Kit and method for multi-analyte determination

• US 7,645,612 B2
• Filed: 04/04/2008
• Issued: 01/12/2010
• Est. Priority Date: 06/02/2000
• Status: Expired due to Fees

• Current Assignee: Bayer Intellectual Property GmbH (Bayer AG)
• Original Assignee: Bayer Technology Services GmbH (Bayer AG)
• Inventors: Pawlak, Michael, Kresbach, Gerhard Matthias, Duveneck, Gert Ludwig, Schick, Eginhard, Bopp, Martin Andreas, Abel, Andreas Peter, Ehrat, Markus, Schurmann-Mader, Eveline
• Primary Examiner(s): Chin; Christopher L
• Application Number:
  US12/062,820
• Publication Number:
  US 20080212070A1
• Time in Patent Office: 648 Days
• Field of Search: 422/50, 422/55, 422/57, 422/82.05, 422/82.11, 435/287.2, 435/287.9, 435/288.7, 435/808, 435/975, 436/164, 436/172, 436/518, 436/524, 436/525, 436/805, 436/808
• US Class Current: 436/164
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/6482 Sample cells, cuvettes
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

The invention is related to different embodiments of a kit for the simultaneous qualitative and/or quantitative determination of a multitude of analytes comprising

• • a sensor platform comprising an optical thin-film waveguide with a layer (a) transparent at least at an excitation wavelength on a layer (b) with lower refractive index than layer (a), also transparent at least at said excitation wavelength, and at least one grating structure (c) modulated in said layer (a), for the incoupling of said excitation light into layer (a),
  • at least one array of biological or biochemical or synthetic recognition elements immobilized in discrete measurement areas (d) directly or by means of an adhesion-promoting layer on layer (a), for specific recognition and/or binding of said analytes and/or for specific interaction with said analytes,
  • means for laterally resolved referencing of the excitation light intensity available in the measurement areas, and optionally
  • means for the calibration of one or more luminescences generated in the near-field of layer (a) as a consequence of the binding of one or more analytes or of the specific interaction with one or more analytes,
    wherein a liquid sample to be analyzed for said analytes is brought into contact with said measurement areas on said sensor platform either directly or after mixture with further reagents. The invention is also related to analytical systems based on a kit according to the invention and to methods for the determination of one or more analytes, based on said kit, and to use thereof.

Claims

1. A method for the simultaneous qualitative and/or quantitative determination of a multitude of analytes comprising:

bringing one or more liquid samples to be analyzed for the analytes into contact with measurement areas of a sensor platform,

the sensor platform comprising

an optical thin film waveguide with a first layer, that is transparent at least at one excitation wavelength, on a second layer that has a lower refractive index than the first layer and is also transparent at least at the excitation wavelength, and at least one grating structure modulated in the first layer for in-coupling of the excitation light into the first layer;

at least one array of biological or biochemical or synthetic recognition elements immobilized in measurement areas for detection of different analytes by specific recognition and/or binding of said analytes and/or by specific interaction with said analytes,

means for laterally resolved referencing of the excitation light intensity available in the measurement areas and, optionally,

means for the calibration of one or more luminescences generated in a near field of the first layer,

the measurement areas for detection of different analytes being usable for detection of analytes to be determined and/or as means for calibration purposes;

irradiating an excitation light towards the measurement areas;

detecting an emission light from the measurement areas;

referencing the excitation light intensity available in the measurement areas in a locally resolved way by means of correcting the emission light intensity from measurement areas for detection of different analytes using the means for laterally resolved referencing of the excitation light intensity available in the measurement areas;

calibrating one or more luminescences generated in a near-field of the first layer from the measurement areas that result from the binding of one or more analytes to biological or biochemical or synthetic recognition elements immobilized in the measurement areas or from interaction between the analytes and the immobilized recognition elements using the measurement areas for detection of different analytes as means for calibration purposes to establish a calibration curve on each platform; and

comparing the emission light intensity from measurement areas for detection of analytes in the sample to be analyzed to the calibration curve for qualitative and/or quantitative determination of the analyte.

2. A method according to claim 1, wherein the excitation light is in-coupled into the optically transparent first layer towards the measurement areas by the grating structure.

3. A method according to claim 1, wherein the sensor platform comprises even, non-modulated regions of the first layer, which are arranged in the direction of propagation of an excitation light in-coupled into the first layer by the grating structure and guided in the first layer.

4. A method according to claim 1, wherein isotropically emitted luminescence or luminescence that is in-coupled into the optically transparent first layer and out-coupled by the grating structure, or luminescence comprising both isotropically emitted luminescence or luminescence that is in-coupled into the optically transparent first layer and out-coupled by the grating structure, is measured simultaneously.

5. A method according to claim 1, 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.

6. A method according to claim 5, wherein 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.

7. A method according to claim 5, wherein a second or more luminescence labels of similar or different excitation wavelength as the first luminescence label and similar or different emission wavelength are used.

8. A method according to claim 7, wherein the second or more luminescence labels can be excited at the same wavelength as the first luminescence label, but emit at other wavelengths.

9. A method according to claim 7, wherein the excitation and emission spectra of the applied luminescent dyes do not overlap or overlap only partially.

10. A method according to claim 7, wherein charge or optical energy transfer from a first luminescent dye, acting as a donor, to a second luminescent dye, acting as an acceptor, is used for the detection of the analyte.

11. A method according to claim 1, wherein 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.

12. A method according to claim 1, wherein, besides determination of one or more luminescences, changes of the effective refractive index on the measurement areas are determined.

13. A method according to claim 1, wherein the density of the recognition elements immobilized in discrete measurement areas for the detection of different analytes on different measurement areas is selected in such a way, that, upon determination of different analytes in a common array, the luminescence signals are of similar order of magnitude, so that the related calibration curves for the analyte determinations to be performed simultaneously can be recorded without a change of the opto-electronic system adjustments.

14. A method according to claim 1, wherein arrays of measurement areas are arranged in segments of one or more measurement areas for analyte determination and of measurement areas for referencing, for the determination of physical parameters and/or of chemical differences between different applied samples.

15. A method according to claim 1, wherein one or more arrays comprise segments of two or more measurement areas with biological or biochemical or synthetic recognition elements for analyte determination or for referencing, that are similar within said segment.

16. A method according to claim 1, wherein one or more measurement areas of a segment or of an array are provided for the same analyte, and wherein the related immobilized biological or biochemical recognition elements have different affinities for said analyte.

17. A method according to claim 1, wherein simultaneously on one or more segments of an array or on one or more arrays different analytes of a common group are determined.

18. A method according to claim 1, wherein simultaneously different analytes of different groups are determined on one or more segments of an array or on one or more arrays.

19. A method according to claim 1, wherein two or more identical measurement areas are provided for the determination of each analyte or for physical or chemical referencing within a segment or an array.

20. A method according to claim 19, wherein said identical measurement areas are arranged in a continuous row or column or diagonal of an array or a segment of measurement areas.

21. A method according to claim 19, wherein said identical measurement areas are distributed statistically within an array or a segment of measurement areas.

22. A method according to claim 1, wherein the laterally resolved referencing of the excitation light intensity available in the measurement areas comprises the simultaneous or sequential generation of an image of the light emanating from the sensor platform at the excitation wavelength.

23. A method according to claim 1, wherein said referencing of the excitation light intensity available in the measurement areas comprises the simultaneous or sequential generation of an image of the light emanating from the sensor platform at another excitation wavelength as used for excitation of a luminescence.

24. A method according to claim 23, wherein the excitation wavelength for said referencing is selected in such a way that molecules capable of luminescence and applied during the assay for the detection of one or more analytes or for purposes of referencing or of calibration have no absorption or absorption as low as possible at said wavelength.

25. A method according to claim 22, wherein the generation of an image of the excitation light emanating from the sensor platform is performed using the same optical path as for the collection of the luminescences emanating from the measurement areas.

26. A method according to claim 1, wherein said referencing of the excitation light intensity available in the measurement areas comprises the simultaneous or sequential generation of an image of the light emanating from the sensor platform at the luminescence wavelength.

27. A method according to claim 1, wherein the local resolution of the image for said referencing the excitation light emanating from the sensor platform is below 100 μ

m.

28. A method according to claim 1, wherein said referencing of the excitation light intensity available in the measurement areas is performed using luminescence marker spots comprising luminescence intensity from measurement areas with pre-immobilized luminescently labeled molecules which have been deposited in the measurement areas before supply of a sample.

29. A method according to claim 28, wherein the luminescence marker spots are provided as a net screen spreading over the whole sensor platform.

30. A method according to claim 1, wherein said referencing of the excitation light intensity available in the measurement areas comprises the determination of an average of multiple locally resolved reference signals.