ° C.) Then solutions of fluorescence-labeled 18-mer oligonucleotides (Cy5-5'-) were used with a commercial spotter (Genetic Microsystems 417 Array er). CCGTAACCTCATGATATT-3'-NH2) (18 * Cy5-NH2) two arrays of 16 x 8 spots (8 rows x 16 columns) each applied (50 pl per spot). The concentration of the applied solutions was alternately 10 ^" per row or 10 ^" M 18 * Cy5-NH2, so that the spots produced (approx. 125 μm diameter in a center-to-center distance of 375 μm) had ruorophore concentrations of approx. 100 or 10 ruorophores per μm ² .

The spot arrays, each about 3.2 mm wide x 5.8 mm long, were arranged one behind the other at a distance of 3.3 mm on the Senso l platforms, so that in the case of the Senso o platform (i), both arrays were a few millimeters apart from the next coupling grids. b) Optical system

The intensity of the fluorescence intensity in the spot arrays on the sensor platforms (i) and (ii) was measured with a commercial scanner (Genetic Microsystems 418 Array Scanner), while the excitation light was irradiated in a reflected light arrangement with a convergent excitation light bundle. The optical axis of the excitation light beam was oriented perpendicular to the sensor platform. The excitation light intensity was about 5 mW. The numerical aperture of the objective lens of the laser scanner is what corresponds to a half opening angle of approximately 53 °. The scan speed corresponded to the information in the product catalog (18 mm / min with a scan width of 22 mm).

For a further comparison, the ruorescence from the measuring ranges of the sensor platform (i) (with grating structures I and II) under coupling conditions, with a parallel excitation light beam and coupling at the right edge of the grating structure 1 (1 mm length x 12 mm width; coupling angle + 3 ° ) measured. An excitation unit according to example 2 / excitation unit a.ii (excitation beam of a helium-neon laser, 0.6 mW expanded with a cylindrical lens) was used in combination with a detection unit according to example 2.b) (i) (I).

c) measurement results

A selection of the measurement results is summarized in Table 3. The signals (net ruorescence signals as the difference between total signals and local background signals), background signals and noise were obtained from four sub-areas with 10 adjacent spots of the same ruorophore concentration (10 ruorophores per μm2) in the case of incident light excitation with the Sensoφlatformaten (i) and (ii) resp two such partial areas in the case of evanescent excitation, ie coupling of the excitation light to the measuring areas on the unstructured part of the sensor platform (i). With the sensor platform (ii), with measurement areas on the monodiffractive grid modulated over the entire platform, significantly higher ruorescence signals are observed in the configuration of the incident light excitation than with the sensor platform (i), which has no grid structure in the area of the measurement areas. Under the test conditions, coupling of excitation light into the optically transparent, wave-guiding layer (a) in the case of the sensor platform (i) can be completely ruled out, in the case of the sensor platform (ii) it can be largely neglected, since only a very small proportion in view of the strongly convergent excitation beam path impinges on the lattice structure at such an angle that it can couple for coupling. The significant increase in the observed ruorescence intensity can be explained by the fact that a considerable proportion of the non-vescent excited ruorescence is coupled into the ruorophores located in the near field of the optically transparent layer (a). Via the continuously modulated lattice structure, however, it is coupled out again after a very short run length, which is dependent on the depth of the lattice structure. Since the decoupling takes place at an angle of approximately -6 ° due to the given parameters of the sensor platform, the decoupled portion is also detected by the detector due to the high numerical aperture of the objective. A certain portion of the observed increase in luminescence may additionally be attributed to a small portion of the coupled excitation light. The high efficiency of the decoupling is shown by the fact that there are no significant differences in the background signals, so that crosstalk of feedback Ruorescence light in neighboring measurement areas can be effectively prevented according to the invention.

In the case of the sensor platform (i), an equally large proportion of luminescent light will be coupled into the layer (a) since it has the same parameters. However, here coupled-in ruorescence light can only couple out via the grating structures located outside the field of view of the detector or exit at the end faces of the sensor platform.

The measurements at the higher Ruoφhor concentration showed with both Sensoφlatforms about ten times higher ruorescence signals and signal / noise ratios. The ratio of net signal to noise can be improved by multiple scanning, with a correspondingly longer measuring time (in the example 10 minutes instead of 1 minute for 10-fold scanning), in this example by a factor of 3. The comparison measurement with the Sensoφlatform (i) under coupling conditions shows that with this arrangement according to the invention, the sensitivity can be increased considerably again with significantly weaker excitation light, namely under these conditions by a factor of 5 to 12, depending on the exposure time. At the same time, as can be seen from the exemplary conditions, much shorter measurement times are necessary for this method.

Table 3:

b) Optical system

For a further comparison, the fluorescence from the measurement areas of the sensor platform (i) (with grating structures I and II) under coupling conditions, with a parallel excitation light bundle and coupling at the right edge of the grating structure 1 (1 mm length x 12 mm width; coupling angle + 3 °) measured. An excitation unit according to example 2 / excitation unit a.ii (excitation beam of a helium-neon laser, 0.6 mW expanded with a cylindrical lens) was used in combination with a detection unit according to example 2.b) (i) (I).

c) measurement results

A selection of the measurement results is summarized in Table 3. The signals (net ruorescence signals as the difference between total signals and local background signals), background signals and noise were obtained from four subareas with 10 adjacent spots of the same ruorophore concentration (10 ruorophores per μm2) in the case of incident light excitation with the Sensoφlatforms (i) and (ii) resp two such sub-areas in the case of evanescent excitation, d. H. Coupling of the excitation light to the measuring areas on the unstructured part of the sensor platform (i).

With the sensor platform (ii), with measurement areas on the monodiffractive grid modulated over the entire platform, significantly higher ruorescence signals are observed in the configuration of the incident light excitation than with the sensor platform (i), which has no grid structure in the area of the measurement areas. Under the test conditions, one Coupling of excitation light into the optically transparent, wave-guiding layer (a) in the case of the sensor platform (i) is completely excluded, in the case of the sensor platform (ii) it is largely neglected, since in view of the strongly convergent excitation beam path, only a very small proportion occurs at such an angle the lattice structure strikes that it can couple to a coupling. The significant increase in the observed fluorescence intensity can be explained by the fact that a significant proportion of the non-vanescent excited ruorescence couples into the ruorophores located in the near field of the optically transparent layer (a). Via the continuously modulated lattice structure, however, it is coupled out again after a very short run length, which is dependent on the depth of the lattice structure. Since the decoupling takes place at an angle of approximately -6 ° due to the given parameters of the sensor platform, the decoupled portion is also detected by the detector due to the high numerical aperture of the objective. A certain portion of the observed increase in luminescence may additionally be attributed to a small portion of the coupled excitation light. The high efficiency of the coupling-out can be seen in the fact that there are no significant differences in the background signals, so that crosstalk from the feedback fluorescent light into neighboring measuring ranges can be effectively prevented according to the invention.

The measurements at the higher Ruoφhor concentration showed with both Sensoφlatforms about ten times higher fluorescence signals and signal / noise ratios. The ratio of net signal to noise can be improved by multiple scanning, with a correspondingly longer measuring time (in the example 10 minutes instead of 1 minute for 10-fold scanning), in this example by a factor of 3.

The comparison measurement with the Sensoφlatform (i) under coupling conditions shows that with this arrangement according to the invention, the sensitivity with considerably weaker excitation light is again considerably, namely under these conditions a factor of 5 to 12, depending on the exposure time, can be increased. At the same time, as can be seen from the exemplary conditions, much shorter measurement times are necessary for this method.

Table 3:

Claims

claims

1. Sensoφlattform for determining one or more luminescence from an array of at least two or more, spatially separated measuring areas (d) or at least two or more spatially separated segments (d '), in which several measuring areas are combined, on this platform, comprising one optical layer waveguide

2. Sensoφlatform according to claim 1, characterized in that the lattice structures (c) and (c ') can be used mutually as a coupling and decoupling grid.

3. Sensoφlattform according to claim 1 or 2, characterized in that the lattice structures (c) and (c ') spatially separate measurement areas (d) or spatially separate segments (d'), in which several measurement areas are combined, preferably enclose in one circular, rectangular or polygonal arrangement around the measuring areas.

4. Sensoφlatform according to claim 1 or 2, characterized in that the grid lines of the grid structures (c) and (c ') are aligned parallel to each other.

5. Sensoφlatform according to claim 4, characterized in that the lattice structures (c) and (c ') form a common continuous lattice structure on which there are several measurement areas or segments.

6. Sensoφlattform for the simultaneous determination of one or more luminescences of at least two or more, spatially separated measuring areas (d) or at least two or more spatially separated segments (d '), in which several measuring areas are combined, on this platform, comprising an optical layer waveguide

- With a grid structure (c) that is continuously modulated in the area of the at least two or more measuring areas or at least two or more spatially separated segments (d '), in which several measuring areas are combined, for coupling excitation light to the measuring areas (d)

7. Sensoφlattform for the simultaneous determination of one or more luminescences of at least two or more, spatially separated measuring areas (d) or at least two or more spatially separated segments (d '), in which several measuring areas are combined, on this platform, comprising an optical layer Waveguide with a first optically transparent layer (a) on a second optically transparent layer (b) with a lower refractive index than layer (a), with one in the region of the at least two or more measuring ranges or at least two or more spatially separated segments (d ') , in which several measuring ranges are combined, consistently modulated lattice structure (c)

Measuring ranges per square centimeter and crosstalk of luminescence generated in the measuring ranges or within a segment and fed back into the optically transparent layer (a) of the layer waveguide into neighboring measuring ranges or segments by coupling this luminescent light through the grating structure that is continuously modulated in the area of the relevant measuring ranges (c ) is prevented.

8. Sensoφlattform according to any one of claims 5-6, characterized in that the grating structure continuously modulated in the area of the one or more measuring areas or segments is 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 to couple excitation light of different wavelengths, the grating lines in the case of two superimposed grating structures preferably being oriented perpendicular to one another.

9. Sensoφlattform according to any one of claims 1-8, characterized in that between the optically transparent layers (a) and (b) and in contact with layer (a), another optically transparent layer (b ') with a lower refractive index than that layer (a) and a thickness of 5 nm - 10,000 nm, preferably of 10 nm - 1000 nm.

10. Sensoφlattform according to any one of claims 1-9, characterized in that for the immobilization of biological or biochemical or synthetic recognition elements (e) on the optically transparent layer (a) an adhesive layer (f) with a thickness of preferably less than 200 nm, especially is preferably applied of less than 20 nm, and that the adhesion-promoting layer (f) preferably comprises a chemical compound from the group consisting of silanes, epoxides and "self-organized functionalized monolayers".

11. Sensoφlattform according to any one of claims 1-10, characterized in that spatially separate measuring areas (d) are generated by spatially selective application of biological or biochemical or synthetic recognition elements on said Sensoφlattform, preferably using one or more methods from the group of methods , which is formed by "InkJet spotting, mechanical spotting, micro contact printing, fluidic contacting of the measuring areas with the biological or biochemical or synthetic recognition elements by supplying them in parallel or crossed microchannels, under the influence of pressure differences or electrical or electromagnetic potentials".

12. Sensoφlattform according to claim 11, characterized in that as said biological or biochemical or synthetic recognition elements components from the group are applied, the nucleic acids (DNA, RNA, ...) and nucleic acid analogs (PNA ...), Antiköφern, aptamers , membrane-bound and isolated receptors, their ligands, antigens for antibodies, "histidine tag components", cavities generated by chemical synthesis for the reception of molecular imprints, etc., or that whole cells or cell fragments are applied as biological or biochemical or synthetic recognition elements become.

13. Sensoφlattform according to any one of claims 11 - 12, characterized in that between the spatially separated measuring areas (d) against the analyte "chemically neutral" compounds, preferably consisting for example of the groups of bovine serum albumin or polyethylene glycol, to reduce non-specific binding or adsorption are upset.

14. Sensoφlattform according to any one of claims 1-13, characterized in that two or more spatially separate measuring areas are each combined into segments on the Sensoφlatform and that different segments preferably by an applied edge, which for fluidic sealing against neighboring areas and / or contributes to a further reduction in optical crosstalk between adjacent segments, are delimited from one another.

15. Sensoφlattform according to any one of claims 1-14, characterized in that up to 100,000 measuring ranges are arranged in a two-dimensional arrangement and a single measuring range occupies an area of 0.001 - 6 mm ² .

16. Sensoφlattform according to any one of claims 1-15, characterized in that the grating structure (c) is a diffractive grating with a uniform period or a multi-diffractive grating.

17. Sensoφlattform according to any one of claims 1-15, characterized in that the grating structure (c) has a spatially varying periodicity perpendicular or parallel to the direction of propagation of the excitation light coupled into the optically transparent layer (a).

18. Sensoφlattform according to any one of claims 1-17, characterized in that the material of the second optically transparent layer (b) consists of glass, quartz or a transparent thermoplastic, for example from the group formed by polycarbonate, polyimide or polymethyl methacrylate .

19. Sensoφlattform according to any one of claims 1-18, characterized in that the refractive index of the first optically transparent layer (a) is greater than 2.

20. Sensoφlattform according to any one of claims 1-18, characterized in that the first optically transparent layer (a) made of Ti0 ₂ , ZnO, Nb ₂ 0 ₅ , Ta ₂ 0 ₅ , Hf0 ₂ , or Zr0 ₂ , particularly preferably made of Ti0 ₂ and Ta ₂ 0 ₅ .

21. Sensoφlattform according to any one of claims 1-20, characterized in that the thickness of the first optically transparent layer (a) is 40 to 300 nm, preferably 70-160 nm.

22. Sensoφlattform according to any one of claims 1-21, characterized in that the grating (c) has a period of 200 nm - 1000 nm and the depth of modulation of the grating (c) is 3 to 100 nm, preferably 10 to 30 nm.

23. Sensoφlattform according to claim 5 or 6 and one of claims 9 - 21, characterized in that by incomplete coupling and decoupling of excitation and / or feedback luminescent light parallel to the direction of propagation of the coupled excitation light, a controllable positive gradient of the intensity of the grating depth guided excitation and / or excited luminescent light is generated within a single and / or over several measuring ranges.

24. Sensoφlattform according to claim 23, characterized in that the grating (c) has a spatially varying grating depth in the direction of propagation of the coupled excitation light.

25. Sensoφlattform according to any one of claims 1-22, characterized in that parallel to the direction of propagation of the coupled excitation light a negative gradient of the intensity of the guided excitation and / or excited luminescent light within a single controllable via its propagation losses in the optically transparent layer (a) and / or is generated over several measuring ranges.

26. Sensoφlatform according to claim 22, characterized in that the ratio of depth of modulation to the thickness of the first optically transparent layer (a) is equal to or less than 0.2.

27. Sensoφlattform according to any one of claims 1-26, characterized in that the grating structure (c) a relief grating with a rectangular, triangular or semicircular profile or a phase or volume grating with a periodic modulation of the refractive index in the substantially planar optically transparent Layer (a).

28. Sensoφlattform according to any one of claims 1-27, characterized in that optically or mechanically recognizable markings are applied to facilitate adjustment in an optical system and / or for connection to sample containers as part of an analytical system.

29. Optical system for determining one or more luminescence, with

- At least one excitation light source

- A Sensoφlatform according to any one of claims 1-28

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

30. Optical system according to claim 29, characterized in that the excitation light is radiated to the measurement areas in an incident light or transmission light arrangement.

31. Optical system according to claim 29 or claim 30, characterized in that the detection of the luminescent light is carried out in such a way that the luminescent light coupled out from a grating structure (c) or (c ') is also detected by the detector.

32. Optical system according to claim 29, characterized in that the excitation light emitted by the at least one light source is coherent and is irradiated onto the one or more measuring ranges at the resonance angle for coupling into the optically transparent layer (a).

33. Optical system according to claim 32, characterized in that the excitation light is expanded by at least one light source with an expansion optics to form a substantially parallel beam and is radiated onto the one or more measurement areas is, this is preferably done at the resonance angle for coupling into the optically transparent layer (a).

34. Optical system according to one of claims 29 - 33, characterized in that the excitation light from the at least one light source through one or, in the case of several light sources, optionally several diffractive optical elements, preferably Dammann grids, or refractive optical elements, preferably microlenses Arrays, is broken down into a plurality of individual beams of the same intensity as possible of the partial beams originating from a common light source, each of which is radiated essentially parallel to one another onto spatially separated measuring areas.

35. Optical system according to one of claims 29 - 34, characterized in that two or more coherent light sources with the same or different emission wavelength are used as excitation light sources.

36. Optical system according to claim 35 with a Sensoφlattfrom according to claim 8, characterized in that the excitation light from 2 or more coherent light sources is simultaneously or sequentially irradiated from different directions onto a grating structure (c) which comprises a superposition of grating structures with different periodicity .

37. Optical system according to one of claims 29 - 36, characterized in that at least one spatially resolving detector is used for the detection, for example from the group consisting of CCD cameras, CCD chips, photodiode arrays, avalanche diode arrays, Multichannel plates and multichannel photomultiplier is formed.

38. Optical system according to one of claims 29-37, characterized in that between the one or more excitation light sources and the sensor platform according to one of claims 1-28 and / or between said sensor platform and the one or more detectors uses optical components from the group are those of lenses or lens systems for shaping the transmitted light bundles, planar or curved mirrors for deflecting and possibly additionally for shaping light bundles, prisms for deflecting and possibly for spectral Distribution of light bundles, dichroic mirrors for spectrally selective deflection of parts of light bundles, neutral filters for regulating the transmitted light intensity, optical filters or monochromators for spectrally selective transmission of parts of light bundles or polarization-selective elements for selecting discrete polarization directions of the excitation or luminescent light.

39. Optical system according to one of claims 29 - 38, characterized in that the irradiation of the excitation light takes place in pulses with a duration between 1 fsec and 10 minutes and the emission light is measured in a temporally resolved manner from the measuring ranges.

40. Optical system according to one of claims 29 - 39, characterized in that for reference purposes light signals from the group are measured which are from excitation light at the location of the light sources or after their expansion or after their subdivision into partial beams, scattered light at the excitation wavelength from the Area of the one or more spatially separated measuring areas, and via the grating structure (c) coupled with the measuring areas coupled light of the excitation wavelength.

41. Optical system according to claim 40, characterized in that the measuring ranges for determining the emission light and the reference signal are identical.

42. Optical system according to one of claims 29 - 41, characterized in that the irradiation of the excitation light and detection of the emission light from one or more measuring ranges takes place sequentially for single or several measuring ranges.

43. Optical system according to claim 42, characterized in that sequential excitation and detection takes place using movable optical components, which is formed from the group of mirrors, deflection prisms and dichroic mirrors.

44. Optical system according to claim 42, characterized in that sequential excitation and detection is carried out using an essentially angle and focus-accurate scanner.

45. Optical system according to one of claims 42-44, characterized in that the sensor platform is moved between steps of sequential excitation and detection.

46. Analytical system for luminescence detection of one or more analytes in at least one sample on one or more measurement areas on a sensor platform, comprising an optical layer waveguide

a sensor platform according to one of claims 1 to 28,

an optical system according to one of claims 29-45,

- Feeding means to bring the one or more samples into contact with the measuring areas on the sensor platform.

47. Analytical system according to claim 46, characterized in that it additionally comprises one or more sample containers, which are open to the sensor platform at least in the area of the one or more measuring ranges or the measuring ranges combined into segments, the sample containers preferably each having a volume of 0.1 have nl - 100 μl.

48. Analytical system according to claim 47, characterized in that the sample containers on the side facing away from the optically transparent layer (a), with the exception of inlet and / or outlet openings for the supply or outlet of the samples and possibly additional reagents, are closed and the supply or discharge of samples and, if necessary, additional reagents take place in a closed flow system, whereby in the case of liquid supply to several measurement areas or segments with common inlet and outlet openings, these are preferably addressed in columns or rows.

49. Analytical system according to claim 47, characterized in that the sample containers on the side facing away from the optically transparent layer (a) have openings for the locally addressed addition or removal of the samples or other reagents.

50. Analytical system according to one of claims 47-49, characterized in that containers are provided for reagents which are wetted during the method for the detection of the one or more analytes and brought into contact with the measurement areas

51. Method for luminescence detection of one or more analytes in one or more samples on at least two or more, spatially separated measuring areas on a sensor platform for determining one or more luminescence from an array of at least two or more, spatially separated measuring areas (d) or at least two or more spatially separated segments (d '), in which several measurement areas are combined, on this platform, comprising an optical layer waveguide

52.Method for the simultaneous detection of luminescence of one or more analytes in one or more samples on at least two or more, spatially separated measurement areas on a sensor platform for the simultaneous determination of one or more luminescence of at least two or more, spatially separated measurement areas (d) or at least two or more spatially separated segments (d '), in which several measurement areas are combined, on this platform, comprising an optical layer waveguide with a first optically transparent layer (a) on a second optically transparent layer (b) with a lower refractive index than layer (a), with one in the region of the at least two or more measuring ranges or at least two or more spatially separated segments (d '), in which several measuring ranges are combined, consistently modulated lattice structure (c) at least two or more spatially separated measuring ranges (d) or at least two or more spatially separated segments (d '), into which several measuring ranges are combined, and immobilized on these measuring ranges, are the same or different biological or biochemical or synthetic recognition elements (e) for the qualitative or quantitative detection of one or more analytes in a sample brought into contact with the measurement areas, characterized in that

53. The method according to claim 52, characterized in that the excitation light is coupled to the measurement areas via the grating structure (c) in the optically transparent layer (a).

54. Method for determining one or more luminescences according to one of claims 51-53, characterized in that (1) the isotropically emitted luminescence or (2) in the optically transparent layer (a) coupled luminescence and coupled out via the grating structure (c) or luminescence of both portions (1) and (2) can be measured simultaneously.

55. A method for luminescence detection of one or more analytes with a sensor platform according to one of claims 23-25, characterized in that by means of a controllable gradient of the guided Excitation and / or excited luminescent light within a single and / or over several measuring ranges, parallel to the direction of propagation of the coupled excitation light, the dynamic range for signal detection and / or quantitative analyte determination can be expanded or limited.

56. The method according to any one of claims 41-55, characterized in that a luminescent dye or luminescent nanoparticle is used as luminescent label to generate the luminescence, which can be excited and emitted at a wavelength between 300 nm and 1100 nm.

57. The method according to claim 56, characterized in that the luminescence label on the analyte or in a competitive assay on an analog of the analyte or in a multi-stage assay on one of the binding partners of the immobilized biological or biochemical or synthetic recognition elements or on the biological or biochemical or synthetic recognition elements is bound.

58. The method according to claim 56 or 57, 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

59. The method according to claim 58, characterized in that the second or even further luminescent label can be excited at the same wavelength as the first luminescent dye, but emit at other wavelengths.

60. The method according to claim 58, characterized in that the excitation spectra and emission spectra of the luminescent dyes used overlap only slightly or not at all.

61. The method according to claim 58, characterized in that charge or optical energy transfer from a first luminescent dye serving as a donor to a second luminescent dye serving as an acceptor is used to detect the analyte.

62. The method according to any one of claims 51-61, 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.

63. The method according to any one of claims 51-62, characterized in that the one or more luminescences and / or determinations of light signals at the excitation wavelength are carried out in a polarization-selective manner, the one or more luminescences preferably being measured at a different polarization than that of the excitation light .

64. The method according to any one of claims 51-63 for the simultaneous or sequential, quantitative or qualitative determination of one or more analytes from the group of antibodies or antigens, receptors or ligands, chelators or "histidine tag components", oligonucleotides, DNA or RNA strands, DNA or RNA analogs, enzymes, enzyme factors or inhibitors, lectins and carbohydrates.

65. The method according to at least one of claims 51-64, 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 liquid or surface water or soil or plant extracts or bio or synthesis process broths or are taken from biological tissue parts.

66. Use of a method according to at least one of claims 51-65 for quantitative or qualitative analyzes for determining chemical, biochemical or biological analytes in screening processes in pharmaceutical research, combinatorial chemistry, clinical and preclinical development, for real-time binding studies and for determining kinetic parameters in Affinity screenmg and in research, for qualitative and quantitative analyte determinations, especially for DNA and RNA analysis, for the preparation of toxicity studies and for the determination of 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 the therapeutic selection of medication, for the detection of pathogens, pollutants and pathogens, in particular salmonella, prions and bacteria, in food and environmental analysis.