EP1281063A1 - Grating optical waveguide structure for multi-analyte determinations and the use thereof - Google Patents

Grating optical waveguide structure for multi-analyte determinations and the use thereof

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Publication number
EP1281063A1
EP1281063A1 EP20010901178 EP01901178A EP1281063A1 EP 1281063 A1 EP1281063 A1 EP 1281063A1 EP 20010901178 EP20010901178 EP 20010901178 EP 01901178 A EP01901178 A EP 01901178A EP 1281063 A1 EP1281063 A1 EP 1281063A1
Authority
EP
European Patent Office
Prior art keywords
light
excitation light
grating
waveguide
coupling
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP20010901178
Other languages
German (de)
French (fr)
Inventor
Martin Bopp
Gert Duveneck
Markus Ehrat
Michael Pawlak
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer Technology Services GmbH
Original Assignee
Zeptosens AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CH8882000 priority Critical
Priority to CH8882000 priority
Priority to CH20952000 priority
Priority to CH20952000 priority
Application filed by Zeptosens AG filed Critical Zeptosens AG
Priority to PCT/EP2001/000605 priority patent/WO2001088511A1/en
Publication of EP1281063A1 publication Critical patent/EP1281063A1/en
Application status is Withdrawn legal-status Critical

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Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/75Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • G01N21/774Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides the reagent being on a grating or periodic structure
    • G01N21/7743Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides the reagent being on a grating or periodic structure the reagent-coated grating coupling light in or out of the waveguide
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • G01N33/54373Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

The invention relates to variable embodiments of a grating optical waveguide structure which enables the determination of locally resolved modifications of the resonance conditions for injecting an excitation light into the wave-guiding layer (a) of a stratified optical waveguide via the grating structure (c) modulated in said layer (a) or for extracting a light guided inside layer (a). The inventive system comprises arrays of measuring areas produced thereupon each having different immobilized biological or biochemical or synthetic identification elements for simultaneously binding and determining one or more analytes. Said excitation light is simultaneously irradiated onto an entire array of measuring areas, and the degree of satisfaction of the resonance condition for the injection of light into the layer (a) is simultaneously measured in said measuring areas. The invention also relates to an optical system comprising at least one excitation light source and at least one locally resolving detector and, optionally, positioning elements for altering the angle of incidence of the excitation light onto the inventive grating optical waveguide structure. The invention additionally relates to a corresponding measuring method and to the use thereof. Surprisingly, it has been found that the inventive method is well-suited as an imaging detection method with high local resolution and sensitivity.

Description

Grating waveguide structure for Multianalytbestimmungen and their use

The invention relates to variable embodiments of a grating waveguide structure, which makes it possible to spatially resolved changes in the resonance conditions for coupling of excitation light into the waveguiding layer (a) of an optical layer waveguide via a in layer (a) modulated grating structure (c) or decoupling of a to determine in the layer (a) guided light having formed thereon arrays of measurement areas, each with different immobilized biological or biochemical or synthetic recognition elements for the simultaneous binding and determining one or more analytes, wherein said excitation light is irradiated at the same time to an entire array of measurement areas and the degree of compliance with the resonance condition for the coupling of light into the layer (a) to said measurement areas is measured simultaneously. The invention also relates to an optical system with at least one excitation light source and at least one position-sensitive detector and optionally positioning elements for changing the angle of incidence of the excitation light to the inventive grating waveguide structure and an associated measurement method and its use. It has been found, surprisingly, that the inventive method is useful as an imaging detection method with high spatial resolution and sensitivity.

As the "spatial resolution" determining a physical parameter of its distribution over a auszumessende, preferably planar measuring surface, is to be understood that this parameter by a respective measurement, a unique value as a function of its x and y coordinates, based on said measurement surface, associated with can be. the maximum achievable spatial resolution is for example limited by the resolution of the detection system.

To determine a plurality of analytes, especially methods are currently widespread, in which this takes place in so-called microtiter plates the detection of different analytes in discrete sample containers or "wells". The most widely used are plates having a grid of 8 x 12 wells on a footprint of typically about 8 cm x 12 cm, whereby the filling of individual wells, a volume of a few hundred microliters is needed. However, for many applications it would be desirable to determine multiple analytes in a single sample compartment, using the smallest possible sample volume.

In US-P 5,747,274 are measuring arrangements and methods for the early detection of myocardial infarction, described by the determination of a plurality of at least three myocardial infarction markers, wherein the determination of these markers can be carried out in the individual or in a common sample container, wherein the following, in the latter case, the description given, a single sample container is designed as a continuous-flow channel, whose one boundary surface, for example, forms a membrane, are immobilized on the antibody for the three different markers. However, there is no evidence of a provision of a plurality of such sample compartments or flow channels on a common carrier. In addition, no geometrical information on the big players in the measurement areas.

In WO 84/01031, US-P 5,807,755, US-P 5,837,551 and US-P 5,432,099, the immobilization of the analyte-specific recognition elements in the form of small "spots" is proposed in some cases significantly less than 1 mm 2 area on solid supports in order to binding of only a small part of existing analyte a dependent only on the incubation time, but - in absence of a continuous flow - to make the absolute sample volume substantially independent determination of the concentration of the analyte. The measuring arrangements described in the respective embodiments based on fluorescence measurements in conventional microtiter plates. In this case, arrangements are also described in which spots are measured by up to three different fluorescently labeled antibodies in the same microtiter plate well. Following the theoretical considerations set forth in these patents, to minimize the spot size would be desirable. However, limiting THAT CONDITION the minimum signal level that can be distinguished from the background signal.

In order to achieve lower detection limits, many measurement arrangements have been developed in recent years in which the detection of the analyte based on its interaction with the evanescent field, which is connected to the light conduit into an optical waveguide, wherein on the surface of the waveguide biochemical or biological recognition elements are immobilized for the specific recognition and binding of the analyte molecules.

Coupled to a lightwave in an optical waveguide of optically thinner media, ie, media is surrounded by a lower refractive index, so it is guided by total internal reflection at the interfaces of the waveguiding layer. provided that no fraction of the electromagnetic energy enters the media of lower refractive. This portion is termed the evanescent field or landscape muted. The strength of the evanescent field is very strongly dependent on the thickness of the waveguiding layer itself as well as the ratio of the refractive indices of the waveguiding layer and of the surrounding media. Case of thin waveguides, ie, layer thicknesses can be distinguished from the same or lower than the thickness to be guided wavelength may be discrete modes of the guided light. Such methods have the advantage that the interaction with the analyte to the penetration depth of the evanescent field into the adjacent medium, in the order of a few hundred nanometers, and interfering signals from the depth of the medium can be largely avoided. The first proposed measurement arrangements were based on highly self-supporting Einschichtwellenleitern, such as fibers or plates of transparent plastic or glass, with thicknesses from several hundred micrometers up to several millimeters.

In WO 94/27137 measuring arrangements are described in which "patches" with different recognition elements are immobilized for the detection of different analytes, on a self-supporting optical substrate waveguide (Einschichtwellenleiter) with Stirnflächenlichteinkopplung, wherein said spatially selective immobilization is effected by means of photoactivatable cross-linker. According to the description given can be more patches may be arranged in series in common parallel flow channels or sample compartments, wherein the parallel flow channels or sample containers over the entire length of the used as the sensor portion of the waveguide extending to avoid deterioration of the light pipe in the waveguide. Notes on a two-dimensional integration of a variety of patches in sample containers of relatively small dimensions, ie considerably less than 1cm base but are not given. In a similar arrangement in WO 97/35203 various embodiments of an assembly will be described in which low in parallel, separate flow channels or sample containers for the sample and calibration solutions and, optionally, high analyte concentration different recognition elements for the determination of various analytes are respectively immobilized. Again, no indication is given as a high integration density of different recognition elements can be achieved in a fed into a common container sample. Also is Susceptability highly multimodal, self-supporting Einschichtwellenleiter for a variety of applications where achieving very low detection limits is necessary not sufficient.

To improve the sensitivity and simultaneously simpler manufacturing in mass production planar thin-film waveguide have been proposed. A planar thin-film waveguide consists in the simplest case of a three-layer system: substrate, waveguiding layer, superstrate (or sample to be investigated), the waveguiding layer having the highest refractive index. Additional intermediate layers can further improve the effect of the planar waveguide.

There are various methods for the coupling of excitation light into a planar waveguide known. The earliest methods used were based on end face coupling or prism coupling, to reduce reflections due to air gaps between generally a liquid prism and the waveguide is applied. These two methods are mainly in connection with waveguides of relatively large layer thickness, ie, in particular self-supporting waveguides, as well as considerably suitable for a refractive index of the waveguide described in 2. the use of coupling gratings in contrast, is a much more elegant method of coupling of excitation light into very thin, highly refractive waveguiding layers.

It can be divided into optical layer waveguides Different methods of analyte guided in the evanescent field of lightwaves. Due to the applied measurement principle, one can distinguish between, for example, fluorescent or general luminescence on the one hand and refractive methods other. Here, method for generating a surface plasmon resonance in a thin metal layer on a dielectric layer of lower refractive index in the group of refractive methods can be included, provided that serves as a basis for determining the measurement parameter of the resonance angle of the incident excitation light for generating surface plasmon resonance. Surface plasmon resonance can be used to amplify a luminescence or for improving the signal-to-background ratio in a luminescence. The conditions for generating a surface plasmon resonance as well as the combination with luminescence as well as the waveguiding structures are widely described in the literature, for example in U.S. Patents US-P 5,478,755, US-P 5,841,143, US-P 5,006,716 and US-P 4,649,280.

By the term "luminescence" the spontaneous emission of photons in the ultraviolet to infrared range is by optical or non-optical, such as electrical or chemical or biochemical or thermal excitation referred to in this application. For example, chemiluminescence, bioluminescence, electroluminescence, and particularly fluorescence and phosphorescence, the term "luminescence".

In the refractive measurement methods, the change of the effective refractive index due to molecular adsorption or desorption is used in the waveguide for detecting the analyte. This change in the effective refractive index is, in the case of grating sensors, determines the from the change in the coupling angle for the input or output coupling of light into or out of the grating sensor, and in the case of interferometric sensors from the change in phase difference between in a sensor arm and a reference arm of the interferometer measurement light guided.

The prior art on the use of one or more coupling gratings for the input and / or output coupling of guided waves by means of one or more coupling gratings is, 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. Tribe, P. White, "Output Grating Couplers on Planar waveguides as Integrated, Optical Chemical Sensors," Sensors and Actuators Bl, 585 (1990), and T. Tamir, ST Peng, "Analysis and Design of Grating Couplers, "Appl. Phys. 14, 235-254 (1977).

In US-P 5,738,825, an arrangement consisting of a microtiter plate completely through holes and a thin-film waveguide as a bottom plate, the latter described consisting of a thin waveguiding film on a transparent self-supporting substrate, hl contact with the pierced from the Mikrotiterplattte and the thin-film waveguide as a base plate formed, open sample containers each diffraction grating for the coupling and decoupling of excitation light are provided to determine the responsible changes in the effective refractive index due to adsorption or desorption of analyte molecules from changes in the observed coupling angle. A detection of multiple analytes in a sample container, by binding to different on the grating structure in the sample container immobilized recognition elements is not provided and the examples following with the used waveguide and grating parameters also difficult to realize. Thus, the achievable with this arrangement density is also independent of one another to be examined measurement areas with different recognition elements for the detection of different analytes in many applications (for example, to determine a plurality of different nucleic acid sequences in a small volume, ie <100 ul sample comprising) is not sufficient.

In US-P 5,991,480 some other form of grating sensors is proposed, in which the angle between the sensor platform is changed with it modulated in a waveguiding layer lattice structure with respect to an excitation light beam is not, but when changing the coupling conditions, the position of the coupling of light on the is substantially to the grating lines, parallel changed grating waveguide structure. This effect is achieved for example by using a so-called "chirped gratings", said "chirped grating" a continuous change in the grating period is substantially parallel to the grating lines. This arrangement has the particular advantage of high potential for miniaturization of the measuring arrangement (including light source, and a spatially resolving detector), as can especially be dispensed with mechanical positioning elements. Here are the dimensions of the discrete areas of "chirped gratings" for light entry or outcoupling however difficult to smaller dimensions to reduce than a few square millimeters.

There are known other phenomena related to grid waveguide structures that have been found as yet little or no sales in analytical measurement technology. In particular, given a suitable choice of the parameters (for example, grating period and the grating depth, thickness of the optically transparent layer (a) of an optical waveguide and whose refractive index and refractive indices of the adjoining media) an almost complete disappearance of the transmitted light and an increase of the light emitted in the direction of reflection light are observed to almost 100%. The physical conditions for the disappearance of the transmission light and the simultaneous occurrence of an extraordinary be "reflection" (as the sum of the regular portion of the reflection, according to the radiation laws and is coupled out by the grating structure), for example in D. Rosenblatt et al., " . resonant Grating waveguide Structures ", IEEE Journal of Quantum Electronics, Vol 33 (1997) 2038 - 2059 described and explained. In all these works, but only the portions of the at the far field of the lattice structure and observed transmitted or reflected light are described and explained with physical models, respectively. There are found no evidence of the distribution of electromagnetic field strength or intensity at the surface of the structure and in particular no evidence of differences in the transmission or "reflection" within an illuminated under the conditions of resonance surface of a coupling grating.

The aforesaid refractive methods have the advantage that they can be used without the use of additional labeling molecules, so-called molecular labels. However, a reference to a spatially resolved detection within an irradiated to a coupling grating light beam is not in any of the above refractive measurement methods using of grating couplers to an analyte by means of determination of changes of the coupling conditions and the coupling angle given on the basis of molecular adsorption or desorption from the coupling grating. Therefore, these methods for detection of a variety of analytes in a small space were previously only slightly or not suitable.

There is a need then to be able to use the advantage of the free analyte detection label for high density arrays, for detecting a plurality of analytes in a small volume sample.

The object of the present invention is to provide a grating waveguide structure, an optical system and a measuring method for label-free analyte detection with high density arrays, the above-mentioned detection.

hn purposes of the present invention are to (d) are defined by the area laterally separated measurement areas, occupy the immobilized biological or biochemical or synthetic recognition elements for detecting one or more analytes in a liquid sample. These areas can have any geometry, for example in the form of dots, circles, rectangles, triangles, ellipses or lines. It is possible, by spatially selective application of biological or biochemical or synthetic recognition elements on the grating waveguide structure laterally separated measurement areas (d) to produce. In contact with an analyte or competing with the analyte for binding to the immobilized recognition elements analogs of the analyte or of a further binding partner in a multistage assay these molecules are only selectively bind waveguide structure in the measurement areas at the surface of the grid, which by the areas are defined, which are occupied by the immobilized recognition elements.

It has now surprisingly been found that with an inventive grating waveguide structure (GWS), for instance modulated with a wave-guiding in the layer and extending over the whole GWS lattice structure, particularly in large-area lighting (ie, with a beam diameter of for example 5 mm) , below or close to the resonance condition for the coupling of light into the layer (a), differences in the degree of fulfillment of the resonance condition for the coupling of light, that is, local differences in the mass occupation of the lattice structure in the form of the applied measurement areas with the biological recognition elements such as oligonucleotides, with high spatial resolution (of 50 microns or less) and with a high contrast, that is, a high sensitivity can be determined for the determination of differences or changes in the mass occupation. Here, spatial resolution and contrast are surprisingly so good that the inventive method even when an image forming process ( "imaging method") for simultaneous topological characterization of the mass density of an extended surface (on the order of several square millimeters to several square centimeters) is suitable, wherein for example for determination of different local mass densities camera images between each of the single beam angle of the excitation light is changed to the GWS so that in dependence on the local area density at different angles minimums in the transmission or be added (for example in transmission or in "reflection") sequentially, maxima result in the "reflection". spatially resolved distribution of mass density can be determined from these sequential images then by numerical methods. Compared to conventional methods of analyte detection from changes in coupling conditions without spatial resolution, the new inventive method has a number of advantages. These relate, for example, a much greater speed, as sequential images can be created at intervals of fractions of a second with millisecond exposure time. Furthermore, omitted any problems of reproducibility in the positioning when the GWS must be moved between sequential local measurement at discrete measurement areas respectively in these new measuring positions, as is necessary when using the conventional method referred to. Furthermore, the method allows advantageously also performing simultaneous kinetic measurements for a plurality of measurement areas within a common sample compartment on the GWS by "angle Scans" may be repeated for the determination of different mass density on the observed surface in rapid succession.

First object of the invention is a grating waveguide structure for the spatially resolved determination of changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on this platform comprising an optical waveguide layer

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

- with one or mehrereren grating structures (c) for the coupling of excitation light to the measurement areas (d) or coupling out in the layer (a) led light in the ranges of at least two or more laterally separated measurement areas (d) on the one or more grating structures (c)

- immobilized in these measurement areas, the same or different biological or biochemical or synthetic recognition elements (e) for the qualitative and / or quantitative detection of one or more analytes in a brought into contact with the measurement areas sample, characterized in that said excitation light simultaneously onto said array of measuring ranges is irradiated and the degree of compliance with the resonance condition for the coupling of light into the layer (a) to the two or more measurement areas is measured simultaneously, and crosstalk from the layer (a) guided excitation light from a measurement range to one or more adjacent measuring ranges by again, this coupling out the excitation light is prevented by means of the grating structure (c).

The inventive grating waveguide structure, it is possible locally resolved at the same time the mass density in a plurality of measurement areas on a grating structure (c) to determine, on the basis of the degree of compliance with the resonance condition for coupling an excitation beam into the optical layer (a) in range of these ranges.

In particular, object of the invention is therefore a grating waveguide structure for the spatially resolved determination of changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light by a two-dimensional array of at least four or more discrete measurement areas (d) on this platform comprising an optical layer waveguide

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

- with one or mehrereren grating structures (c) for the coupling of excitation light to the measurement areas (d) or coupling out in the layer (a) led light in the ranges of at least two or more laterally separated measurement areas (d) on the one or more grating structures (c)

- immobilized in these measurement areas, the same or different biological or biochemical or synthetic recognition elements (e) for the qualitative and / or quantitative detection of one or more analytes in a brought into contact with the measurement areas sample, characterized in that the density of the measurement areas on a common Gitterstruktrur (c) is at least 10 measuring zones per square centimeter, and that said excitation light is simultaneously irradiated on said array of measurement areas and the degree of compliance with the resonance condition for the coupling of light into the layer (a) to said measurement areas is measured simultaneously, and crosstalk from the layer (a) guided excitation light is prevented by a measurement range to one or more adjacent measurement areas by re-extraction of this excitation light by means of the grating structure (c). It is preferred that a continuously modulated grating structure (c) extends substantially over the entire area of ​​the waveguide grating structure.

Are preferred such embodiments of the inventive grating waveguide structure, which are characterized in that the spatial resolution for the determination of the degree of compliance with the resonance condition for the coupling of light into the layer (a) is better than 200 microns. Particularly preferred are embodiments in which the spatial resolution for the determination of the degree of compliance with the resonance condition for the coupling of light into the layer (a) better than 20 .mu.m.

An important parameter for the change in the Orstauflösung or the sensitivity for the determination of changes in mass assignments with reference to corresponding changes in the resonance conditions for the coupling of light is the grating depth. The inventive grating waveguide structure is made possible that the spatial resolution for the determination of the degree of compliance with the resonance condition for the coupling of light into the layer improved (a) by choosing a larger modulation depth of Gitterstrakturen (c), or selection of a smaller modulation depth of said grating structures is reduced can be. It is also possible that the half-width of the resonance angle to fulfill the resonance condition for the coupling of light in the layer can be increased by increasing the modulation depth of said grating structures (a) by decreasing the modulation depth of grating structures (c) reduces or.

Likewise, the Ortsaufösung or sensitivity for the determination of changes in the effective refractive index on the surface of the inventive grating-waveguide structure by choice between transverse magnetic polarized modes (TM) and transverse electric polarized modes can be decisively influenced. For example, comprise TM modes in the case of highly refractive waveguiding layer (a) (for. Example, having a refractive index> 2), which due to their low layer thickness (eg., Between 100 nm and 400 nm), only the fundamental modes of an incident excitation light (TMo or TE 0, see also can lead) below, in a lattice region of a lattice waveguide structure (for example, with grid depths of between 5 nm and 60 nm) a lower attenuation, ie greater run length (within the lattice structure than the corresponding TE modes the same order), ie on TE modes. This means that for the same grating depth, the spatial resolution is lower by using TM modes. On the other hand, the sharpness of the resonance curve to meet the incoupling of excitation light into the waveguiding layer (a) by a grating structure (c) with the same grating parameters (grating period and depth) and layer parameters (refractive indices and layer thicknesses) of the grating waveguide structure for TM modes significantly greater than for TE modes. This means that the resolution of the signal intensity, ie sensitivity, is greater for the determination of the extent of fulfilling the conditions of resonance for TM modes. Accordingly, the choice between the use of TM or TE modes is to be taken in dependence on the particular task.

In order to determine with high sensitivity with an inventive grating waveguide structure of a high spatial resolution to change said resonance conditions, it is desirable that the aforementioned physical parameters such as refractive index and layer thickness of the waveguiding layer, and grating period and the grating depth, as parameters of the grid waveguide structure itself which affect the sensitivity at a determination of a change in the resonance conditions, the waveguide structure corresponding to the surface of an to be examined arrays change within an area on the grid as little as possible in order, apart from the measurement areas, stable to ensure resonance conditions, in particular a uniform coupling angle. Typically, a same time to be inspected array of measurement areas has a size of at least 2 mm x 2 mm. Therefore, it is of advantage if - outside the ranges - the resonance angle for coupling in or out of a monochromatic excitation light ( "coupling angle") within such an area, that is in parallel within an area of at least 4 mm 2, (with the orientation of the pages or not (c)) varies by at most 0.1 ° (as the deviation from a mean) parallel to the lines of the grating structure. of course, it is advantageous if such a high uniformity of the coupling angle can be ensured even over a larger area. therefore, it is preferred that is the coupling angle in an area of ​​at least 10 mm x 10 mm () with alignment of the sides parallel or not (parallel to the lines of the grating structure c) varies by at most 0.1 ° (as the deviation from a mean). particularly preferred, when the coupling angle an area of ​​at least 50 mm x 50 mm (with parallel orientation of the pages or not parallel to the lines of Grating structure (c)) varies by a maximum of 0.1 ° * (as the deviation from a mean). A variety of macroscopic changes in external conditions has an influence on said resonance conditions. For example, the refractive indices of the optically transparent layers change (a) and (b) and in contact with the grid matched waveguide structure samples with changes in temperature. It is preferred, therefore, may be that the temperature of an inventive grating waveguide structure through appropriate precautions held constant or changed in a controlled manner and set.

The inventive grating waveguide structure, the degree of satisfaction of the resonance condition can be determined for light coupling in various ways. An object of the invention is an embodiment of a grating waveguide structure, which is characterized in that the degree of fulfillment of the resonance condition for the coupling of light into the layer (a) to the measurement areas from the intensity of substantially parallel to the reflected light, again decoupled excitation light (ie, the sum of both components) is determined.

Another embodiment is characterized in that the degree of fulfillment of the resonance condition for the coupling of light into the layer (a) is determined to the measurement areas of the intensity of the transmitted excitation light.

A further embodiment is characterized in that the degree of fulfillment of the resonance condition for the coupling of light into the layer (a) to the measurement areas from the intensity of scattered light from by coupling of a grating structure (c) in the layer (a) guided excitation light is determined ,

The inventive grating waveguide structure is characterized in that the sum of the intensities of the reflected and substantially parallel thereto again coupled out excitation light when locally fulfill the resonance condition for the coupling of light into the layer (a) has a maximum in the range of this measuring range. In practice, the decoupled at one and the same measuring range and the reflected there excitation light can not be distinguished from each other because both spread from the same place in the same Direction-. At the same time, the intensity of the transmitted excitation light for local fulfillment of the resonance condition for the coupling of light into the layer (a) in the range of this measurement range to a minimum. In addition, in the layer, the intensity of the scattered light from by coupling of a grating structure (c) in the layer (a) guided excitation light at local meet the resonance condition for coupling the light (a) in the range of this measurement range is a maximum.

The height of the propagation losses of in an optically waveguiding layer (a) guided mode is determined to a large extent on the surface roughness of an underlying support layer as well as to absorption by possibly present in this backing layer chromophores, which additionally the risk of the excitation of undesirable for many applications luminescence in this carrier layer by penetration of the evanescent field of the layer (a) guided mode, contains within itself. Furthermore, it may (a) and come to the occurrence of thermal stresses due to different thermal expansion coefficients of the optically transparent layers (b). In case of a chemically sensitive optically transparent layer (b), if it consists for example of a transparent thermoplastic material, it is desirable to have a penetration of solvents, which may damage the layer (b), by possibly in the optically transparent layer (a) to prevent existing micropores.

Therefore, it is advantageous if an additional optically transparent layers (a) and (b) and in contact with layer (a), a further optically transparent layer (b ') with lower refractive index than that of layer (a) and a thickness of 5 nm - 10 000 nm, preferably 10 nm - 1000 nm, is located. The intermediate layer has the task of reducing the surface roughness of the layer (a) or the reduction of the penetration of the evanescent field of in layer (a) led light in one or more underlying layers, or improve the adhesion of the layer (a) the one or more underlying layers or to the reduction of thermally induced stress within the grating waveguide structure or chemical isolation of the optically transparent layer (a) of the underlying layers by means of sealing of micropores in the layer (a) to underlying layers , The grating structure (c) of the inventive grating waveguide structure may be a diffractive grating with a uniform period or multidiffractive lattice. It is also possible that the grating structure (c) has a perpendicular or parallel to the direction of propagation in the optically transparent layer (a) injected excitation light spatially varying periodicity.

It is preferred that the material of the second optically transparent layer (b) consists of the inventive grating waveguide structure made of glass, quartz or a transparent thermoplastic or moldable plastic, for example from the group which is formed from polycarbonate, polyimide or polymethylmethacrylate.

It is further preferred that the refractive index of the first optically transparent layer (a) is greater than 1.8. For the optical layer (a) are a variety of materials suitable. Without loss of generality it is preferred that the first optically transparent layer (a) a material from the group of TiO 2, ZnO, Nb 2 O 5, Ta 2 O 5, HfO 2, or ZrO 2, more preferably from TiO or Nb 2 O 5 or Ta 2 O 5 comprising.

In addition to the refractive index of the waveguiding optically transparent layer (a) the thickness of which is the second important parameter for the generation of a strong possible evanescent field at their interfaces to adjacent layers of lower refractive index and the highest possible energy density is within the layer (a). The strength of the evanescent field increases with decreasing thickness of the waveguiding layer (a) as long as the layer thickness is sufficient to result in at least one mode of the excitation wavelength. The minimum is "cut-off" layer thickness depends on the wavelength of this mode for guiding a mode. It is larger than for short-wavelength light to longer-wavelength light. By approaching the "cut-off" layer thickness but also unwanted propagation losses increase sharply to, which additionally limits the choice of the preferred layer thickness down. Such layer thicknesses of the optically transparent layer (a) which allow only the guide 1-3 modes at a given excitation wavelength are preferred, very particularly preferred layer thicknesses which lead to monomodal waveguides for this excitation wavelength. It is clear that the discrete nature of the mode guided light refers only to the transverse modes. These requirements mean that advantageously, the product of the thickness of the layer (a) and its refractive index one-tenth to a whole, preferably one-third to two-thirds of the excitation wavelength of the layer (a) is coupled in the excitation light.

For given refractive indices of the waveguiding, optically transparent layer (a) and of the adjacent layers is the resonance angle for coupling in the excitation light corresponding to the above resonance condition depends on the to be coupled diffraction order of the excitation wavelength and the grating period. To increase the coupling efficiency of the coupling of the first diffraction order is advantageous. In addition to the height of the diffraction grating depth is important for the amount of the coupling efficiency. In principle, the coupling efficiency increases with increasing grating depth. Since the process of extraction being completely reciprocal for coupling, but at the same time also increases the extraction efficiency, so that it is a function of the geometry of the measuring portions and the incident excitation light beam, an optimum. Based on these boundary conditions, it is advantageous if the grating (c) a period of 200 nm - 1000 nm and having the modulation depth of the grating (c) 3 to 100 nm, preferably 10 to 30 nm.

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

Besides the already mentioned parameters also the so-called "land-to-groove ratio" in the input and output coupling efficiency to affect. Among land-to-groove ratio is, for example, in a rectangular grid, the ratio of the width of the webs to the width to understand the grooves Preferably, the gratings have a web-to-groove ratio of 0.5 -. 2.

Thereby, the grating structure (c) may be a relief grating with 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).

It may furthermore be advantageous if optically or mechanically recognizable marks for facilitating adjustment in an optical system and / or for connection to sample containers as part of an analytical system are applied to the waveguide grating structure.

The inventive grating waveguide structure is particularly suitable for use in biochemical analytics, for the highly sensitive detection of one or more analytes in one or more of supplied samples. The following set of preferences is particularly focused on this field of use. biological or biochemical or synthetic recognition elements are immobilized tag for recognition and binding to be detected analyte on the grating waveguide structure for these applications. This can be done over a large area, possibly over the entire structure, or in discrete so-called measuring ranges.

For the purposes of the present invention spatially separated measurement areas (d) are to be defined by the area that is occupied immobilized biological or biochemical or synthetic recognition elements for detecting one or more analytes from a liquid sample. These areas can have any geometry, for example in the form of dots, circles, rectangles, triangles, ellipses or lines. It is possible that up to 1 000 000 measurement areas are located on an inventive grating waveguide structure in a 2-dimensional arrangement wherein a single measurement area, for example, an area of ​​0.001 mm - can occupy 6 mm. Typically, the density of measurement areas on a common grating structure (c) can in this case more than 10, preferably be more than 100, more preferably more than 1000 measurement areas per square centimeter.

Furthermore, it is preferred that the external dimensions of their base surface with the base of standard microtiter plates correspond approximately 8 cm x 12 cm (96 or 384 or 1536 wells).

There are a variety of methods for applying the biological or biochemical or synthetic recognition elements on 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 statistically in general. In addition, there is a risk that, for varying composition of the sample containing the analyte or the reagents used in the detection method, a portion of the immobilized recognition elements is rinsed away. Therefore, it may be advantageous if for immobilization of biological or biochemical or synthetic recognition elements (e) on the optically transparent layer (a) an adhesion-promoting layer (f) is applied. This adhesion promoting layer should be optically transparent. In particular, the adhesive layer should not protrude into the overlying medium via the penetration depth of the evanescent field of the waveguiding layer (a). Therefore, the adhesion-promoting layer (f) should have a thickness of less than 200 nm, preferably have less than 20 nm. It can, for example, "self-organized functionalized monolayers" chemical compounds from the group of silanes, epoxides, functionalized, charged or polar polymers and.

For application of the biological or biochemical or synthetic recognition elements, one or more methods may be used from the group of methods, the spotting of "inkjet, mechanical spotting, micro contact printing, fluidic contacting of the measurement areas with the biological or biochemical or synthetic recognition elements upon their supply in parallel or crossed micro-channels are formed under the influence of pressure differences or electrical or electromagnetic potentials. "

As biological or biochemical or synthetic recognition elements, components from the group can be applied, the nucleic acids (e.g. DNA, RNA, oligonucleotides), nucleic acid analogs (eg., PNA), antibodies, aptamers, membrane-bound and isolated receptors, their ligands, antigens for antibodies, "histidine tag components", generated by chemical synthesis to receive molecular cavities hnprints, etc. is formed.

Among the latter type of recognition elements are meant cavities, which are produced in a process which has been described as "molecular imprinting" in the literature. To this end, usually in organic solution, the analyte or an analogue of the analyte, encapsulated in a polymer structure. Then it is called as "Imprint". Then, the analyte or its analog is dissolved out again with the addition of suitable reagents from the polymer structure so that it therein leaves an empty cavity. This empty cavity can then be used as a binding site with high steric selectivity in a subsequent detection method.

It is also likely to be applied as a biochemical or biological recognition elements, whole cells or cell fragments.

hl many cases, the detection limit of the analytical method is limited by signals of so-called non-specific binding, ie signals which are generated by binding of the analyte or other components applied for detection of the analyte compounds which not only in the field of used immobilized biological or biochemical or synthetic recognition elements but also in which uncovered portions of a grating waveguide structure are bound, for example, by hydrophobic adsorption or electrostatic interactions. Therefore, it is beneficial if (d) between the laterally separated measurement areas towards the analyte are "chemically neutral" compounds are applied for reducing non-specific binding or adsorption. As "chemically neutral" compounds are usually referred to, such substances which do not themselves possess specific binding sites for the recognition and binding of the analyte or an analog of the analyte or of a further binding partner in a multi-step assay and by their presence to access the analyte or its analog or block further binding partner to the surface of the grating waveguide structure.

As "chemically neutral" compounds, for example, substances from the groups are used, albumins, especially bovine serum albumin or human serum albumin, is not to be analyzed hybridizing with polynucleotides, fragmented natural or synthetic DNA, such as herring or salmon sperm, or also uncharged, but hydrophilic polymers such as polyethylene glycols or dextrans, are formed.

In particular, the selection of the substances mentioned to reduce nonspecific hybridization in polynucleotide hybridization assays (such as herring or salmon sperm) is determined by the empirical preference for the polynucleotides to be analyzed "from heterologous" DNA, for which no interaction with the detected polynucleotide sequences are known. The invention further provides an optical system for spatially resolved determination of changes in the resonance conditions for coupling a Anregungshchts in a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on this platform, with

- at least one excitation light source

- an inventive grating waveguide structure according to one of the aforementioned embodiments

- at least one spatially resolving detector for detecting the transmitted excitation light on the, with respect to the irradiated excitation light, opposite side of the grating waveguide structure and / or for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or for detecting the scattered light from coupling by means of a grating structure (c) in the layer (a) guided excitation light.

Particularly in the case of the aforementioned embodiment, the detection of the substantially parallel to the reflected light again decoupled light, it may be advantageous if the of the waveguiding layer (a) facing away from the surface of the optically transparent layer (b), ie, with respect to the incident excitation light, opposite side of the grating waveguide structure is provided with an antireflection coating. This can be reduced possible interference reflections and interference phenomena, for example due to Fresnel reflections that can occur regardless of the measurement signals to be detected.

The boundary conditions on the positioning of the at least one laterally resolving detector on the same or opposite side of the grating waveguide structure, with respect to the irradiated excitation light, and in dependence of the detected light component (transmitted excitation light or, parallel to the reflected portion again outcoupled excitation light) listed can be simplified by the use of a suitable in the beam path to be positioned projection screen. A suitable projection screen should be diffusely reflective and / or diffusely transmissive. An essential role in the choice of materials is played by the granularity of the material, particularly its surface. A too coarse granularity leads to a reduction of contrast and for generating an enlarged, blurred contours, ie a reduction of the spatial resolution and sensitivity. Similarly, disadvantageously, a too large run length of the light in the material (eg in a Teflon block) affects. In practice, for example, a piece of fine-grained white paper as a highly suitable diffusely reflecting projection wall which, for the detection of the transmitted excitation light, which is to be positioned on the opposite side of the grating waveguide structure, with respect to the irradiated excitation light. Proves In this example, the at least one spatially resolving detector on the same side of the grating waveguide structure, with respect to the irradiated excitation light is disposed. When using a diffusely transmissive projection screen, the detector may be arranged on both sides of the waveguide grating structure.

Such a projection screen can also be used advantageously for detecting the, substantially parallel to the reflected light again decoupled light. While this light component must be exactly in the propagation direction can be positioned without using such a projection screen, a position-sensitive detector, which can lead to difficulties in the practical implementation due to the spatial dimensions of such a detector, these requirements are waived when using said Proj ektionswand.

It has surprisingly been found that by using a projection screen for the detection of the transmitted excitation light on the opposite side of the grating waveguide structure, with respect to the irradiated excitation light, an especially good contrast in the determination of the degree of compliance with the resonance conditions for coupling light into the inventive grating - waveguide structure could be achieved, for example, compared to the alternative of the detection of the scattered light from the layer (a) led light. For example, the adverse reduction in contrast due to stray light outcoupling guided excitation light by surface defects of the grating waveguide structure can be almost completely avoided with this arrangement. A further advantage of this arrangement is that with the use of a substantially parallel excitation light beam the distance of the screen from the grating waveguide structure in a wide range without significantly impairing the sensitivity and / or the spatial resolution can be varied. For example, also that of the waveguiding layer (a) may be a grating waveguide structure opposite side of a suitable sample container, with the grating waveguide structure as opposed boundary wall may be formed as a projection screen.

Another object of the invention is, therefore, an optical system for spatially resolved determination of changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on this platform, With

- at least one excitation light source

- an inventive grating waveguide structure

- a diffusely reflecting and / or diffusely transmissive projection wall on the, with respect to the irradiated excitation light, opposite side of the grating waveguide structure, to form an image of the transmitted excitation light and at least one spatially resolving detector for detecting the image of the transmitted excitation light on said projection screen.

A possible embodiment is characterized in that arranged at least one spatially resolving detector for detecting the image of the transmitted excitation light on said projection on the wall, with respect to the irradiated excitation light, the same side of the grating waveguide structure.

Another possible variant is characterized in that the at least one spatially resolving detector for detecting the image of the transmitted excitation light on said projection wall on the side of the transmitted excitation light, that is, on the one disposed with respect to the irradiated excitation light, opposite side of the grating waveguide structure, is, said projection screen is at least partially transmissive.

For specific applications, an embodiment of an optical system with a grating waveguide structure with one or more grating structures (c) is preferably spatially varying with a substantially perpendicularly to the direction of propagation in the optically transparent layer (a) excitation light coupled periodicity, which is characterized is that on each grating structure (c) a measuring region is arranged at a substantially perpendicular to the direction of propagation in the optically transparent layer (a) injected excitation light spatially varying periodicity at most, wherein on the grating waveguide structure in the direction of propagation to be coupled and in the layer (a) leading the excitation light waveguide structure adjoins a unstrukurierter area of ​​the grid, and it may be further guided in the direction of propagation in layer (a) excitation light, a further grating structure (c) ange reads, via which said guided excitation light towards a position-sensitive detector coupled out again. Such an embodiment may be designed such that changes in the mass density, or more generally of the local effective refractive index, by adsorption or desorption of molecules from the measurement areas on grating structures (c) a shift of the local position of the fulfillment of the resonant condition for coupling the excitation light into the layer (a) over said grating structure (c) is substantially lead parallel to the grating lines. In this case, such an embodiment of the inventive optical system is preferred which is characterized in that a one-dimensional arrangement of at least two grating structures (c) of the aforementioned embodiment will be simultaneously irradiated with excitation light. Furthermore, the excitation light is irradiated substantially in parallel, and is essentially monochromatic is preferred. In particular, it is advantageous if the excitation light is irradiated linearly polarized, for excitation of a in the layer (a) guided TE 0 - or TMO mode. a large number of such grating structures is advantageous in each case simultaneously irradiated, for example, a two-dimensional array of at least 4 such Gitterstrakturen.

Given layer and lattice parameters of a grating waveguide structure, there are various possibilities, the remaining free parameters to satisfy the resonance conditions of light or in coupling light out of a waveguide grating to vary structure. For a given, fixed wavelength, there are (a), for example in the case of a sufficiently thin waveguiding layer which only monomodal waveguide permits (TM 0 or TE 0) only a well-defined angle (based orthogonal to the plane of the grating waveguide structure plane parallel to the grating lines) at which the resonance condition is satisfied, with only a small, highly dependent on the width of the associated grating depth resonance curve. The change in the angle of incidence of an excitation light to a grating waveguide structure is thus a possible parameter for determining the resonance conditions.

the invention therefore furthermore relates to an optical system for spatially resolved determination of changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light by a two-dimensional array of at least four or more discrete measurement areas (d) on this platform, With

- at least one excitation light source

- an inventive grating waveguide structure

- a positioning member for varying the angle of incidence of the excitation light to the grating waveguide structure

- at least one spatially resolving detector for detecting the transmitted excitation light on the, with respect to the irradiated excitation light, opposite side of the grating waveguide structure and / or for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or for detecting the scattered light from coupling by means of a grating structure (c) in the layer (a) guided excitation light.

As already mentioned earlier, the boundary conditions listed in the positioning of the at least one laterally resolving detector on the same or opposite side of the grating waveguide structure, with respect to the irradiated excitation light, and (in dependence of the detected light component transmitted excitation light can or, parallel to the reflected portion again outcoupled excitation light) can be simplified by using a suitable in the beam path to be positioned projection screen.

Another object of the invention is therefore an optical system for spatially resolved determination of changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light by a two-dimensional array of at least four or more discrete measurement areas (d) on this platform , With

- at least one excitation light source of an inventive grating waveguide structure a positioning element for changing the angle of incidence of the Amegungslichts the grating waveguide structure

- a diffusely reflecting and / or diffusely transmissive projection wall on the, with respect to the irradiated excitation light, opposite side of the grating waveguide structure, to form an image of the transmitted Amegungslichts and at least one spatially resolving detector for detecting the image of the transmitted excitation light on said projection screen.

Often one would like to avoid the use of mechanical movable parts in a low-maintenance system as possible, since these often have a comparatively high wear. In addition, the time required for a high-precision mechanical positioning is not negligible. In pre-enclosed system parameters to a fixed predetermined angle of incidence of a Anregungshchts a grating waveguide structure, which is preferably set close to a suitable angle to meet the resonance conditions, lends itself to a variation of the irradiated excitation wavelength as an alternative.

A preferred embodiment consists in an optical system for spatially resolved determination of changes in the resonance conditions for coupling a Amegungslichts in a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on this platform, with at least a tunable over a certain spectral range

Excitation light source

- a erfindungdgemässen grating waveguide structure according to one of the aforementioned embodiments

- at least one spatially resolving detector for detecting the transmitted excitation light and / or for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or for detecting the scattered light from by coupling via a grating structure (c) in the layer (a) guided excitation light.

Depending on the respective parameters of a grating waveguide structure is well-defined for a particular structure equivalent to a change in the coupling angle or the wavelength of an incident excitation light is obtained. For example, waveguide structure of about 150 nm tantalum pentoxide 2.15 (on glass at 633 nm n =, for a grid (n = 1:52 (at 633 nm) having a lattice structure of 320 nm period grating depth typically 10 nm - 20 nm) a change in the coupling angle corresponding to 0.2 ° a change in a wavelength to be coupled to 1 nm for einzukoppelndes transverse electric polarized light. for such a structure, the resulting change in the coupling angle is in the application of a complete protein monolayer of similar magnitude.

It is preferred that said one tunable light source over a spectral range of at least 1 nm can be tuned at least.

It is particularly advantageous if said one tunable light source over a spectral range of at least 5 nm can be tuned at least.

At said at least one tunable light source may be a laser diode, for example.

Another possible alternative is that a polychromatic over the corresponding spectral light source is preferably used with a continuous spectrum within this region, in place of a tunable over a certain spectral range monochromatic light source. On the one hand it is possible in turn to produce an almost monochromatic, tunable excitation light by combining such a polychromatic light source having a high spectral resolution optical component in the excitation beam path, which can be used in a corresponding manner as the aforementioned variant. However, it is also possible to irradiate the polychromatic light of said spectral simultaneously on the grating waveguide structure. the invention further provides for an embodiment of an optical system for spatially resolved determination of changes in the resonance conditions for coupling a Amegungslichts in a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on this platform polychromatic, with at least one in a certain spectral range

Excitation light source. of an inventive grating waveguide structure according to one of the aforementioned

Embodiments, at least a spatially resolving detector for detecting the transmitted

Excitation light and / or for detecting the reflected light substantially parallel to the back out-coupled light on the, with respect to the irradiated

Excitation light, the same side of the grating waveguide structure and / or

Detecting the scattered light from coupling by means of a grating structure (c) in the

Layer (a) guided excitation light.

Again, it is preferred that said at least one polychromatic light source, an emission bandwidth of at least 1 nm. It is particularly advantageous if said at least one polychromatic light source has an emission band width of at least 5 nm.

This results in different possible variants of a measuring method based on such a novel optical system having a polychromatic light source, which are described further below.

It is preferred such embodiment of the inventive optical system with a polychromatic light source, which is characterized in that placed a spectrally selective optical component with high spectral resolution in said certain spectral range in the beam path between the grating waveguide structure and the at least one position-sensitive detector is. It is advantageous when said spectrally selective component is suitable for the generation of spectrally selective, spatially resolved, two-dimensional representations of the intensity distributions of light emanating from the grating waveguide structure measuring light at different wavelengths within said certain spectral range.

Particularly preferred is such an embodiment of an inventive optical system with a polychromatic within a certain spectral range Lichquelle, which is characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light, of said polychromatic light source in the field of measuring ranges, by simultaneous or sequential detection of the transmitted excitation light and / or by the simultaneous or sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or by the simultaneous or sequential detection of the scattered light from coupling by means of a grating structure (c) in the layer (a) excitation light guided by means within the said certain spectral spectrally selective detection using at least one laterally resolving detector, preferably with constant single beam angle of this excitation light on the grating waveguide structure is performed.

For many embodiments of the inventive optical system, that the excitation light is irradiated substantially in parallel is preferred. By "substantially parallel" light bundle is to be understood to mean that the convergence or divergence is less than 1 °. Accordingly, "substantially orthogonal" or "substantially normal" means a deviation from a corresponding orthogonal or normal orientation of less than 1 ° mean.

For most embodiments (except those that are based on a polychromatic light source) is also preferred that the excitation light is irradiated substantially monochromatic. By a "substantially monochromatic" excitation light is to be understood as meaning that its spectral bandwidth is less than 1 nm.

Furthermore, the excitation light is irradiated linearly polarized is preferred, carried out for exciting a in the layer (a) TE 0 - or TMO mode. In particular, object of the invention is one embodiment of an optical system, which is characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light in the region of the measuring ranges by sequential detection of the transmitted excitation light and / or by sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or by sequential detection of the scattered light from by coupling (via a grid structure c) (in the layer a) guided excitation light is performed with one or more spatially resolving detectors by changing the angle of incidence of the excitation light to the waveguide grating structure.

In addition to the already above-mentioned possibility of changing the angle of incidence using a positioning, z. As to perform a rotational movement of the grid structure with respect to the irradiated Amegungslichts can remove such a change in the angle of incidence by the grating waveguide structure, located in the beam path opto-mechanical components, such as movable mirrors or prisms occur. To execute only very small angle or position changes in particular, such components are suitable, which are driven by piezo actuators.

Another preferred embodiment of an inventive optical system, in particular to avoid mechanical moving parts, characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light in the ranges , by sequential detection of the transmitted excitation light and / or by sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated Amegungslichts, same side of the grating waveguide structure and / or by sequential detection of the scattered light from by coupling a grating structure (c) in the layer (a) guided excitation light each having one or a plurality of spatially resolving detectors while changing the emission wavelength of a tunable light source, preferably under konstan tem angle of incidence on the grating waveguide structure is performed by this excitation light.

For the above-mentioned embodiments of the inventive optical systems that the excitation light is expanded by at least one light source having an optical expansion unit as homogeneously as possible into a substantially parallel beam and is irradiated to the one or more measurement areas is preferred. It is advantageous if the diameter of the irradiated excitation light beam at least in one dimension at least 2 mm, preferably at least 10 mm.

Another preferred embodiment is characterized in that the excitation light from the at least one light source through one or, in case of multiple light sources, optionally a plurality of diffractive optical elements, preferably Dammann grating, or refractive optical elements, preferably microlenses array, into a plurality of individual beams of equal intensity as possible of data originating from a common light source is split sub-beams which are each substantially parallel to each other on grating structures (c) at the resonance angle for in-coupling into layer (a) are irradiated.

Another embodiment of an inventive optical system is characterized in that the excitation light from at least one, preferably monochromatic light source with a beam shaping optics to a beam of homogeneous intensity as possible, and gap-shaped cross-section is expanded (in a plane perpendicular to the optical axis of the optical path), the main axis parallel is aligned to the grating lines, wherein the sub-beams of said beam in a projection plane parallel to the plane of the grating waveguide structure are substantially parallel to each other while said radiation beam in a to the plane of the grating waveguide structure orthogonal plane convergence or divergence with a certain Konvergenzbzw. Divergence angle has.

It is preferred that said angle of convergence or divergence of said radiation beam has a value of up to 5 degrees in a direction orthogonal to the plane of the grating waveguide structure level. That said convergence or divergence angle of said radiation beam has a value of up to 1 ° in a direction orthogonal to the plane of the grating waveguide structure level is especially preferred.

Such an inventive optical system is characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light in the region of the measuring ranges, within a slit-shaped illuminated area according to the above embodiment, by simultaneously detection of the transmitted excitation light and / or by the simultaneous detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or by simultaneously detecting the scattered light from by coupling a Gitterstraktur (c) occurs in the layer (a) guided excitation light each having one or a plurality of spatially resolving detectors, where the local change in the resonance conditions in a measuring range in a shift of the M aximums of light emanating from said measuring area parallel to the reflected light substantially light as well as the maximum of the of said measuring range by coupling of a grating structure (c) in the layer (a) guided excitation light outgoing scattered light and the minimum of the transmitted in the area of ​​said measurement light region (in each case shows upon satisfaction of the conditions of resonance in said measuring range) wherein said displacement of the minimum or maximum in a plane is parallel to the plane of the grating waveguide structure perpendicular to the grating lines.

Such an optical system is also characterized in that the extent of the changes said resonance conditions and the changes in the effective refractive index in the range of said measurement area can be determined from the size of said shift of the minimum or maximum.

For certain applications it is preferred that be used with equal or different emission wavelength than the excitation light sources two or more coherent light sources. For such applications, in which two or more different excitation wavelengths to be used, such an embodiment of the optical system that the excitation light of 2 or more light sources from different directions simultaneously or sequentially on a grating structure (c) is preferred, which is characterized, irradiated and the grating waveguide Straktur is coupled via this in the layer (a) comprising a superposition of grating structures with different periodicity.

It is preferred that a spatially resolving detector is used for detecting at least, for example, from the group formed by CCD cameras, CCD chips, photodiode arrays, avalanche diode arrays, multichannel plates and multichannel photomultipliers.

According to this invention, the optical system includes those embodiments which are characterized in that in between the one or more excitation light sources and the inventive grating waveguide structure and / or between said grating waveguide Straktur and the one or more detectors, optical components the group are used, the lenses or lens systems for shaping the transmitted light bundles, planar or curved mirrors for the deviation and optionally additional shaping of the light bundles, prisms for the deviation and optionally spectral separation of the light bundles, dichroic mirrors for the spectrally selective deviation of parts of the light bundles, neutral filters for controlling the transmitted light intensity, optical filters or monochromators for the spectrally selective transmission of parts of light bundles or polarization-selective elements for selecting discrete Polarisat ionsrichtungen be formed of the excitation or luminescence light.

It is possible that the irradiation of the Amegungslichts fsec in pulses with a duration between 1 and 10 minutes is performed and the emission light is measured from the measurement areas resolved in time. In particular, the binding of one or more analytes to the Erkennunsgelemente in the various measuring ranges, with such embodiments, a spatially resolved be observed in real time. From the time-resolved signals picked up the respective binding kinetics can be determined. In particular, this allows, for example, be determined comparing the affinities of different ligands to a respective immobilized biological or biochemical or synthetic recognition elements. In this case, any binding partner of such an immobilized recognition element will be referred to in this context as a "ligand".

It is possible that the irradiation of the excitation light and detection of the emission light from one or more measurement areas is performed sequentially for one or more measurement areas. This can in particular be realized in that sequential excitation and detection is performed using movable optical components, which is formed from the group of mirrors, deviating prisms, and dichroic mirrors.

Part of the invention is also such an optical system, which is characterized in that sequential excitation and detection is performed using an essentially focus and angle-preserving scanner. Moreover, it is possible that the grating waveguide structure between steps of sequential excitation and detection is moved.

Another part of the invention is an optical system for spatially resolved determination of changes in the resonance conditions for coupling a Amegungslichts in a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on this platform, for the detection one or more analytes in at least one sample on one or more measurement areas on a grating waveguide structure, with

- an inventive grating waveguide Straktur

- an inventive optical system as in one of said Assuführangsformen and additionally

- to bring the feed means one or more samples with the measurement regions on the grating waveguide structure in contact.

The supplemented by the feeding means optical system will be referred to hereinafter as analytical system.

It is preferred that the analytical system in addition comprises one or more sample containers which are open at least in the area of ​​one or more measurement areas or combined to form segments ranges for lattice waveguide Straktur out wherein the sample containers preferably each have a volume of 0.1 nl - have 100 ul.

It is therefore preferred that the temperature of a novel analytical system can be kept constant or changed by appropriate measures in a controlled manner and set. This preferred possibility of temperature control and control comprises addition of an inventive grating waveguide Straktur according to one of the aforementioned embodiments, said sample containers the feeds or feed lines and optionally existing storage containers for samples and / or reagents, as well as optionally their storage places in front of an application in the according to the invention or analytical optical system.

A possible Ausführangsform of the invention the analytical system is that the sample containers on the optically transparent layer (a) side facing away from, with the exception of inlet and / or outlet openings for the supply or discharge of samples and optionally additional reagents are closed take place and the feed or the outlet of samples and optional additional reagents in a closed flow-through system, wherein in the case of liquid supply to a plurality of measurement regions or segments having common inlet and outlet openings preferably this column or addressed row by row.

Another possible Ausführangsform is characterized in that the sample containers remote from the optically transparent layer (a) side openings have the locally-addressed addition or removal of the samples or other reagents.

A further development of the invention the analytical system is designed so that containers are provided for reagents which are wetted during the process for the detection of one or more analytes and brought into contact with the measurement areas

Another object of the invention is a method for the qualitative and / or quantitative detection of one or more analytes in one or more samples in at least two or more discrete measurement areas on a novel grid waveguide Straktur according to one of the aforementioned embodiments, means of determination of changes in the resonance conditions for coupling an excitation light in a waveguide with an array of at least two or more laterally separated measurement areas (d) on this platform, characterized in that the excitation light from at least one excitation light source (c) on a lattice structure having thereon said measurement areas is conducted and the degree of compliance with the resonance condition for the coupling of light into the layer (a) to said measurement areas from the signal of at least one spatially resolving detector for detecting the transmitted excitation light on the, relative to d it incident excitation light, opposite side of the grating waveguide structure and / or for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating-waveguide structure and / or for detecting the scattered light by coupling of a grating structure (c) in the layer (a) guided excitation light is determined.

The invention also relates to a process for the qualitative and / or quantitative detection of one or more analytes in one or more samples in at least two or more discrete measurement areas on a novel grid waveguide structure according to one of the aforementioned embodiments in a novel optical system characterized on that grating waveguide Straktur, by means of determining changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) that the excitation light from at least one excitation light source on a grating structure (c) is conducted thereon with said measurement areas and the degree of compliance with the resonance condition for the coupling of light into the layer (a) to said measurement areas from the sign al of at least one spatially resolving detector for detecting the transmitted excitation light and / or for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated Amegungslichts, same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling of a grating structure (c) in the layer (a) guided excitation light is determined. Another object of the invention is a method for the qualitative and / or quantitative detection of one or more analytes in one or more samples in at least two or more discrete measurement areas on a grating waveguide structure with a substantially perpendicular to the direction of propagation of the optically transparent layer (a) injected excitation light spatially varying periodicity, characterized in that on each grating structure (c) a measuring region is arranged at a substantially perpendicular to the direction of propagation in the optically transparent layer (a) injected excitation light spatially varying periodicity at most, wherein leading to excitation light unstrukurierter a region of the grating waveguide Straktur adjoined on the grating waveguide structure in the direction of propagation to be coupled and in the layer (a), and optionally it further in the direction of propagation in the layer (a) guided excitation light is followed by a further grating structure (c), via which said guided excitation light in the direction of a spatially resolving detector coupled out again.

Such a method is characterized in that changes in the local effective index of refraction, especially in the mass occupation by adsorption or desorption of molecules from the measurement areas on Gitterstrakturen (c), (a shift in the local position of the fulfillment of the resonant condition for coupling the excitation light into the layer (a) over said grid structure c) essentially run parallel to the grating lines. It is preferred that a one-dimensional array of at least 2 such Gitterstrakturen (c) is simultaneously irradiated with excitation light. It is preferred that the excitation light is irradiated substantially in parallel, and is essentially monochromatic. In this case, operations for exciting a in the layer (a) TE 0 is advantageous if the excitation light is irradiated linearly polarized - or TM 0 -Modes. When a two-dimensional array of at least 4 such Gitterstrakturen (c) is simultaneously irradiated with excitation light is particularly preferred.

In particular, the invention also provides a process for the qualitative and / or quantitative detection of one or more analytes in one or more samples in at least two or more discrete measurement areas on a novel grid waveguide Straktur, by means of determining changes in the resonance conditions for coupling an excitation light in a waveguide having a two-dimensional array of at least four or more discrete measurement areas (d) is conducted on this platform, characterized in that the excitation light from at least one excitation light source to a Gitterstraktur (c) having thereon said measurement areas and degree of compliance with the resonance condition for the coupling of light into the layer (a) to said measurement areas from the signal of at least one spatially resolving detector for detecting the transmitted excitation light on the, with respect to the irradiated Anregu ngshchts, opposite side of the grating waveguide Straktur and / or for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light is determined and Straktur is changed by means of a positioning angle of incidence of the excitation light to the grating waveguide, so that said resonance condition at different angles in the range of different ranges on an irradiated Gitterstraktur (c), is a function of the local mass density, are satisfied.

a method for qualitative and / or quantitative detection of one or more analytes in one or more samples in at least two or more discrete measurement areas on a novel grid waveguide Straktur according to one of the aforementioned embodiments is preferable means of determining changes in the resonance conditions for coupling an excitation light into a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) marked on this platform, characterized in that the excitation light from at least one excitation light source to a Gitterstraktur (c) with is conducted thereon said measurement areas and the degree of compliance with the resonance condition for the coupling of light into the layer (a) to said measurement areas from the signal of at least one spatially resolving detector for detecting the transmitted Anregu ngslichts, optionally with the use of a diffusely reflecting and / or diffusely transmissive projection wall on the, with respect to the irradiated excitation light, opposite side of the grating waveguide Straktur, for forming an image of the transmitted Amegungslichts, and / or for detecting the substantially reflected parallel to the light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or for detecting the scattered light from by coupling of a grating structure (c) in the layer (a) guided excitation light is determined, and by means of a positioning element waveguide Straktur changing the irradiation angle of the Amegungslichts on the grid, so that said resonance condition at different angles in the range of different ranges on an irradiated grating structure (c), depending on the local effective refractive Sinde x is satisfied.

Again, it is preferred that the excitation light is irradiated substantially in parallel, and is essentially monochromatic. Particularly advantageous for excitation of a guided in the layer (a) TE 0 is, when the excitation light is irradiated linearly polarized, - or TM 0 -Modes.

A further preferred embodiment of the inventive method is that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) in the region of the measuring ranges by sequential detection of the transmitted excitation light on the, with respect to the irradiated excitation light, opposite side of the grid - waveguide structure and / or by sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or by sequential detection of the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light is performed with one or more spatially resolving detectors by changing the angle of incidence of the excitation light to the grating waveguide Straktur.

A preferred Ausführangsform of the inventive method is characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light in the region of the measuring ranges by sequential detection of the transmitted Amegungslichts and / or by sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or by sequential detection of the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light is performed with one or more spatially resolving detectors by changing the angle of incidence of the excitation light to the waveguide grating structure.

It is preferred that an image of the transmitted Amegungslichts on a diffusely reflective and / or diffusely transmissive projection wall on the, with respect to the irradiated Amegungslichts, opposite side of the grating waveguide Straktur is generated, and this image is captured with at least one position-sensitive detector.

A particularly preferred Ausführangsform this method is characterized in that the angle of incidence of the Amegungslichts is set to the grating waveguide Straktur so that the resonance condition for coupling an excitation light in a waveguide a grating waveguide structure or coupling out a guided in the waveguide light, with an array of at least two or more laterally separated measurement areas (d) on that grating waveguide structure, on one or more of these measurement areas is substantially met with the consequence of a substantially maximum signal of a spatially resolving detector for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light, is satisfied in the region of these ranges, and / or a substantially minimum signal of a spatially resolving detector for detecting the transmitted excitation light in the range of the measurement areas, or between the measurement areas substantially, with the consequence of a substantially maximum signal of a spatially resolving detector for detecting the in substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light, from the areas between these ranges, and / or a substantially minimum signal of a spatially resolving detector for detecting the transmitted excitation light out of the areas between these ranges.

Understanding that these differences to meet the resonance conditions on the irradiated with excitation light the region of the grating waveguide Straktur less than half the width of the resonance curve of the coupling angle, are under the respective conditions, it may be made of the intensity of the jeweiliegen measuring light is a clear correlation between this intensity and the degree of compliance with the resonance condition are derived, so that a sequential recording of the resonance curves, for example by changing the angle of incidence on the grating waveguide Straktur or by changing the irradiated wavelength is not required, but the information on the local degree of fulfillment the resonance conditions and thus can be obtained about the local effective refractive index with a single image capture.

Therefore, it is preferred that local differences in the effective refractive index in the range of different measuring ranges, and in the areas between the measuring ranges from local differences in the intensities of one or more position-sensitive detectors for detecting the transmitted excitation light and / or for detecting the substantially reflected parallel to the light again the light coupled to the respect of the irradiated Amegungslichts, same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light can be determined without the set angle of incidence of the excitation light is changed on the grating waveguide Straktur.

Another preferred Ausführangsform of the inventive method is characterized in that the spatially resolved determination of changes in the resonance conditions for coupling a Amegungslichts in the layer (a) or decoupling of a guided in the waveguide light from at least one over a certain spectral range tunable light source in the region of the measuring ranges by sequential detection of the transmitted excitation light and / or by sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or by sequential detection of the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light each having one or a plurality of spatially resolving detectors by changing the emission wavelength of said at least one tunable Lic , Preferably at a constant angle of incidence of this excitation light on the grating waveguide structure is performed htquelle. The change in emission wavelength of a tunable light source for determining local differences of the resonance condition, instead of a Veränderang the angle of incidence has the designated advantage of avoiding mechanical movable components. This method can offer at a lower system cost also the considerable advantage of a possible higher resolution: In typical commercial laser diodes, the emitted laser wavelength can for example be controlled very precisely on the supplied operating current. Thus, the generation of an extremely precise determinable excitation wavelength may be substantially less expensive than a high-resolution angular adjustment and angle determination with opto-mechanical components.

It is preferred that said one tunable light source over a spectral range of at least 1 nm can be tuned at least.

It is particularly advantageous if said one tunable light source over a spectral range of at least 5 nm can be tuned at least.

At said at least one tunable light source may be a laser diode, for example.

A further preferred embodiment of the method is characterized in that an image of the transmitted excitation light on a diffusely reflecting and / or diffusely transmissive projection wall on the, with respect to the irradiated Amegungslichts, opposite side of the grating waveguide Straktur is generated and this with at least one spatially resolving detector is detected.

A further preferred Ausführangsform of the method is that the emission wavelength of at least one tunable light source, preferably this Amegungslichts is set to the grid waveguide Straktur so at a constant angle of incidence that the resonance condition for coupling an excitation light in a waveguide a grating waveguide Straktur or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) is met on this grating waveguide-Straktur, on one or more of these measuring ranges substantially, with the consequence of a substantially maximum signal of a spatially resolving detector for detecting the reflected light substantially parallel to the back out-coupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from after Einkop plung a Gitterstraktur (c) in the layer (a) guided excitation light, from the region of these ranges, and / or a substantially minimum signal of a spatially resolving detector for detecting the transmitted excitation light in the range of the measurement areas, or between the measurement areas substantially met with the consequence of a substantially maximum signal of a spatially resolving detector for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling a grating structure (c) in the layer (a) guided excitation light from the areas between these ranges, and / or a substantially minimum signal of a spatially resolving detector for detecting the transmitted excitation light out of the areas between these ranges.

Understanding that these differences to meet the resonance conditions on the irradiated with excitation light area of ​​the grating waveguide Straktur less than half the width of the resonance curve of the coupling wavelength (in place of the coupling angle for the case of constant irradiation angle, but variable excitation wavelength), under the respective conditions, are , besides, can in turn from the intensity of jeweiliegen measuring light is a clear correlation between this intensity and the degree of compliance with the resonance condition are derived, so that a sequential recording of the resonance curves, for example by Veränderang the irradiated wavelength is not required, but the information on the local degree of compliance with the resonance conditions and thus can be obtained about the local effective refractive index with a single image capture.

Therefore, it is preferred that local differences in the effective refractive index in the range of different measuring ranges, and in the areas between the measuring ranges from local differences in the intensities of one or more position-sensitive detectors for detecting the transmitted excitation light and / or for detecting the substantially reflected parallel to the light again the light coupled to the be determined with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or for detecting the scattered light from by coupling of a grating structure (c) in the layer (a) led Amegungslicht without the emission wavelength of the tunable light source is changed.

For the above-mentioned embodiments of the inventive method that the Amegungslicht is each substantially parallel irradiated and is essentially monochromatic is preferred. In addition, the excitation light is irradiated linearly polarized is preferred, for excitation of a layer (a) guided TE 0 - 0 or TM -Modes.

Another Ausführangsform of the inventive method is characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light from at least one polychromatic within a certain spectral range of light source in the region of the measuring ranges by detecting of the transmitted excitation light and / or by detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated Amegungslichts, same side of the grating waveguide structure and / or by detecting the scattered light from by coupling of a grating structure (c) each having one or a plurality of spatially resolving detectors, preferably at a constant angle of incidence on the grating waveguide Straktur, takes place in the layer (a) guided excitation light of this excitation light, wherein each of d en regions in which for a particular wavelength of the excitation light from the polychromatic light source, the resonance condition is satisfied for coupling said excitation light into a waveguide of the grating waveguide Straktur or coupling out a guided in the waveguide light of this wavelength, a maximum signal component of that wavelength at the signal of a spatially resolving detector for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer ( a) guided excitation light, from the region of these ranges, and / or a minimum signal component of that wavelength is produced at the signal of a spatially resolving detector for detecting the transmitted Amegungslichts in the range of the measurement areas. Again, it is preferred that said at least one polychromatic light source, an emission bandwidth of at least 1 nm. It is particularly advantageous if said at least one polychromatic light source has an emission band width of at least 5 nm.

It is preferred such Ausführangsform of the inventive method with a polychromatic light source, which is characterized in that there is arranged a spectrally selective optical component with high spectral resolution in said certain spectral range in the beam path between the grating waveguide Straktur and the at least one position-sensitive detector , It is advantageous when said spectrally selective component is suitable for the generation of spectrally selective, spatially resolved, two-dimensional representations of the intensity distributions of the end of the grating waveguide Straktur measuring light at different wavelengths within said certain spectral range.

In order for such Ausführangsform of the inventive method is made possible, which is characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light of said polychromatic light source in the field of measuring ranges, by simultaneous or sequential detection of the transmitted Amegungslichts and / or by the simultaneous or sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or by the simultaneous or sequential detection of the scattered light from by coupling a Gitterstraktur (c) in the layer (a) excitation light guided by means of spectrally selective within said certain spectral detection using at least one it, preferably at a constant angle of incidence of this excitation light on the grating waveguide Straktur, carried out spatially resolving detector.

For the above-mentioned embodiments of the inventive method with a polychromatic light source is also preferred that the excitation light is irradiated in each case substantially parallel. that the excitation light is expanded by at least one light source having an optical expansion unit as homogeneously as possible into a substantially parallel beam and is irradiated to the one or more measurement areas is particularly preferred for a variety of embodiments of the inventive method. It is preferred that the diameter of an incident excitation light beam at least in one dimension at least 2 mm, preferably at least 10 mm.

Another Ausführangsform of the inventive method is characterized, in that the excitation light from the at least one light source through one or, in case of multiple light sources, optionally a plurality of diffractive optical elements, preferably Dammann grating, or refractive optical elements, preferably microlenses array, in a plurality of individual beams of equal intensity as possible of data originating from a common light source is split sub-beams which are each substantially irradiated to each other on laterally separated measurement areas in parallel.

Another Ausführangsform of the inventive method for the qualitative and / or quantitative detection of one or more analytes in one or more samples in at least two or more spatially separated measurement areas on an inventive grating waveguide-Straktur according to one of the aforementioned embodiments in a novel optical system, means of determining changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on that grating waveguide-Straktur, characterized in that the excitation light from at least one, preferably monochromatic light source (with a beam shaping optics to a beam of homogeneous intensity as possible, and gap-shaped cross-section in a plane perpendicular to the optical axis of the beam path ) Is expanded, its main axis is aligned parallel to the grating lines, wherein the sub-beams of said beam in a projection plane parallel to the plane of the grating-waveguide structure are substantially parallel to each other while said radiation beam in a to the plane of the grating waveguide Straktur orthogonal plane convergence or divergence with a certain Konvergenzbzw. Divergence angle has. It is preferred that said angle of convergence or divergence of said radiation beam has a value of up to 5 degrees in a direction orthogonal to the plane of the grating waveguide Straktur level.

That said convergence or Divergenzwinkelsbesagten beam has a value of up to 1 ° in a direction orthogonal to the plane of the grating waveguide Straktur level is especially preferred.

Such an inventive method is characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light, simultaneously in the area of ​​the measuring ranges, within a slit-shaped illuminated area according to the above Ausführangsform by detecting of the transmitted excitation light and / or by the simultaneous detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or by simultaneously detecting the scattered light from by coupling (a Gitterstraktur c) is carried out (in the layer a) guided excitation light each having one or a plurality of spatially resolving detectors, where the local change in the resonance conditions in a measuring range in a shift of the maximum s of light emanating from said measuring area parallel to the reflected light substantially light as well as the maximum of the of said measuring range by coupling a Gitterstraktur (c) in the layer (a) led Amegungslicht outgoing scattered light and the minimum of the said in the range measuring range of transmitted light (in each case shows upon satisfaction of the conditions of resonance in said measuring range) wherein said displacement of the minimum or maximum in a plane is parallel to the plane of the grating waveguide-Straktur perpendicular to the grating lines.

This method is also characterized in that the extent of the changes said resonance conditions and the changes in the effective refractive index in the range of said measurement area can be determined from the size of said shift of the minimum or maximum. This inventive method also comprises an embodiment which is characterized in that the spatially resolved determination of changes said resonance conditions in each case simultaneously in the field of measuring areas within a gap-shaped, convergent with an orthogonal in the plane of the grating waveguide structure level within a certain angular range or divergent beam according to one of the aforementioned embodiments of this method, the illuminated area, by the simultaneous detection of the transmitted Amegungslichts and / or by the simultaneous detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated Amegungslichts, same side of the grating waveguide - Straktur and / or by simultaneously detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light with one or more ren spatially resolving detectors, whereby the local change in the resonance conditions in a measuring region in a shift of the maximum of light emanating from said measuring area parallel to the reflected light substantially light as well as the maximum of the of said measuring range by coupling a Gitterstraktur (c) in the layer (a) guided excitation light outgoing scattered light and the minimum of the transmitted in the area of ​​said measurement light range, showing (in each case in compliance with the resonance conditions in said measuring range) wherein said displacement of the minimum or maximum in a plane perpendicular parallel to the plane of the grating waveguide Straktur is carried to the grid lines, and wherein the grating waveguide Straktur for sequentially spatially resolved determination of changes in said boundary conditions on the entire surface of said grating waveguide Straktur with the thereon ranges between individual process steps after said process perpendicularly and / or parallel to the orientation of the grating lines is shifted to the measurement signals are recorded of all measuring ranges, and a two-dimensional representation of the degree of fulfillment of said resonance conditions can be produced over the entire grating waveguide-Straktur from the recorded signals.

The inventive method according to the aforementioned embodiments, is characterized in that the spatial resolution for the determination of the degree of compliance with the resonance condition for the coupling of light into the layer (a) by choosing a larger modulation depth of Gitterstrakturen (c) improved or selection of a smaller modulation depth of said Gitterstrakturen can be reduced.

Furthermore, the inventive method is characterized in that the half-width of the resonance angle to fulfill the resonance condition for the coupling of light into the layer (a) by decreasing the modulation depth of Gitterstrakturen (c) can be reduced, resulting in increased sensitivity in the spatially resolved determination of changes in the degree of fulfillment of the condition of resonance as a consequence of local changes in the mass occupation, or more generally of the local effective refractive index, has the result or said Gitterstrakturen can be increased by increasing the modulation depth, resulting in reduced sensitivity for the spatially resolved determination of changes of the degree of compliance with the has resonance condition as a result of local changes in the mass occupation, or more generally of the local effective refractive index, result.

In particular, it can improve the sensitivity, ie to reduce the half-width of the resonance angle of advantage that the excitation light linearly polarized for exciting a in the layer (a) to irradiate guided TMO mode, since - with the same grating depth and the same thickness of the waveguiding layer ( a) typically of the resonance angle for the excitation of a TM 0 -Modes by a factor of 5 more sharply defined to 10, ie, the corresponding half-value width is lower by this factor than that half value width of exciting a TEO mode.

A preferred Ausführangsform of the inventive method is characterized in that the degree of fulfillment of the resonance condition for the coupling of light into the layer (a) to the measurement areas from the intensity of substantially (parallel to the reflected light again decoupled excitation light that is the sum of both shares) is determined.

Another preferred Ausführangsform the method is characterized in that the degree of fulfillment of the resonance condition for the coupling of light into the layer (a) is determined to the measurement areas of the intensity of the transmitted excitation light. The former Ausführangsform is characterized in that the local meet the resonance condition for the coupling of light into the layer (a) to a range from a maximum of the sum of the intensities of the reflected and substantially parallel thereto again decoupled excitation light is determined from this measurement range.

The following stated Ausführangsform of the inventive method is characterized in that the local meet the resonance condition for the coupling of light into the layer (a) is determined to be a range from a minimum of the intensity of the transmitted excitation light in this measurement range. In ideal cases, the intensity of the transmitted excitation light can thereby fall almost to zero.

Several embodiments of the inventive method are characterized in that differences in the effective refractive index, especially in the mass assignment, within a measuring range can be resolved. Therefore, it can surprisingly be achieved with a grating-based, image-forming method, a spatial resolution that is equal to the resolution of the best in use today scanner for analyte by fluorescence detection.

In another Ausführangsform of the inventive process is preferred that can be used with the same or different emission wavelength than the excitation light sources two or more coherent light sources.

As mentioned previously, a major advantage of the inventive method is that a use of any label (to the analyte or its analogues or its binding partner binding to tag molecules) is basically not necessary. To increase the sensitivity of a further development of the method may be advantageous, which is characterized in that the change in the mass occupation to enlarge upon binding or dissociation to be detected analyte molecules to this or to one of its binding partner in a multi-step assay, a mass label is attached, which, for example, may be selected from the group of metal colloids (z. B. gold colloids), plastic particles or -Beads or other microparticles having a monodisperse size distribution. Part of the inventive process is also a Ausführangsform, which is characterized in that the change in the effective refractive index at the binding or dissociation to be detected analyte molecules to this or to one of its binding partner in a multistage assay to Vergrösserang is bound a "Absorption label", said " absorption label "having an absorption band of suitable wavelength, which absorption, the imaginary part of the refractive index, resulting in a change of the effective refractive index in the near field of the grating waveguide Straktur. The mathematical and physical methods for the conversion of the effect of absorption at a specific wavelength on the refractive index as a function of wavelength, are known from the literature.

A further development of the inventive method is characterized, in that in addition to the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) of an inventive grating waveguide-Straktur or coupling out a in the layer (a) guided light additionally comprises one or a plurality of guided in the evanescent field of the layer (a) excitation light excited luminescences are determined from one or more measurement areas.

This further development, as a combined imaging method of a spatially resolved determination of the effective refractive index and a spatially resolved luminescence measurement, it allows, for example, the binding of a ligand analyte to an immobilized in one or more measurement areas of biological or biochemical or synthetic recognition element as a receptor based on the local change of the to determine the effective refractive index and to determine a functional response of this ligand-receptor system based on a change in luminescence from said measurement areas.

For example, it may be a transmembrane receptor protein wherein said receptor-ligand system to which a corresponding ligand binds from a supplied sample. A functional response of this receptor-ligand system may consist, for example in the opening of an ion channel, with the consequence of local Veränderang of the pH and / or ionic concentration. Such a local change can be made, for example, by using a luminescent dye with pH-dependent and / or ion-dependent luminescence intensity and / or spectral emission.

Likewise, this inventive combined measuring method enables, for example, the density of the immobilized biological or biochemical or synthetic recognition elements as receptors in one or more measurement areas on the basis of differences between the resonance conditions for coupling an excitation light in the layer (a) of the grating waveguide Straktur or decoupling of a in the layer (a) guided light in the region of these ranges, and the respective resonance conditions in the environment, ie, to determine outside said ranges, and to determine the binding of a ligand analyte to these recognition elements based on a Lumineszenzänderang from said measurement areas.

It is possible that (1) the isotropically emitted luminescence or (2) into the optically transparent layer (a) injected and Gitterstrakturen (c) is coupled out or luminescence comprising both parts (1) and (2) are measured simultaneously.

In the inventive method, a luminescent or fluorescent label can be used to produce the luminescence or fluorescence can be excited at a wavelength between 300 nm and 1100 nm and emitted.

The Lumineszenzoder fluorescence labels can be conventional Lumineszenzoder fluorescent dyes or even 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 mass labels and / or the luminescence labels can be bound to the analyte or, in a competitive assay to an analogue of the analyte, or in a multistage assay to one of the binding partners of the immobilized biological or biochemical or synthetic recognition elements or to the biological or biochemical or synthetic recognition elements. In addition, it may be advantageous if the one or more luminescences and / or determinations of light signals at the excitation wavelengths are performed polarization-selective. Furthermore, the method allows for the possibility that the one or more luminescences are measured at a polarization different from that of the excitation light.

The inventive method according to one of the above embodiments allows a 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 analogues, enzymes, enzyme cofactors or inhibitors, lectins and carbohydrates.

The test samples can be naturally occurring body fluids such as blood, serum, plasma, lymph or urine or egg yolk.

but to be examined sample may also be an optically turbid liquid, surface water, a soil or plant extract, a bio- or synthesis process broth.

The test samples may be taken from biological tissue parts.

The present invention further relates to the use of an inventive grating waveguide-Straktur and / or an inventive optical system and / or an inventive analytical system and / or an inventive method according to one of the above embodiments for the determination of chemical, biochemical or biological analytes in screening methods in pharmaceutical research, combinatorial chemistry, clinical and preclinical development, for qualitative to real-time binding studies and the determination of kinetic parameters in Affinitätsscreenmg and research and quantitative analyte determinations, especially for DNA and RNA analysis, for the generation of toxicity studies and 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 pre-symptomatic plant diagnostics, for patient stratification in pharmaceutical product development and for the therapeutic drug selection, for the detection of pathogens, pollutants and pathogens, especially of salmonella, prions and bacteria, in food and environmental analytics.

With the following Ausführangsbeispielen The invention will be explained in more detail and demonstrated.

Example 1: a) grating waveguide structure

It has a grid waveguide structure with the exterior dimensions of 16 mm width x 48 mm length × 0.7 mm thickness. The substrate material (optically transparent layer (b)) was 45 glass from AF (refractive index n = 1:52 at 633 nm). In the substrate, a continuous structure of a Oberflächenrehefgitters a period of 360 nm and a depth of 25 +/- 5 nm was formed by means of holographic exposure of the layer (b) and subsequent etching with orientation of the grating lines parallel to the stated width of the sensor platform. The waveguiding, optically transparent layer (a) on the optically transparent layer (b) of Ta 2 O 5 was carried reactive, magnetic field-assisted DC sputtering (see DE 4410258) have been generated, and had a refractive index of 2.15 at 633 nm (layer thickness 150 nm ). Under incoupling excitation light of 633 nm can be coupled at an angle of about + 3 ° to the normal of the structure in the layer (a) (and coupled) to.

To prepare for the immobilisation of biochemical or biological or synthetic recognition elements, the grating waveguide structure was cleaned and (with epoxysilane in the liquid phase of 10 ml (2% v / v) 3-glycidyloxypropyl trimethoxysilane and 1 ml (0.2% v / v) N- ethyldiisopropylamine in 500 ml of ortho-xylene silanized (7 hours at 70 ° C) Then, using a commercial Spotter (Genetic Microsystems 417 arrayer), solutions of 18-mer oligonucleotides (5'-CCGTAACCTCATGATATT-3'-NH 2) (18 *. - NH2) two arrays, each 16 x 8 spots (8 rows x 16 columns) is applied (50 pl per spot). the concentration of the applied solution was 5 x 10 "8 M 18 * -NH 2, so that the spots generated ( about 125 microns in diameter in a Zentram-to-Zentram- spacing of 370 microns) as the measurement regions have a mass density of about 600,000 Da microns 2, corresponding to approximately 1 pg / mm 2 exhibited.

b) Optical system

As the excitation light source is a He-Ne laser with 1.1 mW output power (Melles Griot-, 05-LHP-901) was used. The polarization of the laser was aligned parallel to the grating lines of the grating waveguide structure, for exciting the TEO mode under coupling conditions. The laser beam was expanded seven times with a beam expander, and passed through an orifice of 5 mm diameter to diskrminieren outer, weaker portions of the expanded laser beam as well as external diffraction phenomena. The laser light was then strongly attenuated with a neutral density filter (ND 4.7) in order to avoid a saturation of the detector during the measurement of the transmitted light portion. The laser light was (facing substrate side AF45 glass) on the side of the optically transparent layer (b) where the power attenuation amount to about 20 nW.

The grating waveguide Straktur was mounted within an opening located substantially perpendicular to the optical axis of the excitation light plane on a manually adjustable goniometer with the left, the angle of incidence of the excitation light change with respect to the sensor platform, where the grating lines perpendicular to the projection of the excitation light in the plane the grating waveguide Straktur proceeded.

As a position-sensitive detector, a CCD camera (Ultra Pix 0401E, Astrocam, Cambridge, UK) was used with Peltier cooling, with a Kodak CCD chip KAF 0401 El. The camera was for the spatially resolved determination of the intensity of the transmission light, after the passage of the excitation light by the optically transparent waveguiding layer (a) oriented so that the transmitted light substantially perpendicularly fell on the entrance lens of the camera.

c) measurement procedures and results

The measurement method is that no additional sample containers or supplied reagents, carried out in air. The fulfillment of the resonance condition on the free movement of measuring ranges areas of the grating waveguide-Straktur is determine at the almost complete disappearance of the transmitted light (Fig. La), where under the same conditions, the non-fulfillment of the resonance condition in the ranges where there surprisingly clear shows increased transmission signal (FIG. 1a and FIG. lb with a linear section of the signals by two measuring areas). The sharp contrast and high spatial resolution are very surprising, as is shown in Fig. Lb to be removed observation that (an expected after the deposition process inhomogeneous mass distribution within a measurement range, with a maximum approximately in the center) can be also resolved by this measuring method. also is very surprising the extremely high sensitivity, which enables the differences in mass density (between the areas of the spots and the surrounding regions) of lpg / mπT, with an excellent contrast.

Furthermore, it was surprisingly found that upon adjustment of the coupling angle to fulfill the resonance condition in the measurement areas, this can still be detected even at the local minima of the transmission (Figures 2a and 2b;. The two spots are shown in Figures by the distance marked "370 microns" lifted. this observation is surprising however, since the optical system for this measurement was not optimized, as shown by the superimposed strong interference phenomena in Fig. 2a. (these are caused by the not in physical effects of the inventive grating waveguide structure or according to the invention the optical system, but in the provisional nature of the structure).

Example 2: a) grating waveguide structure

It has x 48 mm length × 0.7 mm thickness, a grating waveguide-Straktur with the exterior dimensions 16 mm width. The substrate material (optically transparent layer (b)) was 45 glass from AF (refractive index n = 1.525 at 532 nm). In the substrate, a continuous structure of a Oberflächenrehefgitters a period of 360 nm and a depth of 25 nm was formed again, in parallel with orientation of the grating lines for the designated width of the sensor platform. The then it waveguiding, optically transparent layer (a) on the optically transparent layer (b) of Ta 2 O 5 had a refractive index of 2.137 at 532 nm (layer thickness 150 nm). Under launch conditions Amegungslicht of 532 nm can be at an angle of about + 14.3 ° to the normal of the structure is coupled in the structure (and coupled). To prepare for the immobilization of the biological or biochemical or synthetic recognition elements, the grating waveguide Straktur was purified. Thereafter (GeSim) solutions of NeutrAvidin ™ in an array of 3 x 3 spots (3 rows x 3 columns) were coated with a commercial Spotter applied to the cleaned surface of tantalum pentoxide (500 pl per spot). The concentration of the solutions applied at this time was 1.7 x 10 "5 M NeutrAvidin ™, so that the generated spots (about 430 microns in diameter in a Zentram-to-Zentram- distance of 1 mm) as the measurement regions of an area density of about 4 ng / mm second

b) Optical system

As the excitation light source is a diode-pumped, frequency-doubled NdYag laser with 10 mW output power was used (Laser 2000). The polarization of the laser was ausgereichtet perpendicular to the grating lines of the grating waveguide Straktur, for excitation of the TMO mode under coupling conditions. The laser beam was expanded seven times with a beam expander, and passed through a gap of 4 mm width to diskrminieren outer, weaker portions of the expanded laser beam as well as external diffraction phenomena. The laser light was on the side of the optically transparent layer (b) (substrate side AF45 glass directed).

The grating waveguide Straktur was mounted on a manually adjustable goniometer with which the single beam could be change angle of the excitation light with respect to the sensor platform, that the grating lines were perpendicular to the projection of the excitation light in the plane of the grating waveguide Straktur. A leaflets highly fine white paper was less graininess as a diffusely reflecting projection wall on the, with respect to the irradiated excitation light, opposite side of the grating waveguide-Straktur, for forming an image of the transmitted excitation light is mounted. Since the transmitted excitation light having a practically perfect parallel beam path was the distance from the substantially aligned parallel to grating waveguide-Straktur over a large range, that is between sub-millimeters and decimeters, freely selectable without significant loss of contrast or Konturverzerrangen. As a position-sensitive detector, a CCD camera (Ultra Pix 0401E, Astrocam, Cambridge, UK) was used with Peltier cooling, with a Kodak CCD chip KAF 0401 El. The camera was for the spatially resolved determination of the transmitted excitation light, by detecting the image on the above-mentioned projection screen, and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) led Amegungslicht and / or for detecting the substantially parallel to the reflected light again outcoupled light on the mounted with respect to the irradiated excitation light, the same side of the grating waveguide Straktur.

c) measurement procedures and results

The measurement method is that no additional sample containers or supplied reagents, carried out in air. It was to fulfill the resonance condition in the layer (a) a difference in the coupling angle of 0.124 °, found between coupling and coupling on the measurement areas on the uncoated areas of lattice-wave conductor Straktur.

In Fig. 3, the results of the measurement method for the spatially resolved determination of the transmitted excitation light, represented by means of sensing the image on the aforementioned projection screen and positioning of the camera on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur.

The fulfillment of the resonance condition to the free of measurement areas areas of lattice waveguide structure is wiederam determine where extensive disappearance of the transmission light (at an angle of 14.3 °, Fig. 3, left, and Fig. 3B), wherein. shows under the same conditions, the failure to fulfill the resonance condition in the measurement areas at the higher by a factor of 3 transmission signal (Fig. 3B, and the left part of Fig. 3).

Fig. 3C shows the reverse situation, that meet the resonant condition for coupling light into the layer (a) in the region of the measuring ranges (at an angle of 14.424 °, see Fig. 3 left), with the result minimum transmission at this angle in the ranges and non-fulfillment of the resonance condition in the other areas, with the result of maximum transmission. From Fig. 3C it can be seen with reference to concentrically occur, as a dotted circle-like lines within the dark occurring ranges near its outer edges noticeable, lighter areas that even under these conditions (transverse magnetic polarized excitation guided modes) optionally a spatial resolution significantly below the spot diameter is: the different areas of brightness within the spots show geometric inhomogeneity of the quantities locally adsorbieter or immobilized proteins or recognition elements. The occurrence of such inhomogeneities in the fabrication of arrays of immobilized recognition elements is known from the literature. - With the use of transverse electric-polarized instead of transversely magnetically polarized excitation light of the same wavelength with the same sensor platform, the high spatial resolution showed more pronounced (not shown).

Example 3: Uniformity of the resonance angle for light entry or -auskopplung on a Fläcche corresponding to an array of measurement areas

It is used (with a full area modulated grid) with the same predetermined shift and lattice parameters as in Example a) a grating waveguide Straktur. On a square area of ​​5 mm x 5 mm, corresponding to a typical base is an optionally on such a structure to be formed array of measurement areas, the variability of the coupling angle in the x- and y-direction (x soll: perpendicular to the grating lines, y: be examined parallel to the grating lines).

The parallel excitation light beam of a He-Ne laser (633 nm, 0.8 mm beam diameter) is at an angle close to the resonance angle for light into the layer (a) directed to the structure. In an angular range of about 1 ° above and below the resonance angle of incidence is varied in small steps (step width, for example 0.02 °). The intensity of the scattered light of the by coupling via the Gitterstraktur in the layer (a) is collected guided light with a lens system and focused onto a photomultiplier, as an integral, non-spatially resolving detector, respectively. With an aperture in the intermediate image plane, the size of the area imaged on the detector, the grating waveguide-Straktur be limited to reduce (in this example, on a circle of 1 mm in diameter), in particular to unwanted light scattering effects. The optimum adjustment to meet the resonance condition for the coupling of light into the layer (a) can be recognized by a maximum value of L. For the resonance curves of L as a function of the coupling angle, the half-width of the resonance curves associated may be determined in addition.

The measurement method described above was Messpositio-NEN performed for 25 (5 x 5) on said surface of the grating waveguide-Straktur, in a respective (Zentram-to Zentram-) distance of 1 mm. The resonance angle of the different measurement positions in the said x / y grid are summarized in Table 1 below. On the entire surface of the deviation from the mean value (in this example, 2.15 °) is not more than 0.06 °.

Table 1: variability of the resonance angle for optimum light entry and -auskopplung on a square area of ​​5 mm x 5 mm of an inventive grating waveguide-Straktur (before generation of the measurement areas thereon).

Claims

claims
1. grating waveguide-Straktur for the spatially resolved determination of changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on this platform comprising an optical film waveguide
- with a first optically transparent layer (a) on a second optically transparent layer (b) with lower refractive index than layer (a),
- with one or mehrereren Gitterstrakturen (c) for the coupling of excitation light to the measurement areas (d) or coupling-out of the layer (a) guided light in the ranges
- at least two or more laterally separated measurement areas (d) on the one or more Gitterstrakturen (c)
- immobilized in these measurement areas, the same or different biological or biochemical or synthetic recognition elements (e) for the qualitative and / or quantitative detection of one or more analytes in a brought into contact with the measurement areas sample, characterized in that said excitation light simultaneously onto said array of measuring ranges is irradiated and the degree of compliance with the resonance condition for the coupling of light into the layer (a) to the two or more measurement areas is measured simultaneously, and crosstalk from the layer (a) guided excitation light from a measurement range to one or more adjacent measuring ranges by again, this coupling out the excitation light is prevented by means of the Gitterstraktur (c).
2. grating waveguide-Straktur for the spatially resolved determination of changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light by a two-dimensional array of at least four or more discrete measurement areas (d) on this platform comprising a optical film waveguide
- with a first optically transparent layer (a) on a second optically transparent layer (b) with lower refractive index than layer (a), with one or mehrereren Gitterstrakturen (c) for coupling Amegungslicht to the measurement areas (d) or coupling out in the layer (a) guided light in the ranges
- at least two or more laterally separated measurement areas (d) on the one or more Gitterstrakturen (c)
- immobilized in these measurement areas, the same or different biological or biochemical or synthetic recognition elements (e) for the qualitative and / or quantitative detection of one or more analytes in a brought into contact with the measurement areas sample, characterized in that the density of the measurement areas on a common Gitterstraktrar (c) is at least 10 measuring zones per square centimeter, and that said Amegungslicht is irradiated simultaneously onto said array of measurement areas and the degree of compliance with the resonance condition for the coupling of light into the layer (a) to said measurement areas is measured simultaneously, and crosstalk from the layer (a) guided excitation light is prevented by a measurement range to one or more adjacent measurement areas by re-extraction of this excitation light by means of the Gitterstraktur (c).
3. grating waveguide-Straktur according to any one of claims 1 -2, characterized in that a continuously modulated Gitterstraktur (c) extends substantially over the entire area of ​​the grating waveguide Straktur.
4. grating waveguide-Straktur according to any one of claims 1 - (a) is better than 200 microns 3, characterized in that the spatial resolution for the determination of the degree of compliance with the resonance condition for the coupling of light into the layer.
5. grating waveguide-Straktur according to any one of claims 1-4, characterized in that the spatial resolution for the determination of the degree of compliance with the resonance condition for the coupling of light into the layer (a) is better than 20 microns.
6. grating waveguide-Straktur according to any one of claims 1-5, characterized in that the spatial resolution for the determination of the degree of compliance with the resonance condition for the coupling of light into the layer (a) by choosing a larger modulation depth of Gitterstrakturen (c) improved or selection of a smaller modulation depth of said Gitterstrakturen can be reduced.
7. grating Wellenleiterstraktur according to any one of claims 1-6, characterized in that the half-width of the resonance angle to fulfill the resonance condition for the coupling of light into the layer (a) by decreasing the modulation depth of Gitterstrakturen (c) reduced or eliminated by Vergrösserang the modulation depth of said Gitterstrakturen can be increased.
8. grating waveguide-Straktur according to any one of claims 1 - 7, characterized in that - outside of the measurement areas - (the resonance angle for coupling in or out of a monochromatic excitation light within an area of at least 4 mm 2 in parallel with orientation of the pages or not parallel to the lines of Gitterstraktur (c)) varies by at most 0.1 ° (as the deviation from a mean).
9. grating waveguide-Straktur according to any one of claims 1-8, characterized in that the degree of fulfillment of the resonance condition for the coupling of light into the layer (a) to the measurement areas (1) from the intensity of substantially reflected parallel to the light, again decoupled excitation light (that is from the sum of both parts) or (2) from the intensity of the transmitted Amegungslichts or (3) from the intensity of scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light, or determined from any combination of the light components (1) to (3).
10. grating waveguide structure according to any one of claims 1 - 9, characterized in that (1) the sum of the intensities of the reflected and substantially parallel thereto again decoupled excitation light or (2) the intensity of the scattered light from a to coupling via grating structure (c) in the layer (a) led Amegungslicht or (3) a combination of the light intensities (1) - (2) in local meet the resonance condition for the coupling of light into the layer (a) in the range of this measurement range is a maximum.
has 10, characterized in that the intensity of the transmitted excitation light for local fulfillment of the resonance condition for the coupling of light into the layer (a) in the range of this measuring range a minimum - 11 mesh waveguide Straktur according to any one of Claims. 1
12. grating waveguide-Straktur according to any one of claims 1-11, characterized in that between the optically transparent layers (a) and (b) and in contact with layer (a), a further optically transparent layer (b ') with lower refractive index than that of layer (a) and a thickness of 5 nm - 10 000 nm, preferably 10 nm - 1000 nm, is located.
13, grating-waveguide Straktur according to any one of claims 1-12, characterized in that for immobilization of the biological or biochemical or synthetic recognition elements (e) on the optically transparent layer (a) an adhesion-promoting layer (f) with a thickness of preferably less than 200 nm, is more preferably less than 20 nm is applied, and that the adhesion promoting layer (f) is preferably a chemical compound selected from the group silanes, epoxides, functionalized, charged or polar polymers and comprises "self-organized functionalized monolayers".
14, grating-waveguide Straktur according to any one of claims 1-13, characterized in that spatially separated measurement areas (d) are generated by spatially selective application of biological or biochemical or synthetic recognition elements on said grating waveguide Straktur, preferably using a or more methods from the group of methods, the spotting of "inkjet, mechanical spotting, micro contact printing, fluidic Kontaktierang the measurement areas with the biological or biochemical or synthetic recognition elements upon their supply in parallel or crossed microchannels, under the influence of pressure differences or electrical or is formed electromagnetic potentials. "
15, grating-waveguide Straktur by spoke 14 characterized in that are applied as said biological or biochemical or synthetic recognition elements, components from the group of nucleic acids (DNA, RNA, oligonucleotides) and nucleic acid analogues (eg., PNA), antibodies , aptamers, membrane-bound and isolated receptors, their ligands, antigens for antibodies, "histidine tag components", produced by chemical synthesis cavities to receive molecular imprints, etc. is formed or that as biological or biochemical or synthetic recognition elements, whole cells or cell fragments are applied.
16, grating-waveguide Straktur according to any one of claims 14 - 15, characterized in that between the laterally separated measurement areas (d) compounds are applied to the analyte are "chemically neutral", preferably for example consisting of the groups are formed by albumins, in particular Rinderseramalbumin or human serum albumin, is not to be analyzed with Polynukeotiden hybridizing, fragmented natural or synthetic DNA, such as herring or salmon sperm, or also uncharged, but hydrophilic polymers, such as polyethylene glycols or dextrans, are formed.
17, grating-waveguide Straktur according to any one of claims 1 - 16, characterized in that up to 1 000 000 measurement areas are arranged in a 2-dimensional arrangement and a single measurement area an area of 0.001 - occupies 6 mm 2.
18, grating-waveguide Straktur according to any one of claims 1-17, characterized in that a plurality of measurement areas in a density of more than 10, preferably more than 100, more preferably more than 1000 measurement areas per square centimeter on a common Gitterstraktur (c ) are arranged.
19, grating-waveguide Straktur according to any one of claims 1-18 characterized in that the outside dimensions of their base surface with the Grand area of ​​standard microtiter plates correspond approximately 8 cm x 12 cm (96 or 384 or 1536 wells).
20, grating-waveguide Straktur according to any one of claims 1-19 characterized in that Gitterstrakturen (c) are diffractive grating with a uniform period or multidiffraktive lattice.
21, grating-waveguide Straktur according to any one of claims 1-7 or 10-19, characterized in that one or more Gitterstrakturen (c) substantially perpendicular to the direction of propagation in the optically transparent layer (a) coupled Amegungslichts varying periodicity respectively.
22, grating-waveguide Straktur according to any one of claims 1-21, characterized in that the material of the second optically transparent layer (b) of glass, quartz or a transparent thermoplastic plastic, for example from the group consisting, of polycarbonate, Poly I id or polymethyl methacrylate is formed.
23, grating-waveguide Straktur according to any one of claims 1 - 22 and characterized in that the refractive index of the first optically transparent layer (a) is greater than 1.8.
24, grating-waveguide Straktur according to any one of claims 1-23, characterized in that the first optically transparent layer (a) a material from the group of TiO 2, ZnO, Nb O 5, Ta 2 O 5, HfO 2, or ZrO 2, particularly preferably of TiO 2 or Nb O 5 or Ta 2 O 5 comprising.
25, grating-waveguide Straktur according to any of claim 1-24 characterized in that the product of the thickness of the first optically transparent layer (a) and its refractive index one-tenth to a whole, preferably one-third to two-thirds of the excitation wavelength is one in which layer (a) to be coupled in the excitation light.
26, grating-waveguide Straktur according to any one of claims 1 - 25, characterized in that the grid (c) a period of 200 nm - having 1000 nm and the modulation depth of the grating (c) 3 to 100 nm, preferably 5 to 30 is nm.
27, grating-waveguide Straktur by spoke 25, characterized in that the ratio of modulation depth to the thickness of the first optically transparent layer (a) is equal to or less than 0.2.
28, grating-waveguide Straktur according to any one of claims 1-27, characterized in that the Gitterstraktur (c) a relief grating with rectangular, triangular or semicircular profile, or a phase or volume grating with a periodic modulation of the refractive index in the is essentially planar optically transparent layer (a).
29, grating-waveguide Straktur according to any one of claims 1 - 28, characterized in that optically or mechanically recognizable marks for simplifying Justierang in an optical system and / or for connection to sample containers as part of an analytical system are applied to it.
30. An optical system for spatially resolved determination of changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on this platform, with
- at least one excitation light source
- a grating-waveguide Straktur according to any one of claims 1-29
- at least one spatially resolving detector for detecting the transmitted excitation light on the, with respect to the irradiated excitation light, opposite side of the grating waveguide structure and / or for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light.
31. An optical system for spatially resolved determination of changes in the resonance conditions for coupling an A egungslichts in a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on this platform, with at least one excitation light source of a grating waveguide-Straktur according to any one of claims 1-29 a diffusely reflecting and / or diffusely transmissive projection wall on the, with respect to the irradiated excitation light, opposite side of the grating waveguide Straktur, for forming an image of the transmitted excitation light
- and at least one spatially resolving detector for detecting the image of the transmitted excitation light on said projection screen.
32. The optical system of spoke 31, characterized in that the at least one spatially resolving detector for detecting the image of the transmitted Anregungshchts on said projection wall on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur is arranged.
33. The optical system of spoke 31, characterized in that the at least one spatially resolving detector for detecting the image of the transmitted Amegungslichts on said projection wall on the side of the transmitted excitation light, that is, on the, with respect to the irradiated excitation light, opposite side of the grating waveguide Straktur is arranged, wherein said projection screen is at least partially transmissive.
34. An optical system with a grating waveguide Straktur to spoke 21, characterized in that on each Gitterstraktur (c) spatially varying with a substantially perpendicularly to the direction of propagation in the optically transparent layer (a) excitation light coupled periodicity arranged more than one measurement range is being followed on the grating waveguide Straktur in the propagation direction of the to be coupled and in the layer (a) to leading Amegungslichts a unstrukurierter area of ​​the grating waveguide structure, and it may be further guided in the direction of propagation in layer (a) excitation light is followed by a further Gitterstraktur (c) through which said guided excitation light is coupled again in the direction of a spatially resolving detector.
35. The optical system of spoke 34, characterized in that changes in the mass occupation by adsorption or desorption of molecules from the measurement areas on grating structures (c) a shift of the local position of the fulfillment of the resonant condition for coupling the Amegungslichts in the layer (a) said Gitterstraktur (c) substantially in parallel to the grating lines.
36. Optical system according to any one of claims 34 - 35, characterized in that a one-dimensional arrangement of at least 2 Gitterstrakturen (c) after spoke 21 is simultaneously irradiated with excitation light.
37. Optical system according to any one of claims 34- 36, characterized in that the Amegungslicht is substantially parallel irradiated and is essentially monochromatic.
38. An optical system according to claim 37, characterized in that the excitation light is irradiated linearly polarized, to Amegung one of the layer (a) or performed TEo- TM 0 - Modes.
39. Optical system according to any one of claims 37-38, characterized in that a two-dimensional array of at least 4 Gitterstrakturen (c) is irradiated by spoke 21 simultaneously with excitation light.
40. An optical system for spatially resolved determination of changes in the resonance conditions for coupling a Amegungslichts in a waveguide or coupling out a guided in the waveguide light by a two-dimensional array of at least four or more discrete measurement areas (d) on this platform, with
- at least one excitation light source
- a grating-waveguide Straktur according to any one of claims 1 to 29 a positioning member for varying the angle of incidence of the excitation light on the grating waveguide Straktur at least one spatially resolving detector for detecting the transmitted excitation light on the, with respect to the irradiated excitation light, opposite side of the grid waveguide-Straktur and / or for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling a Gitterstraktur (c ) (in the layer a) guided excitation light.
41. An optical system for spatially resolved determination of changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light by a two-dimensional array of at least four or more discrete measurement areas (d) on this platform, with
- at least one excitation light source
- a grating-waveguide Straktur according to any one of claims 1-29
- a positioning member for Veränderang the angle of incidence of the excitation light on the grating waveguide Straktur - a diffusely reflecting and / or diffusely transmissive projection wall on the, with respect to the irradiated excitation light, opposite side of the grating waveguide Straktur, for forming an image of the transmitted Amegungslichts
- and at least one spatially resolving detector for detecting the image of the transmitted Amegungslichts on said projection screen.
42. An optical system for spatially resolved determination of changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on this platform, with at least one over a certain spectral tuneable excitation light source
- a grating-waveguide Straktur according to any one of claims 1-29 to at least one spatially resolving detector for detecting the transmitted excitation light and / or for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide-Straktur and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light.
43. The optical system of spoke 42, characterized in that said at least can be tuned, a tunable light source over a spectral range of at least 5 nm.
44. An optical system for spatially resolved determination of changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on this platform, with
- at least one polychromatic within a certain spectral range excitation light source of a grating waveguide Straktur according to any one of claims 1 - 29 - at least one spatially resolving detector for detecting the transmitted excitation light and / or for detecting the substantially parallel to the reflected light again the light coupled to the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light.
45. The optical system of spoke 44, characterized in that said at least one polychromatic light source, an emission band width of at least 5 nm.
46. ​​Optical system according to any one of claims 44 - 45, characterized in that a spectrally selective optical component with high spectral resolution in said certain spectral range is arranged in the beam path between the grating waveguide Straktur and the at least one position-sensitive detector.
47. The optical system of spoke 46, characterized in that said spectrally selective component is suitable for the generation of spectrally selective, spatially resolved, two-dimensional representations of the intensity distributions of the end of the grating waveguide Straktur measuring light at different wavelengths within said certain spectral range.
48. Optical system according to any one of claims 44-47, characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light of said polychromatic light source in the region of the measurement areas by simultaneous or sequential detection of the transmitted excitation light and / or by the simultaneous or sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or by the simultaneous or sequential detection of the stray light from coupling by means of a grating structure (c) in the layer (a) by means of guided Amegungslicht spectrally selective within said certain spectral detection using at least one laterally resolving detector, vorzugswe ise at a constant angle of incidence on the grating waveguide Straktur, this is done Amegungslichts.
49. Optical system according to any one of claims 40 - 48, characterized in that the Amegungslicht is substantially parallel irradiated.
50. Optical system according to any one of claims 40-43, characterized in that the excitation light is irradiated substantially monochromatic.
51. Optical system according to any one of claims 40 - 50, characterized in that the excitation light is irradiated linearly polarized, to a Amegung in the layer (a) guided TE 0 - 0 or TM -Modes.
52. Optical system according to any one of claims 40 - 51, characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light in the field of measuring ranges, by sequential detection of the transmitted excitation light and / or (by sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide structure and / or by sequential detection of the scattered light from by coupling a Gitterstraktur c ) (in the layer a) guided excitation light is performed with one or more spatially resolving detectors Veränderang under the angle of incidence of the excitation light on the grating waveguide Straktur.
53. Optical system according to any one of claims 42-51, characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light in the field of measuring ranges, by sequential detection of the transmitted excitation light and / or by sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated Amegungslichts, the same side of the grating waveguide Straktur and / or by sequential detection of the scattered light of (by coupling a Gitterstraktur c ) (in the layer a) led Amegungslicht each having one or more spatially resolving detectors under Veränderang the emission wavelength of a tunable light source, preferably at a constant angle of incidence on the grating waveguide Straktur, this excitation light is performed.
54. Optical system according to any one of claims 30-53, characterized in that the Amegungslicht of at least one light source having an optical expansion unit is expanded homogeneously as possible into a substantially parallel beam and is irradiated to the one or more measurement areas.
55. The optical system of spoke 54, characterized in that the diameter of the irradiated excitation light beam at least in one dimension at least 2 mm, preferably at least 10 mm.
56. Optical system according to any one of claims 30-53, characterized in that the excitation light from the at least one light source through one or, in case of multiple light sources, optionally a plurality of diffractive optical elements, preferably Dammann grating, or refractive optical elements, preferably microlenses - arrays, is decomposed into a plurality of individual beams of equal intensity as possible of data originating from a common light source sub-beams which are each substantially irradiated to each other on laterally separated measurement areas in parallel.
57. Optical system according to any one of claims 30 - 39, characterized in that the Amegungslicht of at least one, preferably monochromatic light source with a beam shaping optics to a beam of homogeneous intensity as possible, and gap-shaped cross-section is expanded (perpendicular to the optical axis of the beam path in a plane) is whose major axis is aligned parallel to the grating lines, wherein the sub-beams of said beam in a projection plane parallel to the plane of the grating waveguide Straktur substantially parallel to each other while said radiation beam a in a direction orthogonal to the plane of the grating waveguide Straktur level having convergence or divergence with a certain convergence or divergence angle.
58. Optical system according to one of claim 57, characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light in the region of the measuring ranges, within a slit-shaped illuminated area by simultaneous detection of the transmitted excitation light and / or by the simultaneous detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated Amegungslichts, the same side of the grating waveguide Straktur and / or by simultaneously detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light each having one or a plurality of spatially resolving detectors, whereby the local change in the resonance conditions in a measuring region in a shift of the maximum of the measurement range of said i m substantially parallel to the reflected light outgoing light and the maximum of the of said measuring range by coupling a Gitterstraktur (c) in the layer (a) guided excitation light outgoing scattered light and the minimum of the transmitted in the area of ​​said measurement light region (in each case in compliance with the resonance conditions in said measurement range), wherein said displacement of the minimum or maximum in a plane is parallel to the plane of the grating waveguide-Straktur perpendicular to the grating lines.
59. Optical system according to any one of claims 30-58, characterized in that are used as excitation light sources two or more coherent light sources with the same or different emission wavelength.
60. The optical system of spoke 59, characterized thereby, that the excitation light of 2 or more coherent light sources simultaneously or sequentially from various directions onto a Gitterstraktur (c) is irradiated, comprising a superposition of Gitterstrakturen with different periodicity.
61. Optical system according to one of claims 30 - 60, characterized in that at least one laterally resolving detector is used for detection, for example, from the group formed by CCD cameras, CCD chips, photodiode arrays, avalanche diode arrays multichannel plates and multichannel photomultipliers is formed.
62. Optical system according to any one of claims 30-61, characterized in that between the one or more excitation light sources and the grating waveguide Straktur according to any one of claims 1-29 and / or between said grating waveguide-Straktur and the one or a plurality of detectors optical components are used from the grappa, by lenses or lens systems for shaping the transmitted light bundles, planar or curved mirrors for the deviation and optionally additional shaping of the light bundles, prisms for the deviation and optionally spectral separation of the light bundles, dichroic mirrors for the spectrally selective deviation of parts of light bundles, neutral filters for controlling the transmitted light intensity, optical filters or monochromators for the spectrally selective transmission of parts of light bundles or polarization-selective elements for selecting discrete polarization directions of the Anregu NGS or luminescence light are formed.
63. Optical system according to any one of claims 30 to 62, characterized thereby, that the irradiation of the excitation light in fsec pulses having a duration between 1 and 10 minutes is performed and the emission light from the measurement areas is measured time-resolved manner.
64. Optical system according to any one of claims 30 to 63, characterized thereby, that the irradiation of the excitation light and the detection of light emanating from one or more measurement areas of light is performed sequentially for one or more measurement areas.
65. The optical system of spoke 64, characterized in that sequential Amegung and detection is performed using movable optical components formed from the Grappas mirrors, deviating prisms, and dichroic mirrors.
66. Optical system according to any one of claims 64-65, characterized in that the grating waveguide Straktur is moved between steps of sequential excitation and detection.
67. An optical system for spatially resolved determination of changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) on this platform, for detecting one or more analytes in at least one sample on one or more measurement areas on a grating waveguide Straktur, with
- a grating-waveguide Straktur according to any one of claims 1-29
- to bring the feed means 66 as well as one or more samples with the measurement areas on the grating waveguide Straktur in contact - an optical system according to any one of claims 30th
68. The optical system of spoke 67, characterized in that it additionally comprises one or more sample containers which are open towards at least in the area of ​​one or more measurement areas or combined to form segments ranges for lattice waveguide Straktur, wherein the sample containers preferably each a volume of 0.1 nl - have 100 ul.
69. The optical system of spoke 68, characterized in that the sample containers on the optically transparent layer (a) side facing away from, with the exception of inlet and / or outlet openings for the supply or discharge of samples and optionally additional reagents, closed are carried out and the feed or the outlet of samples and optional additional reagents in a closed flow-through system, wherein in the case of liquid supply to a plurality of measurement regions or segments having common inlet and outlet openings, these columnar or preferably addressed row by row.
70. Optical system according to any one of claims 67-69, characterized in that containers are provided for reagents which are wetted during the process for the detection of one or more analytes and brought into contact with the measurement areas
71. A method for the qualitative and / or quantitative detection of one or more analytes in one or more samples in at least two or more discrete measurement areas on a grating waveguide-Straktur according to any one of claims 1-29 in an optical system according to any one of claims 34-70, (d) characterized by means of determining changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas on that grating waveguide Straktur, characterized that the excitation light from at least one excitation light source to a Gitterstraktur (c) is passed having thereon said measurement areas and the degree of compliance with the resonance condition for the coupling of light into the layer (a) to said measurement areas from the signal of at least one spatially resolving detector for sensing ung the transmitted Anregungshchts and / or for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling a Gitterstraktur (c ) (in the layer a) guided excitation light is determined.
72. A method for the qualitative and / or quantitative detection of one or more analytes in one or more samples in at least two or more discrete measurement areas on a grating waveguide structure according to claim 21, characterized in that on each Gitterstraktur (c) with a substantially perpendicular to the direction of propagation of the optically transparent layer (a) injected excitation light spatially varying periodicity than one measurement area is arranged to give a on the grating waveguide Straktur in the propagation direction of the to be coupled and in the layer (a) leading the excitation light unstrukurierter area of ​​the grating waveguide Straktur adjoins, and it may be further guided in the direction of propagation in layer (a) Amegungslichts a further grating structure (c) connects, via which said guided excitation light in the direction of a spatially resolving detector ausgekop again pelt is.
73. A method according to claim 72, characterized in that changes in the local effective index of refraction, especially in the mass occupation by adsorption or desorption of molecules from the measurement areas on grating structures (c) a shift of the local position of the fulfillment of the resonant condition for coupling the excitation light into the layer (a) over said Gitterstraktur (c) essentially perform parallel to the grating lines.
74. A method according to any one of claims 72-73, characterized in that a one-dimensional arrangement of at least two grating structures (c) is irradiated according to claim 21 simultaneously with excitation light.
75. A method according to any one of claims 72-74, characterized in that the Amegungslicht is substantially parallel irradiated and is essentially monochromatic.
76. A method according to spoke 75, characterized in that the excitation light is irradiated linearly polarized, guided to the excitation of a in the layer (a) TE 0 - or TMO mode.
77. A method according to any one of claims 75-76, characterized in that a two-dimensional array of at least 4 Gitterstrakturen (c) after spoke 21 is irradiated simultaneously with Amegungslicht.
78. A method for the qualitative and / or quantitative detection of one or more analytes in one or more samples in at least two or more discrete measurement areas on a grating waveguide structure according to any one of claims 1 to 29, by means of determining changes in the resonance conditions for coupling a Amegungslichts in a waveguide or coupling out a guided in the waveguide light by a two-dimensional array of at least four or more discrete measurement areas (d) on this platform, characterized in that the excitation light from at least one excitation light source to a Gitterstraktur (c) is passed thereon said measurement areas and the degree of compliance with the resonance condition for the coupling of light into the layer (a) to said measurement areas from the signal of at least one spatially resolving detector for detecting the transmitted excitation light, optionally under The use of a diffusely reflecting and / or diffusely transmissive projection wall on the, with respect to the irradiated excitation light, opposite side of the grating waveguide-Straktur, for forming an image of the transmitted excitation light, and / or for detecting the substantially parallel to the reflected light again decoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light is determined, and by means of a positioning angle of incidence of the excitation light is changed on the grating waveguide Straktur so that said resonance condition at different angles in the range of different ranges on an irradiated Gitterstraktur (c), is a function of the local effective refractive index is satisfied.
79. A method according to claim 78, characterized in that the A egungslicht is substantially parallel irradiated and is essentially monochromatic
80. A method according to spoke 79, characterized in that the excitation light is irradiated linearly polarized, guided to the excitation of a in the layer (a) TE 0 - or TM 0 - Modes.
81. A method according to any one of claims 78-80, characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light in the region of the measuring ranges by sequential detection of the transmitted excitation light and / or by sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or by sequential detection of the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light is performed with one or more spatially resolving detectors Veränderang under the angle of incidence of the excitation light on the grating waveguide Straktur.
82. A method according to spoke 71, characterized in that the angle of incidence of the excitation light is set to the grating waveguide-Straktur so that the resonance condition for coupling an excitation light in a waveguide a grating waveguide Straktur or coupling out a guided in the waveguide light, with an array of at least two or more laterally separated measurement areas (d) on that grating waveguide Straktur, on one or more of these measurement areas is substantially met with the consequence of a substantially maximum signal of a spatially resolving detector for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light from the area this Messbereic He, and / or a substantially minimum signal of a spatially resolving detector for detecting the transmitted excitation light in the range of the measurement areas, or between the measurement areas substantially met with the consequence of a substantially maximum signal of a spatially resolving detector for detecting the substantially reflected parallel to the light again outcoupled light on the, with respect to the irradiated Amegungslichts, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) led Amegungslicht, in the areas between these measuring ranges, and / or a substantially minimum signal of a spatially resolving detector for detecting the transmitted excitation light out of the areas between these ranges.
83. The method of claim 82, characterized in that local differences in the effective refractive index in the range of different measuring ranges, and in the areas between the measuring ranges from local differences in the intensities of one or more position-sensitive detectors for detecting the hansmittierten excitation light and / or for detecting the in substantially are determined parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating-waveguide Straktur and / or for detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light without the set irradiation angle of the Amegungslichts is changed on the grating waveguide Straktur.
84. A method according to spoke 71, characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light from at least one over a certain spectral range tunable light source in the region of the measuring ranges by sequential detecting the transmitted Amegungslichts and / or by sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating-waveguide Straktur and / or by sequential detection of the scattered light from by coupling a Gitterstraktur (c) in the layer (a) guided excitation light each having one or a plurality of spatially resolving detectors under Veränderang the emission wavelength of said at least one tunable light source, preferably with constant Einstra hlwinkel on the grating waveguide Straktur, this excitation light is performed.
85. A method according to spoke 71, characterized in that the emission wavelength preferably this Amegungslichts is set to the grating waveguide Straktur so at a constant angle of incidence that the resonance condition for coupling an excitation light in a waveguide a grating waveguide at least one tunable light source, -Straktur or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas (d) is met on this grating waveguide structure, on one or more of these measuring ranges substantially, with the consequence of a substantially maximum signal of a spatially resolving detector for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling via ei is satisfied ne grating structure (c) in the layer (a) guided excitation light, from the region of these ranges, and / or a substantially minimum signal of a spatially resolving detector for detecting the transmitted excitation light in the range of the measurement areas, or between the measurement areas Substantially As a result of a substantially maximum signal of a spatially resolving detector for detecting the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling a grating structure (c) in the layer (a) guided excitation light from the areas between these ranges, and / or a substantially minimum signal of a spatially resolving detector for detecting the transmitted excitation light out of the areas between these ranges.
86. A method according to claim 71, characterized in that the spatially resolved determination of changes in the resonance conditions for coupling a Amegungslichts in the layer (a) or decoupling of a guided in the waveguide light from at least one in a certain spectral range polychromatic light source in the region of the measuring ranges by detecting of the transmitted excitation light and / or by detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or by detecting the scattered light from by coupling of a grating structure (c) each having one or a plurality of spatially resolving detectors, preferably at a constant angle of incidence on the grating waveguide Straktur, takes place in the layer (a) guided excitation light of this excitation light, wherein in each case in the areas where best for a immte wavelength of the excitation light from the polychromatic light source dieResonanzbedingung for coupling said excitation light is satisfied in a waveguide of the grating waveguide Straktur or coupling out a guided in the waveguide light of this wavelength, a maximum signal component of that wavelength at the signal of a spatially resolving detector for detecting the substantially parallel again decoupled to the reflected light light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or for detecting the scattered light from by coupling of a grating structure (c) in the layer (a) guided excitation light from the area said measurement areas, and / or a minimum signal component at that wavelength signal of a spatially resolving detector for detecting the transmitted excitation light in the range of the measuring ranges is obtained.
87. A method according to spoke 86, characterized in that a spectrally selective optical component with high spectral resolution in said certain spectral range is arranged in the beam path between the grating waveguide Straktur and the at least one position-sensitive detector.
88. A method according to spoke 87, characterized in that by using said spectrally selective component spectrally selective, spatially resolved, two-dimensional representations of the intensity distributions of the end of the grating waveguide Straktur measuring light can be generated at different wavelengths within said certain spectral range.
89. A method according to any one of claims 86-88, characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light of said polychromatic light source in the field of measuring ranges, by simultaneous or sequential detection of the transmitted Amegungslichts and / or by the simultaneous or sequential detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated Amegungslichts, the same side of the grating waveguide Straktur and / or by the simultaneous or sequential detection of the scattered light from by coupling a Gitterstraktur (c) in the layer (a) by means of guided Amegungslicht spectrally selective within said certain spectral detection using at least one laterally resolving detector, preferably below a constant angle of incidence on the grating waveguide Straktur, this excitation light is performed.
90. A method according to any one of claims 86-89, characterized in that the Amegungslicht is each substantially parallel irradiated.
91. A method according to any one of claims 71-90, characterized in that the excitation light from the at least one light source through one or, in case of multiple light sources, optionally a plurality of diffractive optical elements, preferably Dammann grating, or refractive optical elements, preferably microlenses array, is decomposed into a plurality of individual beams of equal intensity as possible of data originating from a common light source sub-beams which are each substantially irradiated to each other on laterally separated measurement areas in parallel.
92. A method according to spoke 71, characterized in that the excitation light from at least one, preferably monochromatic, light source is expanded with a beam shaping optics to a beam of homogeneous intensity as possible, and gap-shaped cross section (in a plane perpendicular to the optical axis of the optical path), the main axis parallel is aligned to the grating lines, wherein the sub-beams of said beam in a projection plane parallel to the plane of the grating waveguide Straktur substantially parallel to each other while said beam having in a direction orthogonal to the plane of the grating waveguide Straktur plane convergence or divergence has a certain convergence or divergence angle.
93. A method according to spoke 92, characterized in that said convergence or divergence angle of said radiation beam has a value of up to 5 degrees in a direction orthogonal to the plane of the grating waveguide Straktur level.
94. A method according to any one of claims 92-93, characterized in that the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) or decoupling of a guided in the waveguide light in the region of the measuring ranges, within a slit-shaped illuminated area about by the simultaneous detection of the transmitted excitation light and / or by the simultaneous detection of the substantially parallel to the reflected light again outcoupled light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or by simultaneously detecting the scattered light from by coupling a grating structure (c) in the layer (a) is carried guided excitation light each having one or a plurality of spatially resolving detectors, where the local change in the resonance conditions in a measuring region in a shift of the maximum of the measurement range of said substantially parallel to the reflected light outgoing light and the maximum of the of said measuring range by coupling a Gitterstraktur (c) in the layer (a) guided excitation light outgoing scattered light and the minimum of the said in the range measuring range of transmitted light (in each case in compliance with the resonance conditions in said measurement range), wherein said displacement of the minimum or maximum in a plane is parallel to the plane of the grating waveguide-Straktur perpendicular to the grating lines.
95. A method for the qualitative and / or quantitative detection of one or more analytes in one or more samples in at least two or more discrete measurement areas on a grating waveguide-Straktur according to any one of claims 1-29 in an optical system according to any one of claims 34-70, (d) characterized by means of determining changes in the resonance conditions for coupling of excitation light into a waveguide or coupling out a guided in the waveguide light having an array of at least two or more laterally separated measurement areas on that grating waveguide Straktur, characterized that the spatially resolved determination of changes said resonance conditions in each case simultaneously in the field of measuring areas within a slit-shaped illuminated area, by the simultaneous detection of the transmitted excitation light and / or by the simultaneous detection of the reflected substantially parallel to the light Wied he coupled-out light on the, with respect to the irradiated excitation light, the same side of the grating waveguide Straktur and / or by simultaneously detecting the scattered light from by coupling a Gitterstraktur (c) in the layer (a) led Amegungslicht each having one or more spatially resolving detectors takes place, - wherein the local change in the resonance conditions in a measuring region in a shift of the maximum of light emanating from said measuring area parallel to the reflected light substantially light as well as the maximum of the of said measuring range by coupling a Gitterstraktur (c) in the layer ( a) led Amegungslicht outgoing scattered light and the minimum of the transmitted in the area of ​​said measurement light region (shows on fulfillment of the resonance conditions in said measurement range), wherein said displacement of the minimum or maximum in a plane parallel to the plane of the grating waveguide Straktu r is perpendicular to the grating lines, and wherein the grating waveguide Straktur for sequentially spatially resolved determination of changes in said boundary conditions on the entire surface of said grating waveguide Straktur with the thereon ranges between individual process steps, perpendicular and / or parallel to the orientation of the grating lines is deferred until the measurement signals are recorded of all measuring ranges, and a two-dimensional representation of the degree of fulfillment of said resonance conditions can be produced over the entire grating waveguide-Straktur from the recorded signals.
96. A method according to any one of claims 78-95, characterized in that the spatial resolution for the determination of the degree of compliance with the resonance condition for the coupling of light into the layer (a) by choosing a larger modulation depth of Gitterstrakturen (c) improved or selection of a smaller modulation depth said Gitterstrakturen can be reduced.
97. A method according to any one of claims 78-96, characterized in that the half-width of the resonance angle to fulfill the resonance condition for the coupling of light into the layer (a) by decreasing the modulation depth of Gitterstrakturen (c) can be reduced, resulting in increased sensitivity the spatially resolved determination of changes of the degree of compliance with the resonance condition as a result of local changes in the mass occupation result has, or by Vergrösserang the modulation depth of said grating structures can be increased, resulting in reduced sensitivity for the spatially resolved determination of changes of the degree of compliance with the resonance condition as has series of local changes in the mass occupation to follow.
98. A method according to any one of claims 78-97, characterized in that differences in mass density and / or the effective refractive index can be dissolved within a measuring range.
99. A method according to any one of claims 71-98, characterized in that are used as excitation light sources two or more coherent light sources with the same or different emission wavelength.
100. A method according to any one of claims 71-99, characterized in that the change in the mass occupation of Vergrösserang upon binding or dissociation to be detected analyte molecules to this or to one of its binding partner in a multi-step assay, a mass label is bound, which may be for example selected from the grappas of metal colloids (z. B. gold colloids), plastic particles or -Beads or other microparticles having a monodisperse size distribution.
101. A method according to any one of claims 71-100, characterized in that the change in the effective refractive index at the binding or dissociation to be detected analyte molecules to this or to one of its binding partner in a multistage assay to Vergrösserang is bound a "Absorption label", said " absorption label "having an absorption band of suitable wavelength, which absorption, the imaginary part of the refractive index, resulting in a rank changes in the effective refractive index in the near field of the grating waveguide Straktur.
102. A method according to any one of claims 71-101, characterized in that in addition to the spatially resolved determination of changes in the resonance conditions for coupling an excitation light in the layer (a) of a grating waveguide Straktur or coupling out a in the layer (a) guided light one or more operations in the evanescent field of the layer (a) excitation light excited luminescences are determined from one or more measurement regions 29 in addition - to one of claims. 1
103. A method according to spoke 102, characterized in that the binding of a ligand analyte to an immobilized in one or more measurement areas of biological or biochemical or synthetic recognition element as a receptor based on the local change of the effective refractive index is determined and a functional response of this ligand-receptor -systems is determined from a Lumineszenzänderang from said measurement areas.
104. A method according to spoke 102, characterized in that the density of the immobilized biological or biochemical or synthetic recognition elements as receptors in one or more measurement areas on the basis of differences between the resonance conditions for coupling an excitation light in the layer (a) of the grating waveguide Straktur or coupling out a in the layer (a) guided light in the region of these ranges, and the respective resonance conditions in the vicinity thereof, ie is outside said ranges, determined, and the binding of a ligand is determined as the analyte to these recognition elements on the basis of a change in luminescence from said measurement areas ,
105. The method of any of claims 102-104, characterized in that (1) coupled in the isotropically emitted luminescence or (2) into the optically transparent layer (a) and the Gitterstraktur (c) is coupled out or luminescence comprising both parts (1 ) and (2) are measured simultaneously.
106. The method of any of claims 102-105, characterized in that for the generation of luminescence, a luminescent dye or luminescent nanoparticle is used as a luminescence label, which can be excited at a wavelength between 300 nm and 1100 nm and emitted.
107. The method of any of claims 100-106, characterized in that the mass label and / or luminescence label to the analyte or, in a competitive assay to an analogue of the analyte, or in a multistage assay to one of the binding partners of the immobilized biological or biochemical or synthetic is bound to the recognition elements or biological or biochemical or synthetic recognition elements.
108. The method of any of claims 102-107, characterized in that the one or more luminescences and / or determinations of light signals at the excitation wavelengths are performed polarization-selectively, wherein the one or more luminescences preferably at a polarization different from that of the excitation light are measured ,
109. A method according to any one of claims 71-108 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 analogues, enzymes, enzyme cofactors or inhibitors, lectins and carbohydrates.
110. A method according to any one of claims 71-109, characterized in that the to be assayed sample naturally occurring body fluids such as blood, serum, plasma, lymph or urine or egg yolk or optically turbid fluids or surface water or soil or plant extracts or bio- or synthesis process broths or are taken from biological tissue parts.
110 to qualitative and / or quantitative analyzes to determine - 111. The use of a grating waveguide-Straktur according to any one of claims 1 - 29 and / or an optical system according to any one of claims 30 - 70 and / or a method according to one of Claims 71 chemical, biochemical or biological analytes in screening methods in pharmaceutical research, combinatorial chemistry, clinical and preclinical development, to quality for real-time binding studies and the determination of kinetic parameters in Affinitätsscreenmg and research and quantitative analyte determinations, especially for DNA and RNA analysis, for the generation of toxicity studies and 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, the symptomat een and pre-symptomatic plant diagnostics, for patient stratification in pharmaceutical product development and for the therapeutic drug selection, for the detection of pathogens, pollutants and pathogens, especially of salmonella, prions and bacteria, in food and environmental analytics.
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