IPC분류정보
국가/구분 |
United States(US) Patent
등록
|
국제특허분류(IPC7판) |
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출원번호 |
US-0731130
(2010-03-24)
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등록번호 |
US-8236574
(2012-08-07)
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발명자
/ 주소 |
- Duffy, David C.
- Rissin, David M.
- Walt, David R.
- Fournier, David
- Kan, Cheuk
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출원인 / 주소 |
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대리인 / 주소 |
Wolf, Greenfield & Sacks, P.C.
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인용정보 |
피인용 횟수 :
16 인용 특허 :
87 |
초록
▼
The present invention relates to systems and methods for detecting analyte molecules or particles in a fluid sample and in some cases, determining a measure of the concentration of the molecules or particles in the fluid sample. Methods of the present invention may comprise immobilizing a plurality
The present invention relates to systems and methods for detecting analyte molecules or particles in a fluid sample and in some cases, determining a measure of the concentration of the molecules or particles in the fluid sample. Methods of the present invention may comprise immobilizing a plurality of analyte molecules or particles with respect to a plurality of capture objects. At least a portion of the plurality of capture objects may be spatially separated into a plurality of locations. A measure of the concentration of analyte molecules in a fluid sample may be determined, at least in part, on the number of reaction vessels comprising an analyte molecule immobilized with respect to a capture object. In some cases, the assay may additionally comprise steps including binding ligands, precursor labeling agents, and/or enzymatic components.
대표청구항
▼
1. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising: exposing a plurality of capture objects that each include a binding surface having affinity for at least one type of analyte molecule or particle, to a solution containing or s
1. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising: exposing a plurality of capture objects that each include a binding surface having affinity for at least one type of analyte molecule or particle, to a solution containing or suspected of containing the at least one type of analyte molecules or particles;immobilizing analyte molecules or particles with respect to the plurality of capture objects such that at least some of the capture objects associate with at least one analyte molecule or particle and a statistically significant fraction of the capture objects do not associate with any analyte molecule or particle;spatially segregating at least a portion of the capture objects subjected to the immobilizing step into a plurality of separate locations;addressing at least a portion of the plurality of locations subjected to the spatially segregating step and determining the number of said locations containing at least one analyte molecule or particle; anddetermining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the number of locations determined to contain at least one analyte molecule or particle. 2. The method of claim 1, wherein the percentage of capture objects which associate with at least one analyte molecule is less than about 50% of the total number of capture objects. 3. The method of claim 1, wherein the percentage of capture objects which do not associated with any analyte molecules is at least about 20% of the total number of capture objects. 4. The method of claim 1, wherein in the addressing step, the number of said locations containing a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle not containing an analyte molecule or particle is determined. 5. The method of claim 4, wherein the measure of the concentration of analyte molecule or particles in the fluid sample is based at least in part on the ratio of the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle containing at least one analyte molecule or particle, to the total number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle. 6. The method of claim 5, wherein the percentage of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle containing at least one analyte molecule or particle is less than about 50%. 7. The method of claim 4, wherein the measure of the concentration of analyte molecule or particles in the fluid sample is based at least in part on the ratio of the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle containing at least one analyte molecule or particle, to the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle but not to contain any capture objects that include a binding surface having affinity for at least one type of analyte molecule or particle containing at least one analyte molecule or particle. 8. The method of claim 1, wherein the measure of the concentration of analyte molecules or particles in the fluid sample is based at least in part on the ratio of the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle containing at least one analyte molecule or particle, to the number of locations addressed in the addressing step that do not contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle. 9. The method of claim 1, wherein the plurality of capture objects that include a binding surface having affinity for at least one type of analyte molecule or particle comprises a plurality of beads. 10. The method of claim 9, wherein the average diameter of the plurality of beads is between about 0.1 micrometer and about 100 micrometers. 11. The method of claim 9, wherein the average diameter of the plurality of beads is between about 1 micrometer and about 10 micrometers. 12. The method of claim 1, wherein the plurality of locations comprises a plurality of reaction vessels. 13. The method of claim 12, wherein the plurality of reaction vessels is formed on the end of a fiber optic bundle. 14. The method of claim 12, wherein the number of reaction vessels addressed in the addressing step is at least about 5% of the total number of reaction vessels. 15. The method of claim 12, wherein the plurality of reaction vessels are formed upon the mating of at least a portion of a sealing component and at least a portion of a substrate. 16. The method of claim 12, wherein the plurality of reaction vessels are formed in a planar substrate. 17. The method of claim 12, wherein the average volume of the plurality of reaction vessels is between about 10 attoliters and about 100 picoliters. 18. The method of claim 12, wherein the average volume of the plurality of reaction vessels is between about 1 femtoliter and about 1 picoliter. 19. The method of claim 12, further comprising sealing the plurality of reaction vessels. 20. The method of claim 1, wherein at least a portion of the analyte molecules or particles are associated with at least one binding ligand. 21. The method of claim 20, wherein the binding ligand comprises an enzymatic component. 22. The method of claim 20, further comprising exposing the locations to precursor labeling agent. 23. The method of claim 22, wherein the locations are exposed to precursor labeling agent following the spatially segregating step. 24. The method of claim 22, wherein the precursor labeling agent is converted to a labeling agent upon exposure to a binding ligand. 25. The method of claim 24, wherein the number of locations containing a capture object containing at least one analyte molecule or particle is determined by determining the number of locations comprising a labeling agent. 26. The method of claim 24, wherein the labeling agent is a chromogenic, fluorescent, or chemiluminescent. 27. The method of claim 1, wherein the analyte molecules or particles comprise an enzymatic component. 28. The method of claim 1, wherein the concentration of analyte molecules or particles in the fluid sample is less than about 50×10−15 M. 29. The method of claim 1, wherein the measure of the concentration of analyte molecules or particles in the fluid sample is determined at least in part by comparison of a measured parameter to a calibration standard. 30. The method of claim 1, wherein during the immobilizing step, at least about 10% of the analyte molecules or particles are immobilized with respect to a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle. 31. The method of claim 1, wherein during the spatially segregating step, at least about 0.5% of the capture objects subjected to the immobilizing steps are spatially separated into the plurality of locations. 32. The method of claim 1, wherein the portion of the capture objects that include a binding surface having affinity for at least one type of analyte molecule or particle are spatially separated by exposing the plurality of locations to a solution comprising the plurality of capture objects. 33. The method of claim 1, wherein the analyte molecules or particles are proteins. 34. The method of claim 1, wherein the analyte molecules or particles are nucleic acids. 35. The method of claim 1, further comprising performing at least one wash step. 36. The method of claim 1, wherein the plurality of locations is addressed using optical techniques. 37. The method of claim 1, wherein the binding surface comprises a plurality of capture components. 38. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising: exposing a plurality of capture objects that each include a binding surface having affinity for at least one type of analyte molecule or particle, to a solution containing or suspected of containing the at least one type of analyte molecules or particles to form capture objects comprising at least one immobilized analyte molecule or particle;mixing the capture objects prepared in the exposing step to a plurality of binding ligands such that at least some of the capture objects associate with a single binding ligand and a statistically significant fraction of the capture objects do not associate with any binding ligand;spatially segregating at least a portion of the capture objects subjected to the mixing step into a plurality of locations;addressing at least a portion of the plurality of locations subjected to the spatially segregating step and determining the number of locations containing a binding ligand; anddetermining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the number of locations determined to contain a binding ligand. 39. The method of claim 38, wherein in the addressing step, the number of said locations containing a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle not containing a binding ligand is determined. 40. The method of claim 39, wherein the measure of the concentration of analyte molecule or particles in the fluid sample is based at least in part on the ratio of the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle containing a binding ligand, to the total number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle. 41. The method of claim 39, wherein the measure of the concentration of analyte molecule or particles in the fluid sample is based at least in part on the ratio of the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle containing a binding ligand, to the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle but not to contain any capture objects that include a binding surface having affinity for at least one type of analyte molecule or particle containing a binding ligand. 42. The method of claim 38, wherein the measure of the concentration of analyte molecules or particles in the fluid sample is based at least in part on the ratio of the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle containing a binding ligand, to the number of locations addressed in the addressing step that do not contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle. 43. The method of claim 38, wherein the plurality of capture objects that include a binding surface having affinity for at least one type of analyte molecule or particle comprises a plurality of beads. 44. The method of claim 38, wherein the plurality of locations comprises a plurality of reaction vessels. 45. The method of claim 44, wherein the plurality of reaction vessels is formed on the end of a fiber optic bundle. 46. The method of claim 44, wherein the number of reaction vessels addressed in the addressing step is at least about 5% of the total number of reaction vessels. 47. The method of claim 44, wherein the average volume of the plurality of reaction vessels is between about 10 attoliters and about 100 picoliters. 48. The method of claim 44, wherein the average volume of the plurality of reaction vessels is between about 1 femtoliter and about 1 picoliter. 49. The method of claim 38, wherein the binding ligand comprises an enzymatic component. 50. The method of claim 38, wherein the concentration of analyte molecules or particles in the fluid sample is less than about 50×10−15 M. 51. The method of claim 38, wherein the measure of the concentration of analyte molecules or particles in the fluid sample is determined at least in part by comparison of a measured parameter to a calibration standard. 52. The method of claim 38, wherein during the mixing step, at least about 10% of the immobilized analyte molecules or particles associate with a binding ligand. 53. The method of claim 38, wherein during the spatially segregating step, at least about 0.5% of the capture objects subjected to the immobilizing steps are spatially separated into the plurality of locations. 54. The method of claim 38, wherein the portion of the capture objects that include a binding surface having affinity for at least one type of analyte molecule or particle are spatially separated by exposing the plurality of locations to a solution comprising the plurality of capture objects. 55. The method of claim 38, further comprising exposing the locations to precursor labeling agent. 56. The method of claim 55, wherein the precursor labeling agent is converted to a labeling agent upon exposure to a binding ligand. 57. The method of claim 56, wherein the number of locations containing a capture object containing at least one analyte molecule or particle or a labeling agent is determined by determining the number of locations comprising a labeling agent. 58. The method of claim 56, wherein the labeling agent is a chromogenic, fluorescent, or chemiluminescent.
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