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

• US 20030138208A1
• Filed: 11/05/2002
• Published: 07/24/2003
• Est. Priority Date: 05/06/2000
• Status: Abandoned Application

• Current Assignee: Bayer Technology Services GmbH (Bayer AG)
• Original Assignee: Bayer Technology Services GmbH (Bayer AG)
• Inventors: Pawlak, Michael, Bopp, Martin Andreas, Duveneck, Gert Ludwig, Ehrat, Markus
• Application Number:
  US10/275,380
• Publication Number:
  US 20030138208A1
• Time in Patent Office: Days
• US Class Current: 385/37
• CPC Class Codes: G01N 21/7743 the reagent-coated grating coupling light in or out of the waveguide
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

The invention relates to variable embodiments of a grating waveguide structure which enables to determine locally resolved changes of the resonance conditions for the incoupling of an excitation light into the waveguiding layer (a) of a stratified optical waveguide by means of a grating structure (c) modulated in said layer (a) or for outcoupling of a light guided in layer (a). The inventive system comprises arrays of measurement areas produced on the grating waveguide structure having different immobilized biological or biochemical or synthetic recognition elements elements for simultaneously binding and determining one or more analytes, wherein said excitation light is simultaneously irradiated onto an entire array of measurement areas, and the degree of satisfaction of the resonance condition for the incoupling of light into the layer (a) towards said measurement areas is simultaneously measured. 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 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.

Claims

1. Grating waveguide structure for the locally resolved determination of changes of the resonance conditions for the incoupling of an excitation light into a waveguide or for the outcoupling of a light guided in the waveguide, comprising an array of at least two or more, laterally separated measurement areas (d) on said platform, comprising a stratified optical 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 more grating structures (c) for the incoupling of an excitation light towards the measurement areas (d) or for the outcoupling of a light guided in layer (a) in the region of the measurement areas

with at least one or more laterally separated measurement areas (d) on said one or more grating structures (c)

with equal or different biological or biochemical or synthetic recognition elements (e) immobilized on said measurement areas, for the qualitative and/or quantitative determination of one or more analytes in a sample brought into contact with said measurement areas,

wherein said excitation light is irradiated simultaneously onto said array of measurement areas, and the degree of satisfaction of the resonance condition for the incoupling of light into the layer (a) towards said two or more measurement areas is simultaneously measured and a cross-talk of excitation light guided in layer (a), from one measurement area to one or more adjacent measurement areas is prevented by outcoupling said excitation light again by means of the grating structure (c).

3. Grating waveguide structure according to any of claims 1-2, wherein a continuously modulated grating structure (c) extends essentially over the whole area of said grating waveguide structure.

4. Grating waveguide structure according to any of claims 1-3, wherein the lateral resolution for the determination of the degree of satisfaction of the resonance condition for incoupling of light into layer (a) is better than 200 μ

m.

5. Grating waveguide structure according to any of claims 1-4, wherein the lateral resolution for the determination of the degree of satisfaction of the resonance condition for incoupling of light into layer (a) is better than 20 μ

m.

6. Grating waveguide structure according to any of claims 1-5, wherein the lateral resolution for the determination of the degree of satisfaction of the resonance condition for incoupling of light into layer (a) can be improved by choice of a larger modulation depth of grating structures (c) or decreased by choice of a lower modulation depth of said grating structures.

7. Grating waveguide structure according to any of claims 1-6, wherein the halfwidth of the resonance angle for satisfaction of the resonance condition for incoupling of light into layer (a) can be decreased by a decrease of the modulation depth of grating structures (c) or increased by an increase of the modulation depth of said grating structures.

8. Grating waveguide structure according to any of claims 1-7, wherein, outside from the measurement areas, the resonance angle for incoupling or outcoupling of a monochromatic excitation light varies by no more than 0.1°

(as deviation from an average value) within an area of at least 4 mm² (with orientation of the area boundaries in parallel or not in parallel to the lines of the grating structure (c)).

9. Grating waveguide structure according to any of claims 1-8, wherein the degree of satisfaction of the resonance condition for incoupling of light into layer (a) towards the measurement areas is determined (1) from the intensity of the outcoupled excitation light, outcoupled essentially in parallel to the reflected light (i.e. of the sum of both parts) or (2) from the intensity of the transmitted excitation light or (3) from the intensity of the scattered light of excitation light guided in layer (a) after incoupling by means of a grating structure (c), or from any combination of light components (1) to (3).

10. Grating waveguide structure according to any of claims 1-9, wherein (1) the sum of the intensities of the reflected light and of the excitation light outcoupled essentially in parallel thereto or (2) the intensity of scattered light of excitation light guided in layer (a) after incoupling by means of a grating structure (c) or (3) a combination of said light intensities (1) and (2) shows a maximum upon local satisfaction of the resonance condition for incoupling of light into layer (a) in the region of said local measurement area.

11. Grating waveguide structure according to any of claims 1-10, wherein the intensity of the transmitted excitation light shows a mimimum upon local satisfaction of the resonance condition for incoupling of light into layer (a) in the region of said local measurement area.

12. Grating waveguide structure according to any of claims 1-11, wherein a further optically transparent layer (b′

) with lower refractive index than layer (a) and a thickness between 5 nm and 10000 nm, preferably of 10 nm-1000 nm, is provided between layers (a) and (b) and in contact with layer (a).

13. Grating waveguide structure according to any of claims 1-12, wherein an adhesion-promoting layer (f), with a thickness of preferably less than 200 nm, more preferably of less than 20 nm, is deposited on the optically transparent layer (a), for immobilization of biological or biochemical or synthetic recognition elements, and wherein the adhesion-promoting layer preferably comprises chemical compounds of the group comprising silanes, epoxides, functionalized, charged or polar polymers and “

self-organized functionalized monolayers”

.

14. Grating waveguide structure according to any of claims 1-13, wherein laterally separated measurement areas (d) are generated by laterally selective deposition of biological or biochemical or synthetic recognition elements on said grating waveguide structure, preferably using a method of the group of methods comprising ink jet spotting, 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, upon application of pressure differences or electric or electromagnetic potentials.

15. Grating waveguide structure according to claim 14, wherein, as biological or biochemical or synthetic recognition elements, components of the group comprising nucleic acids (DNA, RNA, oligonucleotides) and nucleic acid analogues (e.g. PNA), antibodies, aptamers, membrane-bound and isolated receptors, their ligands, antigens for antibodies, “

histidin-tag components”

, cavities generated by chemical synthesis, for hosting molecular imprints. etc., are deposited, or wherein whole cells or cell fragments are deposited as biological or biochemical or synthetic recognition elements.

16. Grating waveguide structure according to any of claims 14-15, wherein compounds which are “

chemically neutral”

towards the analyte, preferably of the groups comprising, for example, albumines, especially bovine serum albumine or human serum albumine, fragmentated natural or synthetic DNA, such as from herring or salmon sperm, not hybridizing with polynuleotides to be analyzed, or uncharged but hydrophilic polymers, such as polyethyleneglycols or dextranes, are deposited between the laterally separated measurement areas (d).

17. Grating waveguide structure according to any of claims 1-16, wherein up to 1,000,000 measurement areas are provided in a 2-dimensional arrangement and wherein a single measurement area has an area of 0.001 mm²-6 mm².

18. Grating waveguide structure according to any of claims 1-17, wherein a multitude of measurement areas is provided at a density of more than 10, preferably of more than 100, most preferably of more than 1000 measurement areas per square centimeter on a common grating structure (c).

19. Grating waveguide structure according to any of claims 1-18, wherein the exterior dimensions of its footprint are similar to the footprint of standard microtiter plates of about 8 cm×

12 cm (with 96 or 384 or 1536 wells).

20. Grating waveguide structure according to any of claims 1-19, wherein grating structures (c) are diffractive gratings with a common period or multidiffractive gratings.

21. Grating waveguide structure according to any of claims 1-7 or 10-19, wherein one or more grating structures (c) have a laterally varying periodicity essentially perpendicular to the direction of propagation of the exciataion light incoupled into the optically transparent layer (a).

22. Grating waveguide structure according to any of claims 1-21, wherein the material of the second optically transparent layer (b) comprises quartz, glass, or transparent thermoplastic plastics of the group comprising, for example, poly carbonate, poly imide, or poly methylmethacrylate.

23. Grating waveguide structure according to any of claims 1-22, wherein the refractive index of the first optically transparent layer (a) is higher than 1.8

24. Grating waveguide structure according to any of claims 1-23, wherein the first optically transparent layer (a) comprises a material of the group comprising TiO₂, ZnO, Nb₂O₅, Ta₂O₅, HfO₂, or ZrO₂, especially preferably comprising TiO₂, Nb₂O₅, or Ta₂O₅.

25. Grating waveguide structure according to any of claims 1-24, wherein the product of the thickness of the first optically transparent layer (a) and its refractive index is one tenth to a whole, preferably one third to two thirds, of the excitation wavelength of an excitation light to be incoupled into the layer (a).

26. Grating waveguide structure according to any of claims 1-25, wherein the grating (c) has a period of 200 nm-1000 nm and the modulation depth of the grating (c) is 3 nm-100 nm, preferably of 5 nm-30 nm.

27. Grating waveguide structure according to claim 25, wherein the ratio of the modulation depth to the thickness of the first optically transparent layer (a) is equal or smaller than 0.2.

28. Grating waveguide structure according to any of claims 1-27, wherein the grating structure (c) is a relief grating with a rectangular, triangular or semi-circular profile or a phase or volume grating with a periodic modulation of the refractive index in the essentially planar, optically transparent layer (a).

29. Grating waveguide structure according to any of claims 1-28, wherein optically or mechanically recognizable marks for simplifying adjustments in an optical system and/or for the connection to sample compartments as part of an analytical system are provided on it.

30. Optical system for the locally resolved determination of changes of the resonance conditions for the incoupling of an excitation light into a waveguide or for the outcoupling of a light guided in the waveguide, comprising an array of at least two or more, laterally separated measurement areas (d) on said platform, comprising

at least one excitation light source

a grating waveguide structure according to any of claims 1-29

at least one locally resolving detector for determination of the transmitted excitation light located at the opposite side of the grating waveguide structure, with respect to the irradiated excitation light, and/or for the determination of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the direction of irradiation of the excitation light, and/or for the determination of the scattered light of an excitation light guided in layer (a) after incoupling by means of a grating structure (c).

31. Optical system for the locally resolved determination of changes of the resonance conditions for the incoupling of an excitation light into a waveguide or for the outcoupling of a light guided in the waveguide, comprising an array of at least two or more, laterally separated measurement areas (d) on said platform, comprising

at least one excitation light source

a grating waveguide structure according to any of claims 1-29

at least one diffusively reflecting and/or diffusively transmitting projection screen located at the opposite side of the grating waveguide structure, with respect to the direction of irradiation of the excitation light, for generation of an image of the transmitted excitation light,

and at least one locally resolving detector for collection of the image of the transmitted excitation light from said projection screen.

32. Optical system according to claim 31, wherein said at least one locally resolving detector for collection of the image of the transmitted excitation light from said projection screen is located at the same side of the grating waveguide structure, with respect to the direction of irradiation of the excitation light.

33. Optical system according to claim 31, wherein said at least one locally resolving detector for collection of the image of the transmitted excitation light from said projection screen is located at the side of the transmitted excitation light, i.e. at the opposite side of the grating waveguide structure with respect to the direction of irradiation of the excitation light, whereby said projection screen is at least partially transmittant.

34. Optical system with a grating waveguide structure according to claim 21, wherein no more than measurement area is provided on each grating structure (c) with a periodicity locally varying essentially perpendicular to the direction of propagation of the excitation light incoupled into layer (a), and wherein an unstructured area of the grating waveguide structure is provided in direction of propagation of the excitation light to be incoupled into and guided in layer (a), and wherein optionally a further grating structure (c) is provided in direction of the further propagation of the excitation light guided in layer (a), which is used to outcouple said guided excitation light towards a locally resolving detector.

35. Optical system according to claim 34, wherein changes of the mass coverage upon adsorption or desorption of molecules at the measurement areas on grating structures (c) result in a shift, essentially in parallel to the grating lines, of the local position of satisfaction of the resonance condition for the incoupling of the excitation light into layer (a) by means of said grating structure (c).

36. Optical system according to any of claims 34-35, wherein a one-dimensional arrangement of at least two grating structures (c) according to claim 21 is irradiated simultaneously with excitation light.

37. Optical system according to any of claims 34-36, wherein the excitation light is irradiated essentially in parallel and is essentially monochromatic.

38. Optical system according to claim 37, wherein the excitation light is irradiated linearly polarized, for excitation of a TE₀ or TM₀-mode guided in the layer (a).

39. Optical system according to any of claims 37-38, wherein a two-dimensional arrangement of at least four grating structures (c) according to claim 21 is irradiated simultaneously with excitation light.

40. Optical system for the locally resolved determination of changes of the resonance conditions for the incoupling of an excitation light into a waveguide or for the outcoupling of a light guided in the waveguide, comprising a two-dimensional array of at least four or more, laterally separated measurement areas (d) on said platform, comprising

at least one excitation light source

a grating waveguide structure according to any of claims 1-29

a positioning element for the change of the angle of incidence of the excitation light on the grating waveguide structure

at least one locally resolving detector for determination of the transmitted excitation light located opposite side of the grating waveguide structure, with respect to the irradiated excitation light, and/or for the determination of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the direction of irradiation of the excitation light, and/or for the determination of the scattered light of an excitation light guided in layer (a) after incoupling by means of a grating structure (c).

41. Optical system for the locally resolved determination of changes of the resonance conditions for the incoupling of an excitation light into a waveguide or for the outcoupling of a light guided in the waveguide, comprising a two-dimensional array of at least four or more, laterally separated measurement areas (d) on said platform, comprising

at least one excitation light source

a grating waveguide structure according to any of claims 1-29

a positioning element for the change of the angle of incidence of the excitation light on the grating waveguide structure

a diffusively reflecting and/or diffusively transmitting projection screen located at the opposite side of the grating waveguide structure, with respect to the direction of irradiation of the excitation light, for generation of an image of the transmitted excitation light, and at least one locally resolving detector for collection of the image of the transmitted excitation light from said projection screen.

42. Optical system for the locally resolved determination of changes of the resonance conditions for the incoupling of an excitation light into a waveguide or for the outcoupling of a light guided in the waveguide, comprising an array of at least two or more, laterally separated measurement areas (d) on said platform, comprising

at least one excitation light source tunable over a certain spectral range

a grating waveguide structure according to any of claims 1-29

at least one locally resolving detector for determination of the transmitted excitation light located at the same side of the grating waveguide structure, with respect to the irradiated excitation light, and or for the determination of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the direction of irradiation of the excitation light, and/or for the determination of the scattered light of an excitation light guided in layer (a) after incoupling by means of a grating structure (c).

43. Optical system according to claim 42, wherein said at least one tunable light source is tunable over a spectral range of at least 5 nm.

44. Optical system for the locally resolved determination of changes of the resonance conditions for the incoupling of an excitation light into a waveguide or for the outcoupling of a light guided in the waveguide, comprising an array of at least two or more, laterally separated measurement areas (d) on said platform, comprising

at least one excitation light source polychromatic within a certain spectral range

a grating waveguide structure according to any of claims 1-29

at least one locally resolving detector for determination of the transmitted excitation light located at the same side of the grating waveguide structure, with respect to the irradiated excitation light, and/or for the determination of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the direction of irradiation of the excitation light, and/or for the determination of the scattered light of an excitation light guided in layer (a) after incoupling by means of a grating structure (c).

45. Optical system according to claim 44, wherein said at least one polychromatic emission light source has an emission bandwith of at least 5 nm.

46. Optical system according to any of claims 44-45, wherein a spectrally selective optical component of high spectral resolution in said certain spectral range is located in the optical path between the grating waveguide structure and the at least one locally resolving detector.

47. Optical system according to claim 46, wherein said spectrally selective component is suitable for the generation of spectrally selective, locally resolved, two-dimensional illustrations of the intensity distributions of the measurement light emanating from the grating waveguide structure, at different wavelengths within said certain spectral range.

48. Optical system according to any of claims 44-47, wherein the locally resolved determination of changes of the resonance conditions for incoupling of an excitation light into layer (a) or outcoupling of light guided in the waveguide (layer (a)), from said polychromatic light source in the region of the measurement areas, is performed

by simultaneous or sequential collection of the transmitted excitation light and/or

by simultaneous or sequential collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or

by simultaneous or sequential collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c),

by means of spectrally selective detection, within said certain spectral range, using at least one locally resolving detector, preferably under irradiation of the excitation light onto the grating waveguide structure at a constant angle of incidence.

49. Optical system according to any of claims 40-48, wherein the excitation light is irradiated essentially in parallel.

50. Optical system according to any of claims 40-43, wherein the irradiated excitation light is essentially monochromatic.

51. Optical system according to any of claims 40-50, wherein the excitation light is irradiated linearly polarized, for excitation of a TE₀ or TM₀-mode guided in the layer (a).

52. Optical system according to any of claims 40-51, wherein the locally resolved determination of changes of the resonance conditions for incoupling of an excitation light into layer (a) or outcoupling of light guided in the waveguide (layer (a)), in the region of the measurement areas, is performed

by sequential collection of the transmitted excitation light and/or

by sequential collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or

by sequential collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c),

by means of one or more locally resolving detectors upon variation of the angle of incidence of the excitation light irradiated onto the grating waveguide structure.

53. Optical system according to any of claims 42-51, wherein the locally resolved determination of changes of the resonance conditions for incoupling of an excitation light into layer (a) or outcoupling of light guided in the waveguide (layer (a)), in the region of the measurement areas, is performed

by sequential collection of the transmitted excitation light and/or

by sequential collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or

by sequential collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c),

by means of one or more locally resolving detectors upon variation of the emission wavelength of a tunable light source, preferably upon irradiating the excitation light onto the grating waveguide structure at constant angle of incidence.

54. Optical system according to any of claims 30-53, wherein the excitation light from at least one light source is expanded as homogeneously as possible to an essentially light ray bundle by means of an expansion optics and irradiated onto the one or more measurement areas.

55. Optical system according to claim 54, wherein the irradiated excitation light bundle has, at least in one dimension, a diameter of at least 2 mm, preferably of at least 10 mm.

56. Optical system according to any of claims 30-52, wherein the excitation light from the at least one light source is multiplexed to a plurality of individual rays of intensity as uniform as possible by a diffractive optical element, or in case of multiple light sources by multiple diffractive optical elements, which are preferably Dammann gratings, or by refractive optical elements, which are preferably microlens arrays, the individual rays being launched essentially parallel to each other onto laterally separated measurement areas.

57. Optical system according to any of claims 30-39, wherein the excitation light from at least one, preferably monochromatic light source is expanded to a ray bundle of intensity as homogeneous as possible, with a slit-type cross-section (in a plane perpendicular to the optical axis of the optical ray path), the main axis being oriented in parallel to the grating lines, by means of a beam shaping optics, wherein the individual rays of the ray bundle are essentially in parallel to each other in a plane of projection in parallel to the plane of the grating waveguide structure, and wherein said ray bundle has a convergence or divergence with a certain convergence or divergence angle in a plane perpendicular to the plane of the grating waveguide structure.

58. Optical system according to claim 57, wherein the locally resolved determination of changes of the resonance conditions for incoupling of an excitation light into layer (a) or outcoupling of light guided in the waveguide (layer (a)), in the region of the measurement areas, within an irradiated region of slit-type cross-section, is performed

by simultaneous collection of the transmitted excitation light and/or

by simultaneous collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or

by simultaneous collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c),

by means of one or more locally resolving detectors, wherein the local change of the resonance conditions in a measurement area is monitored

by a shift of the intensity maximum of the light emanating essentially in parallel to the reflected light from said measurement area and

by a shift of the intensity maximum of the scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c) and

by a shift of the intensity minimum of the light transmitted in the region of said measurement area

(in each case at the condition of satisfaction of the resonance conditions in said measurement area),

wherein the shift of said intensity maximum respectively intensity minimum occurs in a plane in parallel to the plane of the grating waveguide structure, perpendicular to the grating lines.

59. Optical system according to any of claims 30-58, wherein two or more coherent light sources with equal or different emission wavelength are used as excitation light sources.

60. Optical system according to claim 59, wherein the excitation light of two or more coherent light sources is irradiated simultaneously or sequentially from different directions onto a grating structure (c), which is provided as superposition of grating structures with different periodicity.

61. Optical system according to any of claims 30-60, wherein a laterally resolving detector of the group comprising, for example, CCD cameras, CCD chips, photodiode arrays, avalanche diode arrays, multichannel plates and multichannel photomultipliers, is used for signal detection.

62. Optical system according to any of claims 30-61, wherein optical components of the group comprising lenses or lens systems for the shaping of 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 density filters for the regulation of the transmitted light intensity, optical filters or monochromators for the spectrally selective transmission of parts of the light bundles, or polarization selective elements for the selection of discrete polarization directions of the excitation or luminescence light are located between the one or more excitation light sources and the grating waveguide structure according to any of claims 1-29 and/or between said grating waveguide structure and the one or more detectors.

63. Optical system according to any of claims 30-62, wherein the excitation light is launched in pulses with a duration of 1 fsec to 10 min and the emission light from the measurement areas is measured time-resolved.

64. Optical system according to any of claims 30-63, wherein launching of the excitation light and detection of the light emanating from the one or more measurement areas is performed sequentially for one or more measurement areas.

65. Optical system according to claim 64, wherein sequential excitation and detection is performed using movable optical components of the group comprising mirrors, deviating prisms, and dichroic mirrors.

66. Optical system according to any of claims 64-65, wherein the grating waveguide structure is moved between steps of sequential excitation and detection.

67. Optical system for the locally resolved determination of changes of the resonance conditions for the incoupling of excitation light into a waveguide or outcoupling of a light guided in said waveguide, with an array of at least two or more measurement areas (d) on said platform, for the determination of one or more analytes in at least one sample on one or more measurement areas on a grating waveguide structure, with

a grating waveguide structure according to any of claims 1-29

an optical system according to any of claims 30-66 and

supply means for bringing the one or more samples into contact with the measurement areas on the grating waveguide structure.

68. Optical system according to claim 67, wherein said system additionally comprises one or more sample compartments, which are at least in the area of the one or more measurement areas or of the measurement areas combined to segments open towards the grating waveguide structure, wherein the sample compartments each preferably have a volume of 0. 1 nl-100 μ

l.

69. Optical system according to claim 68, wherein the sample compartments are closed, except for inlet and/or outlet openings for the supply or outlet of samples, at their side opposite to the optically transparent layer (a), and wherein the supply or the outlet of the samples and optionally of additional reagents is performed in a closed flow through system, wherein, in case of liquid supply to several measurement areas or segments with common inlet and outlet openings, these openings are preferably addressed row by row or column by column.

70. Optical system according to any of claims 67-69, wherein compartments for reagents are provided, which reagents are wetted during the assay for the determination of the one or more analytes and contacted with the measurement areas.

71. Method for the qualitative and/or quantitative determination of one or more analytes in one or more samples on at least two or more laterally separated measurement areas on a grating waveguide structure according to any of claims 1-29 in an optical system according to any of claims 34-70, upon determination of changes of the resonance conditions for incoupling of an excitation light into a waveguide or for outcoupling of a light guided in said waveguide, comprising an array of at least two or more laterally separated measurement areas (d) on said grating waveguide structure, wherein the excitation light from at least one excitation light source is irradiated onto a grating waveguide structure (c) with said measurement areas located thereon, and wherein the degree of satisfaction of the resonance condition for the incoupling of light into the layer (a) towards said measurement areas is determined from the signal of at least one locally resolving detector for the collection of the transmitted excitation light and/or for the collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the direction of irradiation of the excitation light, and/or for the collection of the scattered light of an excitation light guided in layer (a) after incoupling by means of a grating structure (c).

72. Method for the qualitative and/or quantitative determination of one or more analytes in one or more samples on at least two or more laterally separated measurement areas on a grating waveguide structure according to claim 21, wherein no more than one measurement area is provided on each grating structure (c) with a periodicity locally varying essentially perpendicular to the direction of propagation of the excitation light incoupled into layer (a), and wherein an unstructured region of the grating waveguide structure is provided in direction of further propagation of the excitation light to be incoupled into and guided in layer (a), and wherein optionally a further grating structure (c) is provided in direction of the still further propagation of the excitation light guided in layer (a), which last grating structure is used to outcouple again said guided excitation light towards a locally resolving detector.

73. Method according to claim 72, wherein changes of the local effective refractive index, especially of the mass coverage upon adsorption or desorption of molecules at the measurement areas on grating structures (c), result in a shift, essentially in parallel to the grating lines, of the local position of satisfaction of the resonance condition for the incoupling of the excitation light into layer (a) by means of said grating structure (c).

74. Method according to any of claims 72-73, wherein a one-dimensional arrangement of at least two grating structures (c) according to claim 21 is irradiated simultaneously with excitation light.

75. Method according to any of claims 72-74, wherein the excitation light is irradiated essentially in parallel and is essentially monochromatic.

76. Method according to claim 75, wherein the excitation light is irradiated linearity polarized, for excitation of a TE₀ or TM₀-mode guided in the layer (a).

77. Method according to any of claims 75-76, wherein a two-dimensional arrangement of at least four grating structures (c) according to claim 21 is irradiated simultaneously with excitation light.

78. Method for the qualitative and/or quantitative determination of one or more analytes in one or more samples on at least two or more laterally separated measurement areas on a grating waveguide structure according to any of claims 1-29, upon determination of changes of the resonance conditions for incoupling of an excitation light into a waveguide or for outcoupling of a light guided in said waveguide, comprising a two-dimensional array of at least four or more, laterally separated measurement areas (d) on said platform, wherein

the excitation light from at least one excitation light source is irradiated onto a grating waveguide structure (c) with said measurement areas located thereon, and wherein the degree of satisfaction of the resonance condition for the incoupling of light into the layer (a) towards said measurement areas is determined from the signal of at least one locally resolving detector for the collection of the transmitted excitation light, optionally upon using a diffusively reflecting and/or diffusively transmitting projection screen located at the opposite side of the grating waveguide structure, with respect to the direction of irradiation of the excitation light, for generation of an image of the transmitted excitation light, and/or from the signal of at least one locally resolving detector for the collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the direction of irradiation of the excitation light, and/or from the signal of at least one locally resolving detector for the collection of the scattered light of an excitation light guided in layer (a) after incoupling by means of a grating structure (c), and wherein the angle of incidence of the excitation light on the grating waveguide structure is changed by means of a positioning element, resulting, dependent on the local refractive index, in satisfaction of said resonance condition at different angles in the regions of different measurement areas irradiated on a grating waveguide structure (c).

79. Method according to claim 78, wherein the excitation light is irradiated essentially in parallel and is essentially monochromatic.

80. Method according to claim 79, wherein the excitation light is irradiated linearly polarized, for excitation of a TE₀ or TM₀-mode guided in the layer (a).

81. Method according to any of claims 78-80, wherein the locally resolved determination of changes of the resonance conditions for incoupling of an excitation light into layer (a) or outcoupling of light guided in the waveguide (layer (a)), in the region of the measurement areas, is performed

by sequential collection of the transmitted excitation light and/or

by sequential collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or

by sequential collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c), by means of one or more locally resolving detectors upon variation of the angle of incidence of the excitation light irradiated onto the grating waveguide structure.

82. Method according to claim 71, wherein the angle of incidence of the excitation light on the grating waveguide structure is adjusted in such a way that the resonance condition for incoupling of an excitation light into a waveguide with a grating waveguide structure or for outcoupling of light guided in the waveguide (layer (a)), comprising an array of at least two or more laterally separated measurement area (d) on said grating waveguide structure, is essentially satisfied

on one or more of said measurement areas, resulting in an essentially maximum signal from a locally resolving detector for collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or for collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c), from the region of said measurement areas and/or resulting in an essentially minimum signal from a locally resolving detector for collection of the transmitted excitation light from the region of the measurement areas

or is essentially satisfied between the measurement areas resulting in an essentially maximum signal from a locally resolving detector for collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or for collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c), from the regions between of measurement areas and/or resulting in an essentially minimum signal from a locally resolving detector for collection of the transmitted excitation light from the regions between the measurement areas.

83. Method according to claim 82, wherein local differences of the effective refractive index in the region of different measurement areas and in the regions between the measurement areas are determined from local differences of the intensities of one or more locally resolving detectors, for of the transmitted excitation light and/or for collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or for collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c), without changing the adjusted angle of incidence of the excitation light on the grating waveguide structure.

84. Method according to claim 71, wherein the locally resolved determination of changes of the resonance condition for the incoupling of an excitation light, from a light source tunable at least over a certain spectral range, into layer (a) or for the outcoupling of a light guided in the waveguide (layer (a)), in the region of the measurement areas, is performed by sequential collection of the transmitted excitation light and/or by sequential collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or by sequential collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c), using one or more locally resolving detectors in each configuration and varying the emission wavelength of said at least one tunable light source, preferably at a constant angle of incidence of the excitation light on the grating waveguide structure.

85. Method according to claim 71, wherein the emission wavelength of at least one tunable light source is adjusted, preferably at a constant angle of incidence of this excitation light on the grating waveguide structure, in such a way that the resonance condition for incoupling of an excitation light into a waveguide of a grating waveguide structure or for outcoupling of light guided in the waveguide (layer (a)), comprising an array of at least two or more laterally separated measurement area (d) on said grating waveguide structure, is essentially satisfied

on one or more of said measurement areas, resulting in an essentially maximum signal from a locally resolving detector for collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or for collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c), from the region of said measurement areas and/or resulting in an essentially minimum signal from a locally resolving detector for collection of the transmitted excitation light from the region of the measurement areas

or is essentially satisfied between the measurement areas resulting in an essentially maximum signal from a locally resolving detector for collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or for collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c), from the regions between of measurement areas and/or resulting in an essentially minimum signal from a locally resolving detector for collection of the transmitted excitation light from the regions between the measurement areas.

86. Method according to claim 71, wherein the locally resolved determination of changes of the resonance condition for the incoupling of an excitation light into layer (a) or for the outcoupling of a light guided in the waveguide (layer (a)), from a polychromatic light source tunable at least over a certain spectral range, in the region of the measurement areas is performed by collection of the transmitted excitation light and/or by collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or by collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c), using one or more locally resolving detectors in each configuration, the excitation light being preferably irradiated at a constant angle of incidence onto the grating waveguide structure, and wherein, upon satisfaction of the resonance condition of incoupling excitation light for a certain wavelength of said excitation light or outcoupling of excitation light of this wavelength guided in the waveguide a maximum signal fraction of this wavelength, as part of the signal from a locally resolving detector for collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or for collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c), from the region of said measurement areas and/or a minimum signal fraction of this wavelength, as part of the signal from a locally resolving detector for collection of the transmitted excitation light from the region of the measurement areas is measured.

87. Method according to claim 86, wherein a spectrally selective optical component of high spectral resolution in said certain spectral range is located in the optical path between the grating waveguide structure and the at least one locally resolving detector.

88. Method according to claim 87, wherein spectrally selective, locally resolved, two-dimensional illustrations of the intensity distributions of the measurement light emanating from the grating waveguide structure, at different wavelengths within said certain spectral range, can be generated using said spectrally selective component.

89. Method according to any of claims 44-47, wherein the locally resolved determination of changes of the resonance conditions for incoupling of an excitation light into layer (a) or outcoupling of light guided in the waveguide (layer (a)), from said polychromatic light source in the region of the measurement areas, is performed

by simultaneous or sequential collection of the transmitted excitation light and/or

by simultaneous or sequential collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or

by simultaneous or sequential collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c),

by means of spectrally selective detection, within said certain spectral range, using at least one locally resolving detector, preferably under irradiation of the excitation light onto the grating waveguide structure at a constant angle of incidence.

90. Method according to any of claims 86-89, wherein the excitation light is irradiated essentially in parallel.

91. Method according to any of claims 71-90, wherein the excitation light from the at least one light source is multiplexed to a plurality of individual rays of intensity as uniform as possible by a diffractive optical element, or in case of multiple light sources by multiple diffractive optical elements, which are preferably Dammann gratings, or by refractive optical elements, which are preferably microlens arrays, the individual rays being launched essentially parallel to each other onto laterally separated measurement areas.

92. Method according to claim 71, wherein the excitation light from at least one, preferably monochromatic light source is expanded to a ray bundle of intensity as homogeneous as possible, with a slit-type cross-section (in a plane perpendicular to the optical axis of the optical ray path), the main axis being oriented in parallel to the grating lines, by means of a beam shaping optics, wherein the individual rays of the ray bundle are essentially in parallel to each other in a plane of projection in parallel to the plane of the grating waveguide structure, and wherein said ray bundle has a convergence or divergence with a certain convergence or divergence angle in a plane perpendicular to the plane of the grating waveguide structure.

93. Method according to claim 92, wherein the angle of convergence of divergence of said ray bundle is smaller than 5°

in a plane perpendicular to the plane of the grating waveguide structure.

94. Method according to any of claims 92-93, wherein the locally resolved determination of changes of the resonance conditions for incoupling of an excitation light into layer (a) or outcoupling of light guided in the waveguide (layer (a)), in the region of the measurement areas, within an irradiated region of slit-type cross-section, is performed

by simultaneous collection of the transmitted excitation light and/or

by simultaneous collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or

by simultaneous collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c),

by means of one or more locally resolving detectors, wherein the local change of the resonance conditions in a measurement area is monitored

by a shift of the intensity maximum of the light emanating essentially in parallel to the reflected light from said measurement area and

by a shift of the intensity maximum of the scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c) and

by a shift of the intensity minimum of the light transmitted in the region of said measurement area (in each case at the condition of satisfaction of the resonance conditions in said measurement area), wherein the shift of said intensity maximum respectively intensity minimum occurs in a plane in parallel to the plane of the grating waveguide structure, perpendicular to the grating lines.

95. Method for the qualitative and/or quantitative determination of one or more analytes in one or more samples on at least two or more laterally separated measurement areas on a grating waveguide structure according to any of claims 1-29 in an optical system according to any of claims 34-70, upon determination of changes of the resonance conditions for incoupling of an excitation light into a waveguide or for outcoupling of a light guided in said waveguide, comprising an array of at least two or more laterally separated measurement areas (d) on said grating waveguide structure,

wherein the locally resolved determination of changes of said resonance conditions) is performed always simultaneously in the region of the measurement areas within an irradiated region of slit-type cross-section

by simultaneous collection of the transmitted excitation light and/or

by simultaneous collection of the light outcoupled again essentially in parallel to the reflected light at the same side of the grating waveguide structure, with respect to the side of irradiation of the excitation light and/or

by simultaneous collection of scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c),

by means of one or more locally resolving detectors, wherein the local change of the resonance conditions in a measurement area is monitored

by a shift of the intensity maximum of the light emanating essentially in parallel to the reflected light from said measurement area and

by a shift of the intensity maximum of the scattered light of excitation light guided in the layer (a) after incoupling by means of a grating waveguide structure (c) and

by a shift of the intensity minimum of the light transmitted in the region of said measurement area

(in each case at the condition of satisfaction of the resonance conditions in said measurement area),

wherein the shift of said intensity maximum respectively intensity minimum occurs in a plane in parallel to the plane of the grating waveguide structure, perpendicular to the grating lines,

and wherein the grating waveguide structure is moved perpendicular and/or in parallel to the direction of the grating lines between sequential measurement process steps, for a sequential locally resolved determination of said resonance conditions on the whole surface of the grating waveguide structure with the measurement areas provided thereon, until the measurement signals from all measurement areas are collected and stored and a two-dimensional representation of the degree of satisfaction of said resonance condition on the whole grating waveguide structure can be generated from the stored signals.

96. Method according to any of claims 78-95, wherein the lateral resolution for the determination of the degree of satisfaction of the resonance condition for incoupling of light into layer (a) can be improved by choice of a larger modulation depth of grating structures (c) or decreased by choice of a lower modulation depth of said grating structures.

97. Method according to any of claims 78-96, wherein the halfwidth of the resonance angle for satisfaction of the resonance condition for incoupling of light into layer (a) can be decreased by a decrease of the modulation depth of grating structures (c), resulting in an increased sensitivity for the laterally resolved determination of the degree of satisfaction of the resonance condition as a consequence from local changes of the mass coverage, or can be increased by an increase of the modulation depth of said grating structures, resulting in .a decreased sensitivity for the laterally resolved determination of the degree of satisfaction of the resonance condition as a consequence from local changes of the mass coverage.

98. Method according to any of claims 78-97, wherein differences of the mass coverage and/or of the effective refractive index can be resolved also within a measurement area.

99. Method according to any of claims 71-98, wherein two or more coherent light sources with equal or different emission wavelengths are used as excitation light sources.

100. Method according to any of claims 71-99, wherein a mass label, which can be selected from the group comprising metal colloids (such as gold colloids), plastic particles or beads or other microparticles with a monodisperse size distribution, is bound to the analyte molecules or to one of its binding partners in a multi-step assay, in order to increase the change of the mass coverage upon the binding to or dissociation of analyte molecules to be determined.

101. Method according to any of claims 71-100, wherein an “

absorption label”

is bound to the analyte molecules or to one of its binding partners in a multi-step assay, in order to increase the change of the effective refractive index upon binding or dissociation of analyte molecules to be determined, the “

absorption label”

having an absorption band of suitable wavelength resulting in a change of the effective refractive index in the near-field of the grating waveguide structure, the absorption being the imaginary part of the refractive index.

102. Method according to any of claims 71-101, wherein one or more luminescences, excited in the evanescent field of an excitation light guided in layer (a), are determined in addition to the locally resolved determination of changes of the resonance conditions for the incoupling of an excitation light into the layer (a) of a grating waveguide structure according to any of claims 1-29 or for the outcoupling of a light guided in said layer (a).

103. Method according to claim 102, wherein the binding of a ligand as an analyte to an immobilized biological or biochemical or synthetic recognition element as a receptor in one or more measurement areas is determined from the local change of the effective refractive index and a functional response of said ligand receptor system is determined from a change of a luminescence emanating from said measurement areas.

104. Method according to claim 102, wherein the density of immobilized biological or biochemical or synthetic recognition elements as receptors in one or more measurement areas is determined from the differences between the resonance conditions for the incoupling of an excitation light into the layer (a) of the grating waveguide structure or for the outcoupling of a light guided in said layer (a), in the region of said measurement areas, and the corresponding resonance conditions in the environment, i..e. outside of said measurement areas, and wherein the binding of a ligand as an analyte to said recognition elements is determined from a change of a luminescence emanating from said measurement areas.

105. Method according to any of claims 102- 104, wherein (firstly) the isotropically emitted luminescence or (secondly) luminescence that is incoupled into the optically transparent layer (a) and out-coupled by a grating structure (c) or luminescence comprising both parts (firstly and secondly) is measured simultaneously.

106. Method according to any of claims 102-105, wherein, for the generation of said luminescence, a luminescent dye or a luminescent nano-particle is used as a luminescence label, which can be excited and emits at a wavelength between 300 nm and 1100 nm.

107. Method according to any of claims 100-106, wherein the mass label and or the luminescence label is bound to the analyte or, in a competitive assay, to an analyte analogue or, in a multi-step 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.

108. Method according to any of claims 102-107, wherein the one or more determinations of luminescences and/or determinations of light signals at the excitation wavelengths are performed polarization-selective, wherein preferably the one or more luminescences are measured at a polarization that is different from the one of the excitation light.

109. Method according to any of claims 71-108 for the simultaneous or sequential, quantitative or qualitative determination of one or more analytes of the group comprising antibodies or antigens, receptors or ligands, chelators or “

histidin-tag components”

, oligonucleotides, DNA or RNA strands, DNA or RNA analogues, enzymes, enzyme cofactors or inhibitors, lectins and carbohydrates.

110. Method according to any of claims 71-109, wherein the samples to be examined are naturally occurring body fluids, such as blood, serum, plasma, lymph or urine or egg yolk or optically turbid liquids or surface water or soil or plant extracts or bio- or process broths or are taken from biological tissue parts.

111. The use of a grating waveguide structure according to any of claims-1-29 and/or of an optical system according to any of claims 30-70 and/or of a method according to any of claims 71-110 for qualitative and/or quantitative analyses for the determination of chemical, biochemical or biological analytes in screening methods in pharmaceutical research, combinatorial chemistry, clinical and preclinical development, for real-time binding studies and the determination of kinetic parameters in affinity screening and in research, for qualitative and quantitative analyte determinations, especially for DNA- and RNA analytics, for the generation of toxicity studies and the determination of expression profiles, and for the determination of antibodies, antigens, pathogens or bacteria in pharmaceutical product development and research, human and veterinary diagnostics, agrochemical product development and research, for symptomatic and pre-symptomatic plant diagnostics, for patient stratification in pharmaceutical product development and for the therapeutic drug selection, for the determination of pathogens, nocuous agents and germs, especially of salmonella, prions and bacteria, in food and environmental analytics.

2. Grating waveguide structure for the locally resolved determination of changes of the resonance conditions for the incoupling of an excitation light into a waveguide or for the outcoupling of a light guided in the waveguide, comprising a two-dimensional array of at least four or more, laterally separated measurement areas (d) on said platform, comprising a stratified optical 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 more grating structures (c) for the incoupling of an excitation light towards the measurement areas (d) or for the outcoupling of a light guided in layer (a) in the region of the measurement areas

with at least one or more laterally separated measurement areas (d) on said one or more grating structures (c)

with equal or different biological or biochemical or synthetic recognition elements (e) immobilized on said measurement areas, for the qualitative and/or quantitative determination of one or more analytes in a sample brought into contact with said measurement areas,

wherein the density of the measurement areas on a common grating structure (c) is at least 10 measurement areas per square centimeter, said excitation light is irradiated simultaneously onto said array of measurement areas, and the degree of satisfaction of the resonance condition for the incoupling of light into the layer (a) towards said two or more measurement areas is simultaneously measured and a cross-talk of excitation light guided in layer (a), from one measurement area to one or more adjacent measurement areas is prevented by outcoupling said excitation light again by means of the grating structure (c).