System for determining analyte concentration

• US 6,287,871 B1
• Filed: 09/18/1998
• Issued: 09/11/2001
• Est. Priority Date: 03/19/1996
• Status: Expired due to Term

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Christensen, Douglas A., Herron, James N.
• Primary Examiner(s): SNAY, JEFFREY R
• Application Number:
  US09/142,947
• Time in Patent Office: 1,089 Days
• Field of Search: 422/82.08, 422/82.11, 422/102, 436/532, 436/165, 436/172, 436/809, 436/527
• US Class Current: 436/172
• 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

Abstract

The present invention relates to a system (80) for determining analyte concentration. The system (80) includes an optical detection system (92) that detects fluorescence from fluorescent binding assays in a biosensor (88). A processing system (96) may be used to determine analyte concentration from the fluorescence detected by the optical detection system (92). The optical detection system (92) may include photodetectors with or without in series lenses. Alternatively, a CCD camera (146) may be used.

Claims

1. A method of simultaneously detecting light emanating from a plurality of discrete areas of a biosensor and passing through a waveguide, said method comprising:

simultaneously emanating light from a plurality of discrete areas of a biosensor;

segregating the light simultaneously emanating from each discrete area of said plurality of discrete areas of the biosensor from other light simultaneously emanating from other discrete areas of the plurality of discrete areas of the biosensor by concurrently channeling the light from each said discrete area;

directing the segregated light simultaneously emanating from each said discrete area of said plurality of discrete areas of the biosensor to a respective photodetector for each said discrete area of said plurality of discrete areas; and

detecting the segregated light simultaneously emanating from each said discrete area of said plurality of discrete areas of the biosensor with said respective photodetector.

2. The method of claim 1, wherein the light simultaneously emanating from each said discrete area of said plurality of discrete areas of the biosensor is segregated from other light simultaneously emanating from other discrete areas of said plurality of discrete areas of the biosensor by means of a structure which defines an inlet opening therein and a channel associated with said inlet opening, said inlet opening being positioned adjacent each said discrete area of said plurality of discrete areas of the biosensor, whereby light emanating from each said discrete area of said plurality of discrete areas of the biosensor passes through said inlet opening and thereafter through said channel to said respective photodetector.

3. The method of claim 1, wherein the segregated light emanating from each said discrete area of said plurality of discrete areas of the biosensor is directed to the respective photodetector by at least one lens associated optically and interposed between each said discrete area of said plurality of discrete areas of the biosensor and the respective photodetector.

4. The method of claim 2, wherein the segregated light emanating from each said discrete area of said plurality of discrete areas of the biosensor is directed to the respective photodetector by at least one mirror.

5. The method of claim 4, wherein the mirror is a parabolic mirror.

6. The method of claim 1, wherein the photodetector is a CCD camera.

7. The method of claim 6, wherein the CCD camera is coupled to a grating spectrograph for spectral analysis of the detected light, and further including the step of detecting a total collected fluorescence of a sample.

8. An apparatus for detecting fluorescent light emanating from a plurality of discrete areas of a biosensor, said apparatus comprising:

a light source for simultaneous emanation of fluorescent light through a plurality of discrete areas of a biosensor;

a grate, optically associated with said plurality of discrete areas of the biosensor and said light source, for concurrently segregating fluorescent light simultaneously emanating from each discrete area of said plurality of discrete areas of the biosensor from fluorescent light simultaneously emanating from other discrete areas of said plurality of discrete areas of the biosensor; and

a structure for focusing the segregated fluorescent light onto a respective photodetector.

9. The apparatus of claim 8, wherein the structure for focusing the segregated fluorescent light is selected from the group of a lens, mirror, fiber optic cable, and combinations thereof.

10. A method for determining analyte concentration, said method comprising:

activating a light source to simultaneously emanate fluorescent light through a plurality of discrete areas of a biosensor, with

a grate, optically associated with said plurality of discrete areas of the biosensor and said light source, for concurrently segregating fluorescent light simultaneously emanating from each discrete area of said plurality of discrete areas of the biosensor from fluorescent light simultaneously emanating from other discrete areas of said plurality of discrete areas of the biosensor; and

a structure for focusing the segregated fluorescent light onto a respective photodetector; and

detecting fluorescent light emanating from a plurality of discrete areas of a biosensor.

11. The method of claim 10 further comprising focusing the segregated fluorescent light is selected from the group of a lens, mirror, fiber optic cable, and combinations thereof.

12. A method of simultaneously determining the presence of a plurality of analytes in a sample, said method comprising:

providing a biosensor having a waveguide and a plurality of patches disposed within a well defined in said waveguide, a first patch of said plurality of patches having a first type of capture molecule associated therewith, and a second patch of said plurality of patches having a second type of capture molecule associated therewith;

introducing a sample believed to contain a plurality of analytes into said well;

introducing at least one type of tracer molecule into said well, said tracer molecule comprising a fluorescent label bonded to a molecule that binds with either one of said first type and said second type of capture molecules or to at least one analyte of said plurality of analytes;

directing light through said waveguide, said light having a wave length which will excite said fluorescent label;

segregating fluorescent light emanating from said first patch from light simultaneously emanating from said second patch and light simultaneously emanating from a remainder of said biosensor by channeling said fluorescent light emanating from said first patch;

segregating fluorescent light emanating from said second patch from light simultaneously emanating from said first patch and light simultaneously emanating from said remainder of said biosensor by channeling said fluorescent light emanating from said second patch;

detecting said segregated fluorescent light emanating from said first patch with a first photodetector;

detecting said segregated fluorescent light emanating from said second patch with a second photodetector;

analyzing said segregated fluorescent light emanating from said first patch to determine a presence of a first analyte; and

analyzing said segregated fluorescent light emanating from said second patch to determine a presence of a second analyte.

13. The method of claim 12, wherein each said first and second patches of said plurality of patches is associated with a respective unique capture molecule.

14. The method of claim 12, further including the step of introducing a plurality of types of tracer molecules into said well, wherein each said type of tracer molecule of said plurality of types of tracer molecules has an affinity for a respective type of analyte being investigated.

15. The method of claim 12, wherein said sample and said plurality of types of tracer molecules are introduced simultaneously into said well.

16. A method of simultaneously determining the individual concentration of several analytes in a sample, said method comprising:

providing a biosensor having a waveguide which defines a first well and a second well and a plurality of patches disposed within said first and second wells, each said first and second wells containing a first patch of said plurality of patches having a first type of capture molecule associated therewith and a second patch of said plurality of patches having a second type of capture molecule associated therewith;

introducing a sample believed to contain a first analyte and a second analyte into said first well;

introducing a first liquid containing first known quantities of said first analyte and said second analyte into said second well;

introducing at least one type of tracer molecule into said first well and into said second well, said tracer molecule comprising a fluorescent label bonded to a molecule that binds with either one of said first and second types of capture molecules or at least one of said first and second analytes;

directing light through said waveguide, said light having a wave length which will excite said fluorescent label;

segregating fluorescent light emanating from said first patch in said first well from fluorescent light simultaneously emanating from said first patch in said second well, from fluorescent light simultaneously emanating from said second patches in said first well and said second well, and from fluorescent light simultaneously emanating from a remainder of said biosensor by channeling said fluorescent light emanating from said first patch in said first well;

segregating fluorescent light emanating from said first patch in said second well from fluorescent light simultaneously emanating from said first patch in said first well, from fluorescent light simultaneously emanating from said second patches in said first well and said second well, and from fluorescent light simultaneously emanating from a remainder of said biosensor by channeling said fluorescent light emanating from said first patch in said second well;

segregating fluorescent light emanating from said second patch in said first well from fluorescent light simultaneously emanating from said second patch in said second well, from fluorescent light simultaneously emanating from said first patches in said first well and said second well, and from fluorescent light simultaneously emanating from a remainder of said biosensor by channeling said fluorescent light emanating from said second patch in said first well;

segregating fluorescent light emanating from said second patch in said second well from fluorescent light simultaneously emanating from said second patch in said first well, from fluorescent light simultaneously emanating from said first patches in said first well, and said second well, and from fluorescent light simultaneously emanating from a remainder of said biosensor by channeling said fluorescent light emanating from said second patch in said second well;

detecting said segregated fluorescent light emanating from said first patch in said first well with a first photodetector;

detecting said segregated fluorescent light emanating from said first patch in said second well with a second photodetector;

detecting said segregated fluorescent light emanating from said second patch in said first well with a third photodetector;

detecting said segregated fluorescent light emanating from said second patch in said second well with a fourth photodetector;

analyzing said segregated fluorescent light emanating from said first patch in said first well detected by said first photodetector in view of said segregated fluorescent light simultaneously emanating from said first patch in said second well detected by said second photodetector to determine a concentration of said first analyte in said sample;

analyzing said segregated fluorescent light emanating from said second patch in said first well detected by said third photodetector in view of said segregated fluorescent light simultaneously emanating from said second patch in said second well detected by said fourth photodetector to determine a concentration of said second analyte in said sample.

17. The method of claim 16, wherein said first liquid contains no said first analytes and no said second analytes.

18. The method of claim 16, wherein said biosensor defines a third well and a plurality of patches disposed within said third well, said third well containing a first patch of said plurality of patches having said first type of capture molecule associated therewith and a second patch of said plurality of patches having said second type capture molecule associate therewith, said method further comprising the steps of:

introducing a second liquid having second known quantities of said first analyte and said second analyte into said third well;

introducing said at least one type of tracer molecule into said third well;

segregating fluorescent light emanating from said first patch in said third well from fluorescent light simultaneously emanating from said second patch in said third well, from fluorescent light emanating from said first patches in said first well and said second well, from light simultaneously emanating from said second patch in said second well, and from fluorescent light simultaneously emanating from the remainder of said biosensor by channeling said fluorescent light emanating from said first patch in said third well;

segregating fluorescent light emanating from said second patch in said third well from fluorescent light simultaneously emanating from said first patch in said third well, from fluorescent light simultaneously emanating from said first patches in said first well and said second well, from light simultaneously emanating from said second patches in said first well and said second well and a remaining portion of said biosensor by channeling said fluorescent light emanating from said second patch in said third well;

detecting said segregated fluorescent light emanating from said first patch in said third well with a fifth photodetector;

detecting said segregated fluorescent light emanating from said second patch in said third well with a sixth photodetector;

analyzing said segregated fluorescent light emanating from said first patch in said first well detected by said first photodetector in view of said light simultaneously emanating from said first patch in said second well detected by said second photodetector and said segregated fluorescent light simultaneously emanating from said first patch in said third well detected by said fifth photodetector to determine a concentration of said first analyte in said sample; and

analyzing said segregated fluorescent light emanating from said second patch in said first well detected by said third photodetector in view of said segregated fluorescent light simultaneously emanating from said second patch in said second well detected by said fourth photodetector and said fluorescent light simultaneously emanating from said second patch in said third well detected by said sixth photodetector to determine a concentration of said second analyte in said sample.

19. The method of claim 18, wherein said at least one type of tracer molecule is introduced into said third well simultaneously with said second liquid.

20. The method of claim 16, 17, 18 or 19, wherein said at least one type of tracer molecule is introduced into said well simultaneously with said sample.

21. The method of claim 16, 17, 18 or 19, wherein said at least one type of tracer molecule is introduced into said second well simultaneously with said first liquid.

22. The method of claim 16, 17, 18 or 19, wherein said at least one type of tracer molecule is introduced into said third well simultaneously with said second liquid.

23. The method of claim 20, wherein said at least one type of tracer molecule is introduced into said third well simultaneously with said second liquid.