Tagged microparticle compositions and methods

• US 20030134333A1
• Filed: 11/08/2002
• Published: 07/17/2003
• Est. Priority Date: 04/28/2000
• Status: Active Grant

• Current Assignee: Virologic, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Virologic, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat, Kirakossian, Hrair, Dehlinger, Peter
• Granted Patent: US 7,160,735 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: Y10S 436/823 Immunogenic carrier or carrier per se
• CPC Class Codes: Y10S 436/824 Immunological separation techniques

Abstract

Compositions and methods are disclosed for detecting multiple target analytes in a sample using microparticles having molecular tags attached by cleavable linkages. Generally, an assay mixture is formed comprising a sample and a reagent comprising multiple such microparticles under conditions that permit stable complexes to form between binding moieties on the surfaces of the microparticles and the analytes. In one aspect of the invention, the a second binding composition is added so that complexes form among the microparticle-bound binding moieties, the analytes, and second binding moieties of the second binding composition. Such second binding moieties have cleavage-inducing moieties attached that upon activation cause the cleavage of the cleavable linkages and the release of molecular tags. Released molecular tags are separated and the presence and/or amount of the target analytes are determined based on the analysis of the released and separated molecular tags.

Claims

1. A composition comprising:

a mixture of more than one microparticle, each microparticle in the mixture having molecular tags attached by cleavable linkages such that different molecular tags are attached to different microparticles, the molecular tags being selected from a plurality of molecular tags such that each molecular tag of the plurality has one or more physical and/or optical characteristics distinct from those of the other molecular tags of the plurality so that each molecular tag forms a distinguishable peak upon cleavage and separation based on such one or more physical and/or optical characteristics.

2. The composition of claim 1 wherein said separation is electrophoretic separation or chromatographic separation, and wherein said molecular tag has a molecular weight in the range of from 100 to 2500 daltons.

3. The composition of claim 2 wherein each of said molecular tags attached to said microparticles are selected from a group defined by the formula:

-L-(M,D)

wherein;

L is a cleavable linkage;

D is a detection moiety; and

M is a bond or a water soluble organic compound consisting of from 1 to 100 atoms, not including hydrogen, that are selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

4. The composition of claim 3 wherein D is a fluorescent label, a chromogenic label, or an electrochemical label.

5. The composition of claim 4 wherein M is a polymer selected from any one of polyethers, polyesters, polypeptides, oligosaccharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof.

6. The composition of claim 5 wherein D is a fluorescein.

7. The composition of claim 6 wherein said fluorescein is selected from the group consisting of 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxyfluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, and 2′

,4′

,5′

,7′

-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein.

8. The composition of claim 3 wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles.

9. The composition in accordance with claims 1, 2, 3, 4, 5, 6, 7, or 8 wherein said plurality of molecular tags is in the range of from 2 to 100, and wherein said separation is electrophoretic separation.

10. The composition of claim 9 wherein said plurality of molecular tags is in the range of from 3 to 50.

11. The composition of claim 10 wherein said microparticles each have attached a binding moiety such that different pairs of said molecular tags and binding moieties are attached to different microparticles.

12. The composition of claim 11 wherein said binding moiety is an antibody binding composition.

13. The composition of claim 11 wherein said binding moiety is an oligonucleotide.

14. A composition comprising:

a mixture of more than one microparticle, each microparticle having a surface with binding moieties and molecular tags attached, each binding moiety being specific for an analyte and the molecular tags being attached by cleavable linkages, such that different pairs of molecular tags and binding moieties are attached to different microparticles, the molecular tags being selected from a plurality of molecular tags such that each molecular tag of the plurality has one or more physical and/or optical characteristics distinct from those of the other molecular tags of the plurality so that each molecular tag forms a distinguishable peak upon cleavage and separation based on such one or more physical and/or optical characteristics; and

a second binding composition comprising at least one binding moiety specific for each of the analytes, each such binding moiety having a sensitizer for generating an active species capable of cleaving the cleavable linkage.

15. The composition of claim 14 wherein said separation is electrophoretic separation or chromatographic separation, and wherein said molecular tag has a molecular weight in the range of from 100 to 2500 daltons.

16. The composition of claim 15 wherein said sensitizer is a photosensitizer capable, in its excited state, of activating oxygen to its singlet state, and wherein said first and second binding moieties are both antibody binding compositions.

17. The composition of claim 15 wherein sensitizer is a photosensitizer capable, in its excited state, of activating oxygen to its singlet state, and wherein said first binding moiety is a ligand for a cell surface membrane receptor and said second binding moiety is a receptor in a cell surface membrane of a biological cell, the photosensitizer being anchored in the cell surface membrane of the biological cell.

18. The composition in accordance with claims 15, 16, or 17 wherein each of said molecular tags attached to said microparticles are selected from a group defined by the formula:

-L-(M,D)

wherein;

L is a cleavable linkage;

D is a detection moiety; and

M is a bond or a water soluble organic compound consisting of from 1 to 100 atoms, not including hydrogen, that are selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

19. The composition of claim 18 wherein D is a fluorescent label, a chromogenic label, or an electrochemical label.

20. The composition of claim 19 wherein M is a polymer selected from any one of polyethers, polyesters, polypeptides, oligosaccharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof.

21. The composition of claim 20 wherein D is a fluorescein.

22. The composition in accordance with claims 18, 19, 20, or 21 wherein (i) L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles, (ii) said plurality of molecular tags is in the range of from 2 to 100, and (iii) said separation is electrophoretic separation.

23. The composition of claim 22 wherein said plurality of molecular tags is in the range of from 3 to 50.

24. A composition comprising:

one or more target analytes;

a mixture of more than one microparticle, each microparticle having a surface with first binding moieties and molecular tags attached, at least one first binding moiety being specifically bound to a target analyte and the molecular tags being attached by cleavable linkages to the surface, such that different pairs of molecular tags and first binding moieties are attached to different microparticles and the molecular tags are selected from a plurality of molecular tags such that each molecular tag of the plurality has one or more physical and/or optical characteristics distinct from those of the other molecular tags of the plurality so that each molecular tag forms a distinguishable peak upon cleavage and separation based on such one or more physical and/or optical characteristics; and

a second binding composition comprising at least one second binding moiety specific for each target analyte, each second binding moiety having a sensitizer for generating an active species within an effective proximity and at least one second binding moiety specifically bound to a target analyte so that cleavable linkage of a molecular tag of a first binding moiety specifically bound to the same target analyte is within the effective proximity of the sensitizer of the second binding moiety.

25. The composition of claim 24 wherein said separation is electrophoretic separation or chromatographic separation, and wherein said molecular tag has a molecular weight in the range of from 100 to 2500 daltons.

26. The composition of claim 25 wherein said sensitizer is a photosensitizer capable, in its excited state, of activating oxygen to its singlet state, wherein said target analytes are each proteins, and wherein said first and second binding moieties are both antibody binding compositions.

27. The composition of claim 25 wherein said sensitizer is a photosensitizer capable, in its excited state, of activating oxygen to its singlet state, wherein said target analytes are each polynucleotides, and wherein said first and second binding moieties are both oligonucleotides.

28. The composition in accordance with claims 24, 25, 26, or 27 wherein each of said molecular tags attached to said microparticles are selected from a group defined by the formula:

-L-(M,D)

wherein;

L is a cleavable linkage;

D is a detection moiety; and

M is a bond or a water soluble organic compound consisting of from 1 to 100 atoms, not including hydrogen, that are selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

29. The composition of claim 28 wherein D is a fluorescent label, a chromogenic label, or an electrochemical label.

30. The composition of claim 29 wherein M is a polymer selected from any one of polyethers, polyesters, polypeptides, oligosaccharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof.

31. The composition of claim 30 wherein D is a fluorescein.

32. The composition in accordance with claims 28, 29, 30, or 31 wherein (i) L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles, (ii) said plurality of molecular tags is in the range of from 2 to 100, and (iii) said separation is electrophoretic separation.

33. The composition of claim 32 wherein said plurality of molecular tags is in the range of from 3 to 50.

34. A method for detecting more than one target analytes in a sample, the method comprising the steps of:

providing a mixture of at least one microparticle for each target analyte, each microparticle having a surface with first binding moieties and molecular tags attached, each first binding moiety being specific for a target analyte and the molecular tags being attached by cleavable linkages, such that different pairs of molecular tags and first binding moieties are attached to different microparticles, the molecular tags being selected from a plurality of molecular tags such that each molecular tag of the plurality has one or more physical and/or optical characteristics distinct from those of the other molecular tags of the plurality so that each molecular tag forms a distinguishable peak upon cleavage and separation based on such one or more physical and/or optical characteristics;

providing a second binding composition comprising at least one second binding moiety specific for each of the target analytes, each such second binding moiety having a sensitizer for generating an active species capable of cleaving the cleavable linkage;

combining with the sample the mixture of microparticles and the second binding composition such that in the presence of a target analyte a complex is formed between the target analyte and at least one first binding moiety and at least one second binding moiety specific therefor, and such that the sensitizer of the second binding moiety causes the generation of an active species and the cleavage of one or more cleavable linkages to release one or more molecular tags from at least one microparticle; and

separating and identifying the released molecular tags by the one or more physical and/or optical characteristics to determine the target analytes in the sample.

35. The composition of claim 34 wherein said separation is electrophoretic separation or chromatographic separation, and wherein said molecular tag has a molecular weight in the range of from 100 to 2500 daltons.

36. The composition of claim 35 wherein said sensitizer is a photosensitizer capable, in its excited state, of activating oxygen to its singlet state, and wherein said first and second binding moieties are both antibody binding compositions.

37. The composition in accordance with claims 34, 35, or 36 wherein each of said molecular tags attached to said microparticles are selected from a group defined by the formula:

-L-(M,D)

wherein;

L is a cleavable linkage;

D is a detection moiety; and

M is a bond or a water soluble organic compound consisting of from 1 to 100 atoms, not including hydrogen, that are selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

38. The composition of claim 37 wherein D is a fluorescent label, a chromogenic label, or an electrochemical label.

39. The composition of claim 38 wherein M is a polymer selected from any one of polyethers, polyesters, polypeptides, oligosaccharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof.

40. The composition of claim 39 wherein D is a fluorescein.

41. The composition in accordance with claims 38, 39, or 40 wherein (i) L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles, (ii) said plurality of molecular tags is in the range of from 2 to 100, and (iii) said separation is electrophoretic separation.

42. The composition of claim 41 wherein said plurality of molecular tags is in the range of from 3 to 50.