Membrane-based assay devices that utilize time-resolved fluorescence

• US 20040043502A1
• Filed: 11/01/2002
• Published: 03/04/2004
• Est. Priority Date: 08/27/2002
• Status: Active Grant

• Current Assignee: Kimberly-Clark Worldwide Incorporated (Kimberly-Clark Corporation)
• Original Assignee: Kimberly-Clark Worldwide Incorporated (Kimberly-Clark Corporation)
• Inventors: Wei, Ning, Song, Xuedong, Kaylor, Rosann, Knotts, Michael
• Granted Patent: US 7,632,653 B2
• Time in Patent Office: Days
• US Class Current: 436/172
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/274 Calibration, base line adjustment, drift correction
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 33/525 Multi-layer analytical elements
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/824 Immunological separation techniques
• CPC Class Codes: Y10T 436/13 Tracers or tags

Abstract

A membrane-based assay device for detecting the presence or quantity of an analyte residing in a test sample is provided. The device utilizes time-resolved fluorescence to detect the signals generated by excited fluorescent labels. Because the labels can have relatively long emission lifetime, short-lived background interference can be practically eliminated through delayed fluorescence detection. In addition, the resulting fluorescent reader can have a simple and inexpensive design. For instance, in one embodiment, the reader can utilize a silicon photodiode and a pulsed light-emitting diode (LED) to accurately excite labels and detect fluorescence on a membrane-based assay device without requiring the use of expensive components, such as monochromators or narrow emission band width optical filters.

Claims

1. A method for detecting the presence or quantity of an analyte residing in a test sample, said method comprising:

i) providing a flow-through assay device that comprises a porous membrane in fluid communication with a fluorescent label, said fluorescent label having a fluorescence emission lifetime of greater than about 1 microsecond, said porous membrane defining a detection zone;

ii) contacting said fluorescent label with the test sample to form a mixture;

iii) allowing said mixture to flow to said detection zone;

iv) placing a time-resolved fluorescence reader proximate to said detection zone, said fluorescence reader comprising a pulsed excitation source and a time-gated detector;

v) exciting said fluorescent label at said detection zone with said pulsed excitation source, wherein said excitation causes said fluorescent label to emit a detection signal; and

vi) measuring the intensity of the detection signal with said time-gated detector.

2. A method as defined in claim 1, wherein said fluorescent label has an emission lifetime of greater than about 10 microseconds.

3. A method as defined in claim 1, wherein said fluorescent label has an emission lifetime of from about 100 to about 1000 microseconds.

4. A method as defined in claim 1, wherein said fluorescent label has a Stokes shift greater than about 50 nanometers

5. A method as defined in claim 1, wherein said fluorescent label has a Stokes shift of greater than about 100 nanometers.

6. A method as defined in claim 1, wherein said fluorescent label has a Stokes shift of from about 250 to about 350 nanometers.

7. A method as defined in claim 1, wherein said fluorescent label includes a lanthanide chelate of samarium, dysprosium, europium, terbium, or combinations thereof.

8. A method as defined in claim 1, wherein said fluorescent label is europium chelate.

9. A method as defined in claim 1, wherein said fluorescent label is used in conjunction with a microparticle, a nanoparticle, a liposome, a dendrimer, a polymer, or combinations thereof.

10. A method as defined in claim 9, wherein said fluorescent label is used in conjunction with a microparticle or nanoparticle modified with a specific binding member for the analyte.

11. A method as defined in claim 1, wherein said detection zone includes multiple detection regions.

12. A method as defined in claim 11, wherein said detection regions contains multiple capture reagents for binding to multiple analytes.

13. A method as defined in claim 1, wherein said porous membrane further defines a calibration zone, wherein said mixture is also allowed to flow to said calibration zone.

14. A method as defined in claim 13, wherein said calibration zone includes multiple detection regions.

15. A method as defined in claim 14, wherein said calibration regions contains multiple capture reagents for binding to multiple fluorescent labels.

16. A method as defined in claim 13, further comprising:

placing said time-resolved fluorescence reader adjacent to said calibration zone;

exciting said fluorescent label at said calibration zone with said pulsed excitation source, wherein said excitation causes said fluorescent label to emit a calibration signal;

measuring the intensity of the calibration signal with said time-gated detector; and

comparing the intensity of the detection signal to the calibration signal, wherein the amount of the analyte within the test sample is proportional to the intensity of the detection signal calibrated by the intensity of the calibration signal.

17. A method as defined in claim 16, wherein said fluorescent label at said detection zone is excited simultaneously with said fluorescent label at said calibration zone.

18. A method as defined in claim 16, wherein said detection signal and said calibration signal are measured simultaneously.

19. A method as defined in claim 16, wherein said pulsed excitation source is a light-emitting diode.

20. A method as defined in claim 16, wherein said time-gated detector is a silicon photodiode.

21. A method as defined in claim 16, wherein said fluorescence reader contains timing circuitry in communication with said pulsed excitation source and said time-gated detector, said timing circuitry controlling pulsed excitation and detection.

22. A method as defined in claim 16, wherein an optical filter is positioned adjacent to said pulsed excitation source, said time-gated detector, or combinations thereof.

23. A method for detecting the presence or quantity of an analyte residing in a test sample, said method comprising:

i) providing a flow-through assay device that comprises a porous membrane in fluid communication with a conjugated probe that contains a fluorescent label, said fluorescent label having a fluorescence emission lifetime of greater than about 10 microseconds, said porous membrane defining a detection zone and a calibration zone; and

ii) contacting said conjugated probe with the test sample to form a mixture;

iii) allowing said mixture to flow to said detection zone and said calibration zone;

iv) placing a time-resolved fluorescence reader proximate to said detection zone and said calibration zone, said fluorescence reader comprising a pulsed excitation source and a time-gated detector;

v) exciting said fluorescent label at said detection zone and said calibration zone with said pulsed excitation source, wherein said excitation causes said fluorescent label to emit a detection signal at said detection zone and a calibration signal at said calibration zone;

vi) measuring the intensity of the detection signal and said calibration signal with said time-gated detector; and

vii) comparing the intensity of the detection signal to the calibration signal, wherein the amount of the analyte within the test sample is proportional to the intensity of the detection signal calibrated by the intensity of the calibration signal.

24. A method as defined in claim 23, wherein said fluorescent label has an emission lifetime of from about 100 to about 1000 microseconds.

25. A method as defined in claim 23, wherein said fluorescent label has a Stokes shift of greater than about 50 nanometers

26. A method as defined in claim 23, wherein said fluorescent label has a Stokes shift of from about 250 to about 350 nanometers.

27. A method as defined in claim 23, wherein said fluorescent label includes a lanthanide chelate of samarium, dysprosium, europium, terbium, or combinations thereof.

28. A method as defined in claim 27, wherein said fluorescent label is europium chelate.

29. A method as defined in claim 23, wherein said fluorescent label at said detection zone is excited simultaneously with said fluorescent label at said calibration zone.

30. A method as defined in claim 23, wherein said detection signal and said calibration signal are measured simultaneously.

31. A method as defined in claim 23, wherein said pulsed excitation source is a pulsed light-emitting diode.

32. A method as defined in claim 23, wherein said time-gated detector is a silicon photodiode.

33. A method as defined in claim 23, wherein said fluorescence reader contains timing circuitry in communication with said pulsed excitation source and said time-gated detector, said timing circuitry controlling pulsed excitation and detection.

34. A method for detecting the presence or quantity of an analyte residing in a test sample, said method comprising:

i) providing a flow-through assay device that comprises a porous membrane in fluid communication with a conjugated probe that contains a lanthanide chelate, said lanthanide chelate having a fluorescence emission lifetime of greater than about 50 microseconds and a Stokes shift greater than about 100 nanometers, said porous membrane defining a detection zone and a calibration zone; and

ii) contacting said conjugated probe with the test sample to form a mixture;

iii) allowing said mixture to flow to said detection zone and said calibration zone;

iv) placing a time-resolved fluorescence reader proximate to said detection zone and said calibration zone, said fluorescence reader comprising a pulsed light-emitting diode and a time-gated detector that comprises a silicon photodiode;

v) exciting said lanthanide chelate at said detection zone and said calibration zone with said pulsed light-emitting diode, wherein said excitation causes said lanthanide chelate at said detection zone to emit a detection signal and said lanthanide chelate at said calibration zone to emit a calibration signal;

vi) measuring the intensity of the detection signal and the calibration signal with said time-gated detector;

vii) comparing the intensity of the detection signal to the calibration signal, wherein the amount of the analyte within the test sample is proportional to the intensity of the detection signal calibrated by the intensity of the calibration signal.

35. A method as defined in claim 34, wherein said lanthanide chelate is selected from the group consisting of lanthanide chelates of samarium, dysprosium, europium, terbium, or combinations thereof.

36. A method as defined in claim 34, wherein said lanthanide chelate is europium chelate.

37. A method as defined in claim 34, wherein said pulsed light-emitting diode is an ultraviolet light-emitting diode.

38. A method as defined in claim 34, wherein said fluorescence reader contains timing circuitry in communication with said pulsed light-emitting diode and said time-gated detector, said timing circuitry controlling pulsed excitation and detection.

39. A method as defined in claim 34, wherein said fluorescent label at said detection zone is excited simultaneously with said fluorescent label at said calibration zone.

40. A method as defined in claim 34, wherein said detection signal and said calibration signal are measured simultaneously.

41. A method as defined in claim 34, wherein said fluorescent label has an emission lifetime of from about 100 to about 1000 microseconds.

42. A method as defined in claim 34, wherein said fluorescent label has a Stokes shift of from about 250 to about 350 nanometers.