Oscillation apparatus and methods for multi-analyte homogeneous fluoro-immunoassays

• US 6,242,267 B1
• Filed: 09/18/1998
• Issued: 06/05/2001
• 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: Miles, Scott D., Christensen, Douglas A., Herron, James N.
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): PADMANABHAN, KARTIC
• Application Number:
  US09/142,946
• Time in Patent Office: 991 Days
• Field of Search: 422/55, 422/56, 422/57, 422/58, 422/102, 435/5, 435/7.1, 435/7.2, 435/7.5, 435/287.1, 435/287.2, 435/808, 435/288.5, 435/288.7, 436/164, 436/165, 436/172, 436/518, 436/527, 436/528, 436/524, 436/531, 436/807, 356/427, 356/426
• US Class Current: 436/527
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures

Abstract

An apparatus and method for rapidly analyzing samples for analytes of interest by an homogeneous immunofluorescence assay. The apparatus includes a sample test cartridge having a high control sample section, a low control sample section, and at least one test sample section. Each of these sections contain at least one pre-loaded reagent housed in a well within the cartridge wherein the low control sample section contains a known low amount of an analyte of interest and the high control sample section contains a known high amount of an analyte of interest. The cartridge includes a biosensor comprising a planar waveguide having first and second parallel plane surfaces and an edge extending between them, the edge having a receiving region for receiving a light beam. Each of the high control sample section, the low control sample section, and the test sample control sections have a well which includes a waveguide surface, wherein the contents of each section contacts capture molecules immobilized on the waveguide surface. The capture molecules are configured to specifically bind a chosen analyte and fluoresce when interacting with light passing through the waveguide surface. The concentration of said analyte of interest in said sample fluid is determined by a comparison of intensities of fluorescence of between said capture molecule areas of said sample capture molecule well, said low control capture molecule well, and said high control capture molecule well.

Claims

1. An assay cartridge for conducting homogeneous immunofluorescence assays comprising:

a housing comprising;

a sample apparatus having a port for receiving a fluid sample containing an unknown concentration of at least one analyte of interest, said port in fluid communication with a sample reagent well containing a predetermined sample reagent that interacts with said fluid sample and capture molecules, said sample reagent well in fluid communication with a sample capture molecule well, and a sample oscillation well for receiving, holding and releasing fluid during oscillation, said sample oscillation well in fluid communication with said sample capture molecule well; and

a low control apparatus having a low control reagent well containing a predetermined low control reagent having a known low concentration, relative to the fluid sample, of at least one analyte of interest, said predetermined low control reagent interacting with capture molecules, said low control reagent well in fluid communication with a low control capture molecule well, and a low control oscillation well for receiving, holding and releasing fluid during oscillation, said low control oscillation well in fluid communication with said low control capture molecule well;

said sample capture molecule well and said low control capture molecule well each having an adjacent wall including waveguide material; and

at least one area containing capture molecules within each of said sample capture molecule well and said low control capture molecule well, wherein each of said capture molecule areas is attached to respective waveguide material.

2. The assay cartridge of claim 1 further comprising:

a high control apparatus having a high control reagent well containing a predetermined high control reagent, which interacts with capture molecules, having a known high concentration, relative to said fluid sample, of at least one analyte of interest, said high control reagent well in fluid communication with a high control capture molecule well, and a high control oscillation well for receiving, holding and releasing fluid during oscillation, said high control oscillation well in fluid communication with said high control capture molecule well, said high control capture molecule well having a wall comprising waveguide material, and having at least one area containing capture molecules within said high control capture molecule well, wherein each of said capture molecule areas is attached to respective waveguide material.

5. The assay cartridge of claim 1 or claim 3, wherein said port is a sample cup.

6. The assay cartridge of claim 2 or claim 4, further comprising:

a sample transfer means for transferring said fluid sample to said sample reagent well, and for transferring said fluid sample and said sample reagent from said sample reagent well to said sample capture molecule well;

a low control transfer means for transferring said low control reagent to said low control capture molecule well; and

a high control transfer means for transferring said high control reagent to said high control capture molecule well.

7. The assay cartridge of claim 6, further comprising:

a first valve set between said sample cup and said sample reagent well, and between said sample reagent well and said sample capture molecule well;

a second valve between said low control reagent well and said low control capture molecule well; and

a third valve between said high control reagent well and said high control capture molecule well.

8. The assay cartridge of claim 2, further comprising:

a sample oscillation means for oscillating said fluid sample and said sample reagent between said sample reagent well and said sample oscillation well through said sample capture molecule well;

a low control oscillation means for oscillating said low control reagent between said low control reagent well and said low control oscillation well through said low control capture molecule well; and

a high control oscillation means for oscillating said high control reagent between said high control reagent well and said high control oscillation well through said high control capture molecule well.

9. The assay cartridge of claim 2 or claim 4, wherein the waveguide material of each of said sample capture molecule well, said low control capture molecule well, and said high control capture molecule well comprises a single waveguide, and said capture molecule wells include an opaque and rough wall surface opposing said waveguide.

10. The assay cartridge of claim 1 or claim 3, wherein said waveguide material comprises a single waveguide, said waveguide further comprising light receiving means on an edge thereof, said light receiving means configured to receive a light beam from a light source.

11. The assay cartridge of claim 10, wherein said waveguide further comprises light output means configured to output a portion of said beam received by said light receiving means.

12. The assay cartridge of claim 1 or claim 3, wherein said waveguide materials comprise a single waveguide detachably attached to said housing.

13. The assay cartridge of claim 1 or claim 3, wherein said capture molecules are selected from the group consisting of antibodies, antibody fragments, whole antigenic molecules, antigenic molecule fragments, oligopeptides, nucleic acids, oliogonucleotides, membrane receptors, peptides, and mixtures thereof.

14. The assay cartridge of claim 2 or claim 4, wherein said sample apparatus further includes a second sample reagent well containing a predetermined second reagent, said second sample reagent interacting with said fluid sample, said capture molecules and said sample reagent, in fluid communication with said sample reagent well and a sample waste well in fluid communication with said second sample reagent well and in fluid communication with said sample cup;

wherein said low control apparatus further includes a second low control reagent well containing a predetermined second low control reagent, that interacts with said capture molecules and said low control reagent, said second low control reagent well in fluid communication with said low control reagent well; and

wherein said high control apparatus further includes a second high control reagent well containing a predetermined second high control reagent, that interacts with said capture molecules and said high control reagent, said second high control reagent well in fluid communication with said high control reagent well.

15. The assay cartridge of claim 2, further comprising turbulence inducing means positioned in at least one of the following locations, between said sample reagent well and said sample capture molecule well, between said low control reagent well and said low control capture molecule well, and between said high control reagent well and said high control capture molecule well.

3. An assay cartridge for conducting homogeneous immunofluorescence assays comprising:

a housing comprising;

a sample apparatus having a port for receiving a fluid sample containing an unknown concentration of at least one analyte of interest, said port in fluid communication with a sample reagent well containing a predetermined sample reagent, which interacts with said fluid sample and capture molecules, said sample reagent well in fluid communication with a sample capture molecule well, and a turbulence inducing means positioned between said sample reagent well and said sample capture molecule well; and

a low control apparatus having a low control reagent well containing a predetermined low control reagent, which predetermined low control reagent interacts with capture molecules, having a known low concentration, relative to said fluid sample, of at least one analyte of interest, said low control reagent well in fluid communication with a low control capture molecule well, and a turbulence inducing means positioned between said low control reagent well and said low control capture molecule well;

said sample capture molecule well and said low control capture molecule well each having an adjacent wall including waveguide material; and

at least one area containing capture molecules within each of said sample capture molecule well and said low control capture molecule well, wherein each of said capture molecule areas is attached to respective waveguide material.

4. The assay cartridge of claim 3 further comprising:

a high control apparatus having a high control reagent well containing a predetermined high control reagent, which predetermined high control reagent interacts with capture molecules, having a known high concentration, relative to said fluid sample, of at least one analyte of interest, said high control reagent well in fluid communication with a high control capture molecule well, and a turbulence inducing means positioned between said high control reagent well and said high control capture molecule well, said high control capture molecule well having a wall comprising waveguide material, and having at least one area containing capture molecules within said high control capture molecule well, wherein each of said capture molecule areas is attached to respective waveguide material.

16. The assay cartridge of claim 4, further comprising:

a sample oscillation well for receiving, holding and releasing fluid during oscillation, in fluid communication with said sample capture molecule well;

a low control oscillation well for receiving, holding and releasing fluid during oscillation, said low control oscillation well in fluid communication with said low control capture molecule well; and

a high control oscillation well for receiving, holding and releasing fluid during oscillation, said high control oscillation well in fluid communication with said high control capture molecule well.

17. The assay cartridge of claim 2 or claim 16, further comprising turbulence inducing means positioned in at least one of the following locations, between said sample capture molecule well and said sample oscillation well, between said low control capture molecule well and said low control oscillation well, and between said high control capture molecule well and said high control oscillation well.

37. The assay cartridge of claim 16, further comprising:

a sample oscillation means for oscillating said fluid sample and said sample reagent between said sample reagent well and said sample oscillation well through said sample capture molecule well;

a low control oscillation means for oscillating said low control reagent between said low control reagent well and said low control oscillation well through said low control capture molecule well; and

a high control oscillation means for oscillating said high control reagent between said high control reagent well and said high control oscillation well through said high control capture molecule well.

18. A method for conducting homogeneous immunofluorescence assays comprising:

providing an assay cartridge comprising;

a housing comprising;

a sample apparatus having a port for receiving a fluid sample containing an unknown concentration of at least one analyte of interest;

said port in fluid communication with a first sample reagent well containing a predetermined sample reagent comprising tracer molecules having a fluorescent label thereon, said first sample reagent well in fluid communication with a sample capture molecule well, and a sample oscillation well for receiving, holding and releasing fluid during oscillation, said sample oscillation well in fluid communication with said sample capture molecule well; and

a low control apparatus having a first low control reagent well containing a predetermined low control reagent having a known low concentration, relative to said fluid sample, of at least one analyte of interest and tracer molecules having a fluorescent label thereon, said first low control reagent well in fluid communication with a low control capture molecule well, and a low control oscillation well for receiving, holding and releasing fluid during oscillation, said low control oscillation well in fluid communication with said low control capture molecule well;

waveguide means forming an adjacent wall of each of said sample capture molecule well and said low control capture molecule well wherein said waveguide means includes a receiving lens on an edge thereof, configured to receive a beam from a light source; and

at least one area containing capture molecules within each of said sample capture molecule well and said low control capture molecule well, wherein said capture molecule areas are attached to said waveguide means;

introducing a predetermined quantity of a sample fluid in said port;

transferring said sample fluid into said first sample reagent well to mix with said predetermined sample reagent;

transferring said sample fluid/sample reagent mixture into said sample capture molecule well to contact the sample fluid/sample reagent mixture with said sample capture molecule well capture molecule areas;

transferring said low control reagent mixture into said low control capture molecule well to contact said low control reagent mixture with said low control capture molecule well capture molecule areas;

transmitting a light beam into said receiving lens of said waveguide means, wherein said light beam travels a length of said waveguide means, and interacts with tracer molecules which have interacted with said capture molecules causing them to fluoresce; and

determining the concentration of said at least one analyte of interest in said sample fluid by a comparison of intensities of fluorescence between said capture molecule areas of said sample capture molecule well and said low control capture molecule well.

19. The method of claim 18 wherein the housing further comprises:

a high control apparatus having a first high control reagent well containing a predetermined high control reagent having a known high concentration, relative to said fluid sample, of at least one analyte of interest, and tracer molecules having a fluorescent label thereon, said first high control reagent well in fluid communication with a high control capture molecule well, and a high control oscillation well for receiving, holding and releasing fluid during oscillation, said high control oscillation well in fluid communication with said high control capture molecule well, said first high control capture molecule well having a wall comprising waveguide material, and having at least one area containing capture molecules within said first high control capture molecule well;

and the method further comprises;

transferring said high control reagent mixture into said high control capture molecule well to contact said high control reagent mixture with said high control capture molecule well capture molecule areas.

28. The method of claim 19 or claim 21, further comprising:

oscillating said fluid sample/second sample reagent/first sample reagent mixture between said first sample reagent well and said sample oscillation well through said sample capture molecule well prior to fluorescence detection;

oscillating said second low control reagent/first low control reagent mixture between said first low control reagent well and said low control oscillation well through said low control capture molecule well prior to fluorescence detection; and

oscillating said second high control reagent/first high control reagent mixture between said first high control reagent well and said high control oscillation well through said high control capture molecule well prior to fluorescence detection.

29. The method of claim 19, claim 21, claim 23, or claim 25, wherein each of said sample capture molecule well, said low control capture molecule well, and said high control capture molecule well include an opaque and rough wall surface opposing said waveguide.

30. The method of claim 18, claim 20, claim 22, or claim 24, wherein said waveguide further comprises an output lens configured to output a portion of said beam received from said receiving lens.

31. The method of claim 18, claim 20, claim 22, or claim 24, wherein said capture molecules are selected from the group consisting of antibodies, antibody fragments, whole antigenic molecules, antigenic molecule fragments, oligopeptides, nucleic acids, oliogonucleotides, membrane receptors, peptides, and mixtures thereof.

32. The method of claim 19 or claim 21, wherein said assay cartridge further comprises turbulence inducing means positioned in at least one of the following locations, between said first sample reagent well and said sample capture molecule well, between said first low control reagent well and said low control capture molecule well, and between said first high control reagent well and said high control capture molecule well.

33. The method of claim 19 or claim 21, wherein said assay cartridge further comprises turbulence inducing means positioned in at least one of the following locations, between said sample capture molecule well and said sample oscillation well, between said low control capture molecule well and said low control oscillation well, and between said high control capture molecule well and said high control oscillation well.

34. The method of claim 18 or claim 22, wherein the step of transferring said sample fluid into said first sample reagent well to mix with said sample reagent further includes shifting at least a portion of the sample fluid/sample reagent mixture between said first sample reagent well and said port to mix the sample fluid/sample reagent mixture.

20. A method for conducting homogeneous immunofluorescence assays comprising:

providing an assay cartridge comprising;

a housing comprising;

a sample apparatus having a port for receiving a fluid sample containing an unknown concentration of at least one analyte of interest;

said port in fluid communication with a sample waste well, said sample waste well in fluid communication with a second sample reagent well containing a predetermined second sample reagent, which second sample reagent interacts with said fluid sample, a first sample reagent and capture molecules, said second sample reagent in fluid communication with a first sample reagent well containing a predetermined first sample reagent comprising tracer molecules, said first sample reagent well in fluid communication with a sample capture molecule well, and a sample oscillation well for receiving, holding and releasing fluid during oscillating, said sample oscillation well in fluid communication with said sample capture molecule well; and

a low control apparatus having a second low control reagent well containing a predetermined second low control reagent having a known low concentration, relative to said fluid sample, of at least one analyte of interest, said second low control reagent interacting with capture molecules and a first low control reagent, said second low control reagent well in fluid communication with a first low control reagent well containing a predetermined first low control reagent comprising tracer molecules, said first low control reagent well in fluid communication with a low control capture molecule well, and a low control oscillation well in fluid communication with said low control capture molecule well, said low control oscillation well for receiving, holding and releasing fluid during oscillation;

a waveguide forming an adjacent wall of each of said sample capture molecule well and said low control capture molecule well wherein said waveguide includes a receiving lens on an edge of said waveguide configured to receive a beam from a light source; and

at least one area containing capture molecules within each of said sample capture molecule well and said low control capture molecule well, wherein each of said capture molecule areas is attached to said waveguide;

depositing a predetermined quantity of a sample fluid in said assay cartridge port;

transferring said sample fluid into said second sample reagent well to mix with said second sample reagent;

transferring said sample fluid/second sample reagent mixture into said first sample reagent well to mix with said first sample reagent;

transferring said sample fluid/second sample reagent/first sample reagent mixture into said sample capture molecule well to contact the sample fluid/second sample reagent/first sample reagent mixture with said sample capture molecule well capture molecule areas;

transferring said second low control reagent into said first low control reagent well to contact said second low control reagent with said first low control reagent;

transferring said second low control reagent/first low control reagent mixture into said low control capture molecule well to contact said second low control reagent/first low control reagent mixture with said low control capture molecule well capture molecule areas;

transmitting a light beam into said receiving lens of said waveguide means, wherein said light beam travels a length of said waveguide, and interacts with tracer molecules which have been captured by said capture molecules, causing them to fluoresce; and

determining the concentration of said analyte of interest in said sample fluid by comparing intensities of fluorescence between said capture molecule areas of said sample capture molecule well and said low control capture molecule well.

21. The method of claim 20 wherein the housing of the assay cartridge further comprises:

a high control apparatus having a second high control reagent well containing a predetermined second high control reagent having a known high concentration, relative to said sample fluid, of at least one analyte of interest, said second high control reagent interacting with a first high control reagent and capture molecules, said second high control reagent well in fluid communication with a first high control reagent well containing a predetermined first high control reagent comprising tracer molecules, said first high control reagent well in fluid communication with a high control capture molecule well, and a high control oscillation well in fluid communication with said high control capture molecule well, said high control oscillation well for receiving, holding and releasing fluids during oscillation, said first high control capture molecule well having a wall comprising waveguide material, and having at least one area containing capture molecules within said first high control capture molecule well;

and the method further comprises;

transferring said second high control reagent into said first high control reagent well to contact said second high control reagent with said first high control reagent; and

transferring said second high control reagent/first high control reagent mixture into said high control capture molecule well to contact said second high control reagent first high control reagent mixture with said high control capture molecule well capture molecule areas; and

the concentration of the analyte of interest in said sample fluid is compared with the intensity of fluorescence in the high control capture molecule well.

26. The method of claim 21 or claim 25, further comprising:

transferring said sample fluid/second sample reagent/first sample reagent mixture between the first sample reagent well and the second sample reagent well to mix the sample solution/first reagent/second reagent mixture;

transferring said second low control reagent/first low control reagent mixture between the first low control reagent well and the second low control reagent well to mix the second low control reagent/first low control reagent mixture; and

transferring said second high control reagent/first high control reagent mixture between the first high control reagent well and the second high control reagent well to mix the second high control reagent/first high control reagent mixture.

22. A method for conducting homogeneous immunofluorescence assays comprising:

providing an assay cartridge comprising;

a housing comprising;

a sample apparatus having a port for receiving a fluid sample containing an unknown concentration of at least one analyte of interest;

said port in fluid communication with a first sample reagent well containing a predetermined sample reagent comprising tracer molecules having a fluorescent label thereon, said first sample reagent well in fluid communication with a sample capture molecule well, and a turbulence inducing means positioned between said first sample reagent well and said sample capture molecule well; and

a low control apparatus having a first low control reagent well containing a predetermined low control reagent having a known low concentration, relative to the fluid sample, of at least one analyte of interest, said low control reagent comprising tracer molecules having a fluorescent label thereon, said first low control reagent well in fluid communication with a low control capture molecule well, and a turbulence inducing means positioned between said first low control reagent well and said low control capture molecule well;

waveguide means forming an adjacent wall of each of said sample capture molecule well and said low control capture molecule well wherein said waveguide means includes a receiving lens on an edge thereof, configured to receive a beam from a light source; and

at least one area containing capture molecules within each of said sample capture molecule well and said low control capture molecule well, wherein said capture molecule areas are attached to said waveguide means;

introducing a predetermined quantity of a sample fluid in said port;

transferring said sample fluid into said first sample reagent well to mix with said sample reagent;

transferring said sample fluid/sample reagent mixture into said sample capture molecule well to contact the sample fluid/sample reagent mixture with said sample capture molecule well capture molecule areas;

transferring said low control reagent mixture into said low control capture molecule well to contact said low control reagent mixture with said low control capture molecule well capture molecule areas;

transmitting a light beam into said receiving lens of said waveguide means, wherein said light beam travels a length of said waveguide, and interacts with tracer molecules which have interacted with said capture molecules causing them to fluoresce; and

determining the concentration of said analyte of interest in said sample fluid by a comparison of intensities of fluorescence between said capture molecule areas of said sample capture molecule well and said low control capture molecule well.

23. The method of claim 22 wherein the housing further comprises:

a high control apparatus having a first high control reagent well containing a predetermined high control reagent having a known high concentration, relative to said fluid sample, of at least one analyte of interest, said high control reagent comprising tracer molecules having a fluorescent label thereon, said first high control reagent well in fluid communication with a high control capture molecule well, and a turbulence inducing means positioned between said first high control reagent well and said high control capture molecule well, said first high control capture molecule well having a wall comprising waveguide material, and having at least one area having capture molecules within said first high control capture molecule well;

and the method further comprises;

transferring said high control reagent mixture into said high control capture molecule well to contact said high control reagent mixture with said high control capture molecule well capture molecule areas.

27. The method of claim 23 or claim 25, wherein said assay cartridge used in said method further comprises:

a sample oscillation well for receiving, holding and releasing fluid during oscillation, said sample oscillation well in fluid communication with said sample capture molecule well;

a low control oscillation well for receiving, holding and releasing fluid during oscillation, said low control oscillation well in fluid communication with said low control capture molecule well; and

a high control oscillation well for receiving, holding and releasing fluid during oscillation, said high control oscillation well in fluid communication with said high control capture molecule well.

24. A method for conducting homogeneous immunofluorescence assays comprising:

providing an assay cartridge comprising;

a housing comprising;

a sample apparatus having a port for receiving a fluid sample containing an unknown concentration of at least one analyte of interest;

said port in fluid communication with a sample waste well, said sample waste well in fluid communication with a second sample reagent well containing a predetermined second sample reagent, which second sample reagent reacts with said fluid sample, a first sample reagent and capture molecules, said second sample reagent in fluid communication with a first sample reagent well containing a predetermined first sample reagent comprising tracer molecules, said first sample reagent well in fluid communication with a sample capture molecule well, and a turbulence inducing means positioned between said first sample reagent well and said sample capture molecule well; and

a low control apparatus having a second low control reagent well containing a predetermined second low control reagent having a known low concentration, relative to said fluid sample, of at least one analyte of interest, said second low control reagent reacts with a first low control reagent and capture molecules, said second low control reagent well in fluid communication with a first low control reagent well containing a predetermined first low control reagent comprising tracer molecules, said first low control reagent well in fluid communication with a low control capture molecule well, and a turbulence inducing means positioned between said first low control reagent well and said low control capture molecule well;

a waveguide forming an adjacent wall of each of said sample capture molecule well and said low control capture molecule well wherein said waveguide includes a receiving lens on an edge of said waveguide configured to receive a beam from a light source; and

at least one area containing capture molecules within each of said sample capture molecule well and said low control capture molecule well, wherein each of said capture molecule areas is attached to said waveguide;

depositing a predetermined quantity of a sample fluid in said port;

transferring said sample fluid into said second sample reagent well to mix with said second sample reagent;

transferring said sample fluid/second sample reagent mixture into said first sample reagent well to mix with said first sample reagent;

transferring said sample fluid/second sample reagent/first sample reagent mixture into said sample capture molecule well to contact the sample fluid/second sample reagent/first sample reagent mixture with said sample capture molecule well capture molecule areas;

transferring said second low control reagent into said first low control reagent well to contact said second low control reagent with said first low control reagent;

transferring said second low control reagent/first low control reagent mixture into said low control capture molecule well to contact said second low control reagent/first low control reagent mixture with said low control capture molecule well capture molecule areas;

transmitting a light beam into said receiving lens of said waveguide, wherein said light beam travels a length of said waveguide, and interacts with tracer molecules which have been captured by said capture molecules, causing them to fluoresce; and

determining the concentration of said analyte of interest in said sample fluid by comparing intensities of fluorescence between said capture molecule areas of said sample capture molecule well and said low control capture molecule well.

25. The method of claim 24 wherein the housing of the assay cartridge further comprises:

a high control apparatus having a second high control reagent well containing a predetermined second high control reagent having a known high concentration, relative to the fluid sample, of at least one analyte of interest, said second high control reagent interacting with a first high control reagent and capture molecules, said second high control reagent well in fluid communication with a first high control reagent well containing a predetermined first high control reagent comprising tracer molecules, said first high control reagent well in fluid communication with a high control capture molecule well, and a turbulence inducing means positioned between said first high control reagent well and said high control capture molecule well, said first high control capture molecule well having a wall comprising waveguide material, and having at least one area containing capture molecules within said first high control capture molecule well;

and the method further comprises;

transferring said second high control reagent into said first high control reagent well to contact said second high control reagent with said first high control reagent; and

transferring said second high control reagent/first high control reagent mixture into said high control capture molecule well to contact said second high control reagent/first high control reagent mixture with said high control capture molecule well capture molecule areas; and

the concentration of the analyte of interest in said sample fluid is compared with the intensity of fluorescence in the high control capture molecule well.

35. A method for conducting homogeneous immunofluorescence assays comprising providing an assay cartridge including a housing comprising an opening for receiving a fluid sample and at least one area containing capture molecules attached to a waveguide material, and introducing said fluid sample containing an unknown concentration of at least one analyte of interest such that said fluid sample contacts said capture molecule, an improvement comprising:

oscillating said fluid sample over said capture molecules.

36. A method for conducting homogeneous immunofluorescence assays comprising providing an assay cartridge including a housing comprising an opening for receiving a fluid sample and at least one area containing capture molecules attached to a waveguide material, and introducing a fluid sample containing an unknown concentration of at least one analyte of interest such that said fluid sample contacts said capture molecule, an improvement comprising:

inducing turbulent flow of said fluid sample over said capture molecules.