Analyte assay using particulate labels

• US 6,586,193 B2
• Filed: 10/17/1997
• Issued: 07/01/2003
• Est. Priority Date: 04/25/1996
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated), Regents of the University of California (University of California)
• Original Assignee: Genicon Sciences Corporation, Regents of the University of California (University of California)
• Inventors: Kohne, David E., Jackson, Jeffrey T., Yguerabide, Evangelina E., Yguerabide, Juan
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): Gabel, Gailene R.
• Application Number:
  US08/953,713
• Publication Number:
  US 20020028519A1
• Time in Patent Office: 2,083 Days
• Field of Search: 435/5, 435/6, 435/7.1, 435/7.21, 435/7.4, 435/7.92, 435/7.95, 436/500, 436/510, 436/517, 436/518, 436/525, 436/527, 422/50, 422/51, 422/73, 356/335, 356/336, 356/338, 356/339, 356/340
• US Class Current: 506/3
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/1425 using an analyser being characterised by its control arrangement
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/0038 Investigating nanoparticles
• CPC Class Codes: G01N 2015/1493 Particle size
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/817 Steroids or hormones

Abstract

Method for specific detection of one or more analytes in a sample. The method includes specifically associating any one or more analytes in the sample with a scattered-light detectable particle, illuminating any particle associated with the analytes with light under conditions which produce scattered light from the particle and in which light scattered from one or more particles can be detected by a human eye with less than 500 times magnification and without electronic amplification. The method also includes detecting the light scattered by any such particles under those conditions as a measure of the presence of the analytes.

Claims

1. A method for specific detection of one or more analytes in a sample, comprising the steps of:

(a) providing a plurality of distinct populations of scattered-light detectable particles of a size between 1 and 500 nm inclusive, wherein each population specifically binds to a different predetermined analyte, and has a particle type configuration distinguishable from other populations by its predetermined scattered-light detectable property;

(b) contacting the sample with said particles;

(c) illuminating said particles using non-evanescent wave light under conditions which produce scattered light from said particle and in which light scattered from one or more said particles can be detected by a human eye with less than 500 times magnification and without electronic amplification; and

(d) detecting light scattered by (i) said populations of particles bound with analyte under said conditions;

or (ii) said populations of particles not bound with analyte under said conditions;

or (iii) both (i) and (ii), as a measure of the presence of said bound analyte.

2. The method of claim 1 wherein each said particle has a size which produces a specific colored light when observed by said human eye and illuminated with polychromatic light.

3. The method of claim 2 wherein the color of said specific colored light provides a means to distinguish the different analytes.

4. The method of claim 1, wherein said detecting comprises measurement of the intensity of scattered light as a measurement of the presence or amount of bound analyte.

5. The method of claim 1, wherein said detecting comprises measurement of the color of scattered light as a measurement of the presence or amount of said bound analyte.

6. The method of claim 1 wherein each said particle has a composition which produces a specific colored light when observed by said human eye and illuminated with polychromatic light.

7. The method of claim 1 wherein said particles are bound to a solid phase bound analyte.

8. The method of claim 1 wherein said particles are in a liquid phase during said detecting step.

9. The method of claim 1 wherein said one or more analytes are bound to a solid phase.

10. The method of claim 1 wherein said one or more analytes are in a liquid phase.

11. The method of claim 1 wherein said one or more analytes is specifically bound onto a microarray or array chip comprising separately addressable assay sites.

12. The method of claim 1 wherein the light used to illuminate the particles is polychromatic light.

13. The method of claim 1 wherein the light used to illuminate the particles is monochromatic light.

14. The method of claim 1 wherein said plurality of different populations of particles each has a different visual appearance when observed by said human eye.

15. The method of claim 1,

wherein after step(b), particles that are bound to analytes are brought in close proximity to one another such that the scattered-light detectable property of at least one said particle is altered, and

wherein an alteration in scattered light is detected in step (d) as a measure of the presence or amount of said bound analytes.

16. The method of claim 1,

wherein after step(b), particles that are bound to analytes are brought in close proximity to one another such that the scattered-light detectable property of said particles can be resolved from single particles.

17. The method of claim 1,

wherein after step(b), particles that are in close proximity are separated due to the binding to said one or more analytes such that the scattered-light detectable property of at least one said particle is altered, and

wherein an alteration in scattered light is detected in step (d) as a measure of the presence or amount of said bound analytes.

18. The method of claim 1, wherein said particles are linked by one or more molecular interactions, wherein the molecular interactions are disrupted in the presence of one or more analytes resulting in the release of one or more particles from the molecular interaction, and wherein the light scattered by said released particle or particles is detected in step (d) as a measure of the presence or amount of said one or more analytes.

19. The method of claim 1 wherein at least one said population of particles is gold or silver particles.

20. The method of claim 1 wherein said non-evanescent wave light for illuminating said particles is directed toward said particles by a prism, lens or light guide system.

21. The method of claim 1, wherein said particles bound with analyte form multi-particle structures.

22. The method of claim 1, wherein light scattered by said particles are detected while flowing in a capillary, microchannel, spectrophotometric flow cell, or flow cytometric flow cell.

23. The method of claim 1, wherein said analytes each comprises a nucleic acid molecule and the plurality of said particles are individually attached to one of at least two nucleic acid probes that hybridizes to different regions of said nucleic acid molecules;

and

wherein light scattered by multi-particle structures formed by hybridzation of at least two nucleic acid probes to said nucleic acid molecules which is distinguishably detected in step (d) from light scattered by single particles is indicative of the presence of said nucleic acid sequence.

24. The method of claim 1, wherein said sample comprises a cell or a cellular constituent and said detecting further comprises discriminating light scattering of said scattered-light detectable particles from light scattering from said cell or said cell constituent.

25. The method of claim 24, wherein said discriminating comprises providing two detectors such that the relative levels of light scattered by said cell or said cell constituent, and light scattered by said particles differ for said two detectors.

26. The method of claim 1, wherein said light scattered provides an integrated scattered light signal, light signals from a group of particles, light signals from individual particles, or a combination thereof.

27. The method of claim 1, wherein said light scattered provides measurable characteristics comprising at least one of intensity, polarization, wavelength, and angle of observation.

28. The method of claim 1, wherein prior to said detecting step, said sample and said particles are subjected to electrophoresis or a magnetic field.

29. The method of claim 1, wherein said one or more analytes are deposited on a membrane and said detecting is performed on said membrane.

30. The method of claim 29, wherein said particles are in a medium, and the refractive index of said medium is selected to reduce non-specific light scattering.

31. The method of claim 1, wherein said method further comprises placing said sample in a gel and said detecting is performed with said particles in said gel.

32. The method of claim 1, wherein said sample comprises a chromosome preparation.

33. The method of claim 1, wherein said binding comprises in situ hybridization.

34. The method of claim 33, wherein said at least one scattered-light detectable particle comprises a plurality of different scattered-light detectable particles.

35. The method of claim 1, wherein a probe is labeled with at least one scattered light detectable particle and said specific binding comprises binding of said probe with said analyte.

36. The method of claim 1, wherein said specific binding comprises binding of a probe with one of the analytes and binding of a secondary binding pair member with said probe, wherein said secondary binding pair member is labeled with at least one scattered-light detectable particle.

37. The method of claim 1, wherein said specific binding comprises binding of a probe with one of the analytes to form a probe/analyte complex, and binding of an agent with said complex, wherein said agent has specific binding properties for said complex and is labeled with at least one scattered light detectable particle.

38. The method of claim 1, wherein said analytes are selected from the group consisting of proteins, peptides, hormones, protein-lipid complexes, lipids, carbohydrates, carbohydrate-containing compounds, nucleic acids, pharmaceutical agents, pharmaceutical drug targets, antibodies, antigenic substances, viruses, bacteria, and cells.

39. The method of claim 1, wherein said specific binding comprises nucleic acid hybridization with a heterogeneous ribonucleic acid (hnRNA), transfer ribonucleic acid (tRNA), messenger ribonucleic acid (mRNA), or ribosomal ribonucleic acid (rRNA).

40. The method of claim 1, wherein said specific binding comprises nucleic acid hybridization and said detecting provides a determination of the presence of gene expression, a measurement of the level of gene expression, and/or the presence of genetic polymorphism.

41. The method of claim 40, wherein said specific binding is performed using a nucleic acid array.

42. The method of claim 1, wherein said analytes are nucleic acid molecules.

43. The method of claim 42, wherein said nucleic acid sequence is an oligonucleotide, a complementary DNA (cDNA), a ribonucleic acid (RNA), or a product of a polymerase chain reaction.

44. The method of claim 1, wherein the presence of said one or more analytes provides detection of an infectious disease, determination of a genotype, analysis of a gene mutation, or analysis of a genetic polymorphism.

45. The method of claim 1, wherein the presence of said one or more analytes is detected in a method of sequence by hybridization.

46. The method of claim 1, wherein at least one step is performed using a solid phase which comprises one or more internal calibration zones.

47. The method of claim 46, wherein said one or more internal calibration zones are utilized for purposes selected from the group consisting of setting calibration parameters for detection of the light scattering intensities, setting calibration parameters for identification of light scattering particles, adjusting the amount of illumination light, adjusting the gain of a detector used in said detecting step, and normalizing the data on light scattered by scattered light detectable particles in multiple assay sites.

48. The method of claim 46, wherein said internal calibration zone is on a slide or array chip.

49. The method of claim 1, wherein the particles arc indirectly bound to said analytes using a secondary binding pair.

50. The method of claim 49, wherein said secondary binding pair comprises a hapten, biotin, fluorescein, or digoxigenin.

51. The method of claim 1, wherein said detecting is performed on an array and further comprises interpreting the data by pattern recognition.

52. The method of claim 1, wherein said particles are in a medium and wherein no critical angle exists for said non-evanescent wave light illuminating said particles.

53. The method of claim 1, wherein said particles are in a medium and a critical angle exists for said incident illuminating light passing through a solid phase surface into said medium and said detecting is at an angle to the perpendicular to said surface less than said critical angle.

54. The method of claim 1, wherein said illuminating and said detecting are from opposite sides of a solid phase surface.

55. The method of claim 1, wherein the particle type configuration of each of said populations of particles is based on the size, composition, and/or shape of the particles.

56. The method of claim 55, wherein at least one said populations of particles is oval, and said oval shape provides a distinguishable scattered-light detectable property.

57. The method of claim 55, wherein at least one said population of particles is asymmetric, and said asymmetric shape provides a distinguishable scattered-light property.

58. The method of claim 1, wherein refractive index matching is utilized to reduce non-specific light scattering.

59. The method of claim 58, wherein said particles are in a medium on a surface, and the refractive index of said medium is selected to reduce non-specific light scattering.

60. The method of claim 59, wherein the refractive index of said medium is increased by addition of a higher refractive index liquid.

61. The method of claim 1, wherein said illuminating is with unpolarized light.

62. The method, of claim 1, wherein said illuminating is with polarized light.

63. The method of claim 1, wherein said illuminating is not with an epi-illumination arrangement or a laser.

64. The method of claim 1, wherein said illuminating is with an epi-illumination arrangement or with a laser.

65. The method of claim 1, wherein said detecting involves formation of an image and analysis of said image.

66. The method of claim 65, wherein said image analysis provides an integrated light intensity measurement for at least a portion of said image.

68. The method of claim 67 or 21 wherein said light scattered is detected by flowing said particle or said multi-particle structure individually by at least one dectector.

69. The method of claim 67 or 21, wherein said detecting further comprises measuring changes in scattered light as said particles or said multi-particle structures move in liquid phase.

67. A method for specific detection of one or more analytes in a sample, comprising the steps of:

(a) providing a plurality of distinct populations of scattered-light detectable particles of a size between 1 and 500 nm inclusive, wherein each population specifically binds to a different predetermined analyte, and has a particle type configuration distinguishable from other populations by its predetermined scattered-light detectable property;

(b) contacting the sample with said particles;

(c) illuminating any said particles with non-evanescent wave light under conditions which produce scattered light from said particle and in which light scattered from one or more said particles can be detected by a human eye with less than 500 times magnification and without electronic amplification; and

(d) detecting light scattered by (i) said populations of particles bound with analyte under said conditions;

or (ii) said populations of particles not bound with analyte under said conditions;

or (iii) both (i) and (ii), as a measure of the presence of said bound analytes,

wherein said detecting comprises flowing said particles by at least one detector.

70. The method of claim 67, wherein the plurality of particles comprises different populations of particles of different sizes, shape and/or compositions.

71. The method of claim 67, wherein the light scattering signal consists of light scattered from the particles in a single multi-particle structure containing a plurality of particles.

72. The method of claim 67, wherein said detecting involves formation of an image and analysis of said image.

73. The method of claim 72, wherein said image analysis provides an integrated light intensity measurement for at least a portion of said image.