Up-converting reporters for biological and other assays using laser excitation techniques

• US 5,674,698 A
• Filed: 03/30/1995
• Issued: 10/07/1997
• Est. Priority Date: 09/14/1992
• Status: Expired due to Term

• Current Assignee: Sarnoff Corporation (SRI International, Inc.)
• Original Assignee: SRI International, Inc.
• Inventors: Zarling, David A., Rossi, Michel J., Peppers, Norman A., Kane, James, Faris, Gregory W., Dyer, Mark J., Ng, Steve Y., Schneider, Luke V.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): PORTNER, VIRGINIA ALLEN
• Application Number:
  US08/416,023
• Time in Patent Office: 922 Days
• Field of Search: 422/52, 422/57, 422/82.05, 422/82.08, 422/82.09, 435/5, 435/6, 435/7.1, 435/7.5, 435/7.92-7.95, 435/808, 435/968, 435/969, 435/971, 435/973, 436/518, 436/519, 436/523, 436/526, 436/531, 436/532, 436/538, 436/540, 436/546, 436/169, 436/172, 436/800, 436/805-806, 436/824, 252/301
• US Class Current: 435/7.92
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/00 Nucleic acid detection characterized by the use of physical, structural and functional properties
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/588 with semiconductor nanocrystal label, e.g. quantum dots

Abstract

The invention provides methods, compositions, and apparatus for performing sensitive detection of analytes, such as biological macromolecules and other analytes, by labeling a probe molecule with an up-converting label. The up-converting label absorbs radiation from an illumination source and emits radiation at one or more higher frequencies, providing enhanced signal-to-noise ratio and the essential elimination of background sample autofluorescence. The methods, compositions, and apparatus are suitable for the sensitive detection of multiple analytes and for various clinical and environmental sampling techniques.

Claims

1. A method for detecting an analyte in a sample, comprising the steps of:

contacting a sample containing a target analyte with a labeled binding component to specifically bind the target analyte and form a labeled binding component-target complex, wherein the labeled binding component comprises a binding component attached to an up-converting inorganic phosphor particle comprising at least one rare earth element and a phosphor host material and being capable of converting excitation radiation to emission radiation of a shorter wavelength;

separating any unbound labeled binding component from the labeled binding component-target complex;

illuminating the labeled binding component-target complex with excitation radiation; and

detecting emission radiation of at least one label emission wavelength, wherein the emission radiation has a shorter wavelength than the excitation radiation.

2. A method according to claim 1, wherein said up-converting inorganic phosphor comprises ytterbium and erbium in a phosphor host material.

3. A method according to claim 2, wherein the up-converting inorganic phosphor comprises sodium yttrium fluoride ytterbium erbium or yttrium ytterbium erbium oxysulfide.

4. A method of claim 1, further comprising, before the contacting step, the step of attaching the up-converting inorganic phosphor particle to the binding component to form the labeled binding component.

5. A method according to claim 4, wherein the binding component is attached to the label by covalent or noncovalent binding.

6. A method according to claim 5, wherein the binding component is streptavidin or avidin and the target analyte is a biotinylated target analyte.

7. A method of claim 1, wherein the labeled binding component-target complex is separated from the unbound labeled probe by immobilization on a solid support.

8. A method according to claim 7, wherein the step of separating unbound labeled binding component from the labeled binding component-target complex in the sample is performed by washing the sample with an aqueous solution to remove suspendible or soluble unbound labeled binding component.

9. A method of claim 7, wherein the labeled binding component target complex is bound to a first binding component on the solid support to form a sandwich complex.

10. A method according to claim 1, wherein the target analyte is selected from the group consisting of:

polynucleotides, polypeptides, viruses, microorganisms, haptens, mammalian cells, steroid hormones, glycoproteins, lipoproteins, biotinylated magnetic beads, prescribed or over-the-counter drugs, illegal substances, intoxicants and drugs of abuse.

11. A method according to claim 1, wherein the step of illuminating with a label excitation wavelength is performed with an infrared laser diode or light-emitting diode.

12. A method according to claim 1, wherein the infrared laser diode or light-emitting diode emits pulsed illumination.

13. A method according to claim 12, wherein the infrared laser diode or light-emitting diode is pulsed through direct current modulation.

14. A method according to claim 12, wherein the step of detecting light emission of at least one label emission wavelength is performed by time-gated or lock-in detection.

15. A method according to claim 11, wherein the step of detecting light emission is performed with phase-sensitive detection.

16. A method according to claim 11, wherein the laser diode or light-emitting diode has peak emissions in the range of 960-980 nm and at approximately 1500 nm.

17. A method according to claim 1, wherein said step of detecting light emission is performed with a photomultiplier, photodiode, a charge coupled device, a charge injection device, or photographic film emulsion.

18. A method According to claim 1, wherein the target analyte is immobilized in a histological tissue section or a solid support.

19. A method according to claim 1, wherein the binding component is selected from the group consisting of:

antibodies, polynucleotides, polypeptide hormones, streptavidin, Staphylococcus aureus Protein A, lectins, and antigens.

20. A method for detecting an analyte in a sample, comprising the steps of:

contacting a sample containing a target analyte with a binding component to specifically bind the target analyte and form a binding component-target complex;

contacting the binding component-target complex with a label to form a labeled binding component-target complex, the label comprising an up-converting inorganic phosphor particle comprising at least one rare earth element and a phosphor host material and being capable of converting excitation radiation to emission radiation of a shorter wavelength;

separating any unbound label from the labeled binding component-target complex;

illuminating the labeled binding component-target complex with excitation radiation; and

detecting emission radiation of at least one label emission wavelength, wherein the emission radiation has a shorter wavelength than the excitation radiation.

21. A method according to claim 20, wherein the binding component is a primary antibody and the up-converting inorganic phosphor is bound to the binding component through a secondary antibody.

22. A method according to claim 21, wherein said secondary antibody is biotinylated and said up-converting inorganic phosphor is bound to streptavidin.

23. A method according to claim 20, wherein the target analyte is a polynucleotide and the binding component is a biotinylated polynucleotide which hybridizes to the target polynucleotide under binding conditions.

24. A method according to claim 23, wherein the up-converting inorganic phosphor comprises an up-converting phosphor particle and streptavidin.

25. A method of claim 20 further comprising, before the contacting step, the step of attaching the up-converting inorganic phosphor particle to the binding component to form the labeled binding component.

26. A method for detecting a biotinylated analyte in a sample, comprising the steps of:

contacting a sample containing a biotinylated analyte with a labelled binding component to specifically bind the biotinylated analyte and form a labeled binding component-target complex, wherein the labeled binding component comprises a streptavidin-coated up-converting inorganic phosphor particle, the up-converting inorganic phosphor particle comprising at least one rare earth element and a phosphor host material and being capable of converting excitation radiation to emission radiation of a shorter wavelength;

separating any unbound labeled binding component from the labeled binding component-target complex;

illuminating the labeled binding component-target complex with excitation radiation; and

detecting emission radiation of at least one label emission wavelength, wherein the emission radiation has a shorter wavelength than the excitation radiation.

27. A method according to claim 26, wherein the target is a biotinylated magnetic bead.

28. A method of claim 26 further comprising, before the contacting step, the step of attaching the up-converting inorganic phosphor particle to the binding component to form the labeled binding component.

29. A method for detecting an analyte in a sample, comprising the steps of:

contacting a sample containing a target analyte with a labeled binding component to specifically bind the target analyte and form a labeled binding component-target complex, wherein the labeled binding component comprises a binding component attached to an up-converting inorganic phosphor particle comprising at least one rare earth element and a phosphor host material and being capable of converting excitation radiation to emission radiation of a shorter wavelength;

differentiating the labeled binding component-target complex from any unbound labeled binding component in the sample;

illuminating the labeled binding component-target complex with excitation radiation; and

detecting emission radiation of at least one label emission wavelength from the labeled binding component-target complex, wherein the emission radiation has a shorter wavelength than the excitation radiation.

30. The method of claim 29, wherein the labeled binding component-target complex is illuminated with a confocal beam having a focal point at the contact surface and being divergent at points other than the contact surface.

31. A method of claim 29, further comprising, before the contacting step, the step of attaching the up-converting inorganic phosphor particle to the binding component to form the labeled binding component.

32. A method of claim 29, wherein the differentiating step comprises contacting the labeled binding component-target complex with a contact surface wherein the labeled binding component-target complex is localized at the contact surface as compared to the unbound labeled binding component;

and the illuminating step comprises illuminating the labeled binding component-target complex at the contact surface with excitation radiation.

33. The method of claim 32, wherein binding component-target complexes are localized to the contact surface by a method selected from the group consisting of:

magnetic localization of magnetic beads to said contact surface, wherein said binding component-target complexes are localized on the magnetic beads relative to unbound labeled binding component;

gravitational sedimentation of binding component-target complexes from unbound labelled binding component, wherein said sedimented binding component-target complexes are localized on the contact surface relative to unbound labeled binding component;

filtration over a contact surface wherein said binding component-target complexes are localized on the contact surface relative to unbound labeled binding component;

antibody capture;

affinity adsorption; and

nucleic acid hybridization.

34. A method of claim 29, wherein the differentiating and illuminating steps are accomplishied by confocal excitation.

35. A method of claim 29 wherein the differentiating, illuminating, and detecting steps are accomplished by confocal excitation and confocal detection.

36. A method of claim 29, wherein the differentiating step is accomplished by size discrimination.