Rapid and sensitive detection of molecules

• US 10,000,788 B2
• Filed: 09/06/2002
• Issued: 06/19/2018
• Est. Priority Date: 09/06/2001
• Status: Active Grant

• Current Assignee: First Light Diagnostics, Inc.
• Original Assignee: First Light Biosciences, Inc.
• Inventors: Straus, Don
• Primary Examiner(s): Brown, Melanie Yu
• Assistant Examiner(s): MOERSCHELL, RICHARD P
• Application Number:
  US10/236,105
• Publication Number:
  US 20030143580A1
• Time in Patent Office: 5,765 Days
• Field of Search: 436169, 436172, 436531-534, 436525-526, 436523, 422 82, 422 8207, 422 8208, 209576-582, 209636, 356335-343, 356 72, 356 73
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 20/00 Nanooptics, e.g. quantum optics or photonic crystals
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/06 Quantitative determination
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: G01N 15/1429 Signal processing
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2015/1488 Methods for deciding
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6471 Special filters, filter wheel
• 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 2333/195 from bacteria
• CPC Class Codes: G01N 2333/245 Escherichia (G)
• CPC Class Codes: G01N 2333/31 from Staphylococcus (G)
• CPC Class Codes: G01N 2333/32 from Bacillus (G)
• CPC Class Codes: G01N 2333/33 from Clostridium (G)
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/56916 : Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
• CPC Class Codes: G01N 33/56938 : Staphylococcus
• CPC Class Codes: G01N 33/56966 : Animal cells
• CPC Class Codes: G01N 33/56983 : Viruses
• CPC Class Codes: G01N 33/58 : involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/582 : with fluorescent label

Abstract

The invention features methods for rapidly and sensitively identifying molecular targets in medical, industrial, and environmental samples. The invention labels target molecules and then images them using large area imaging. Diagnostic tests based on the invention can be rapid, ultrasensitive, quantitative, multiplexed, and automated. A broad range of infectious agents (e.g., bacteria, viruses, fungi, and parasites) and molecules (e.g., proteins, DNA, RNA, hormones, and drugs) can be detected by the methods. The invention enables rapid, ultra-sensitive, cost-effective, and portable assays. The ability of the invention to detect low levels of target molecules rapidly and cost-effectively results from the combination of high intensity labeling, formats that facilitate rapid reaction kinetics, and large area imaging based using either instrumentation made from off-the-shelf commercial components or no instrumentation at all.

Claims

1. A method for detecting target molecules in a sample, wherein said method comprises the steps of:

a. contacting said target molecules with labeling particles at a labeling ratio of less than 100 to form individual target molecule;

labeling particle complexes, wherein said labeling particles have photonic signaling character,

b. concentrating the target molecule;

labeling particle complexes in a liquid phase in a container comprising an optically transparent detection surface having a longest linear dimension of greater than 1 mm so that the complexes are deposited in a detection zone that is a volume with one face defined by the detection surface and an opposite face spaced from the detection surface by the depth of field of an optical detection system, thereby separating the complexes from unbound labeling particles lying outside of said detection zone, wherein said concentrating comprises either (i) binding said complexes to said detection surface or (ii) applying a selection force to said complexes, wherein said complexes further comprise a selection moiety bound to said target molecule; and

c. simultaneously optically detecting individual target molecule;

labeling particle complexes in the detection zone with the optical detection system, thereby detecting said target molecules, wherein said detecting does not entail magnification of more than 5×

, wherein said labeling particles that do not bind to said targets are not removed from said container prior to said detecting wherein the container comprises a colloidal or soluble substance that absorbs the photonic signal emitted by said labeling particles, and wherein said substance is present at a concentration sufficient to prevent the detection of unbound labeling particles not in said detection zone and sufficient to allow the detection of the labeling particles in the complexes deposited in the detection zone.

2. The method of claim 1, wherein said complexes are randomly dispersed in said detection zone at a density of less than 10 complexes per mm² of the detection surface.

3. The method of claim 2, wherein said complexes are randomly dispersed in said detection zone at a density of less than 1 complex per mm² of the detection surface.

4. The method of claim 1, wherein said detecting does not entail magnification of more than 2×

.

5. The method of claim 4, wherein said detecting does not entail magnification of more than 1×

.

6. The method of claim 5, wherein said detecting does not entail magnification.

7. The method of claim 1, wherein said target molecules are proteins.

8. The method of claim 1, wherein said target molecules are nucleic acids.

9. The method of claim 1, wherein said target molecules have a molecular weight of less than 100 kD.

10. The method of claim 9, wherein said target molecules have a molecular weight of less than 10 kD.

11. The method of claim 10, wherein said target molecules have a molecular weight of less than 1 kD.

12. The method of claim 1, wherein said detecting detects and identifies more than one non-overlapping category of target molecules.

13. The method of claim 1, wherein said target molecules comprise category-specific binding sites and said category-specific binding sites on said target molecules are sites that bind specifically to natural or recombinant antibodies or aptamers.

14. The method of claim 1, wherein said target molecules comprise category-specific binding sites and said category-specific binding sites on said target molecules are sites that bind specifically to DNA, RNA, or PNA probes.

15. The method of claim 1, wherein said target molecules comprise category-specific binding sites and said the category-specific binding sites on said target molecules are or are immediately adjacent to nucleic acid polymorphisms including single nucleotide polymorphisms.

16. The method of claim 1, wherein said sample comprises a fluid or tissue obtained from a multicellular organism.

17. The method of claim 16, wherein said sample comprises the bodily fluids or tissues of an animal.

18. The method of claim 17, wherein said sample is derived from a human.

19. The method of claim 17, wherein said sample is derived from a non-human vertebrate.

20. The method of claim 17, wherein said sample is selected from the group consisting of:

respiratory, urogenital, reproductive tract, central nervous system, urine, blood, dermal, plasma, serum, saliva, wound tissue, wound exudate, biopsy, feces, and solid tissue samples.

21. The method of claim 1, wherein said sample is derived from a plant.

22. The method of claim 1, wherein said sample is obtained by sampling environmental air or water, or surfaces, objects, or organisms exposed to the environment.

23. The method of claim 1, wherein said sample is selected from the group consisting of:

raw, finished or in-process material in the manufacture pharmacological, cosmetic, blood, or other products for topical or internal use in humans or animals;

raw, in-process or finished material in the manufacture of foods or beverages; and

chemical products.

24. The method of claim 1, wherein in step (b) said complexes are deposited on a detection surface by magnetic selection, centrifugation, or settling.

25. The method of claim 24, wherein said magnetic selection comprises contacting said sample with magnetic particles that are conjugated to category-binding molecules.

26. The method of claim 24, wherein said target molecules are contacted in the liquid phase with target-molecule specific selection moieties that have an average density greater than the average density of said liquid phase and are subsequently deposited on said detection surface using gravitational, centrifugal, or centripetal force.

27. The method of claim 1, wherein said sample is treated to liquefy and/or homogenize said sample.

28. The method of claim 1, wherein said contacting occurs in a liquid phase.

29. The method of claim 1, wherein said contacting occurs at an interface between a liquid and solid phase.

30. The method of claim 1, wherein said sample is treated to remove substances or objects other than said target molecules.

31. The method of claim 1, wherein said target molecules are immobilized on the detection surface prior to said contacting.

32. The method of claim 1, wherein said target molecules are specifically bound in the detection zone by category-binding molecules that are bound to the matrix or substrate of the detection zone.

33. The method of claim 1, wherein said target molecules are specifically bound in the detection zone by forming chemical bonds to matrix or substrate of the detection zone.

34. The method of claim 1, wherein said target molecules are immobilized in said detection zone by a process selected from the group consisting of air drying, heat fixation, and chemical fixation.

35. The method of claim 1, wherein said target molecules comprise category-specific binding sites and said sample is treated so that the category-specific binding sites on said target molecules become accessible to contact by said labeling particles.

36. The method of claim 1, wherein said sample is subdivided into individual aliquots that are tested, in parallel, for the presence of different non-overlapping categories of target molecules.

37. The method of claim 36, wherein each of said aliquots is contacted with a population of labeling particles that is conjugated to a different non-overlapping family of category-binding molecules.

38. The method of claim 36, wherein said sample is contacted successively with distinct families of category-binding molecules that specifically bind to non-overlapping categories of target-entities.

39. The method of claim 1, wherein said detection surface is selected from the group consisting of solid glass, solid plastic, the surface of the wells of microtiter plates, bibulous membranes, plastic strips, the surfaces of capillary tubes, the surfaces of microfluidic chambers, and the surfaces of microfluidic channels.

40. The method of claim 1, wherein said method is automatically repeated on a series of samples.

41. The method of claim 40, wherein said samples are automatically loaded into an instrument that contains a detector for detecting said complexes.

42. The method of claim 40, wherein said samples are automatically deposited in a series of detection zones that are physically associated and that are automatically and successively loaded into an instrument that contains a detector for detecting said complexes.

43. The method of claim 1, wherein said complexes are illuminated to generate a detectable signal.

44. The method of claim 43, wherein said method detects light emitted, scattered, reflected, or absorbed as a result of said illumination of said complexes.

45. The method of claim 43, wherein said detecting detects fluorescence.

46. The method of claim 1, wherein said detecting detects chemiluminescence.

47. The method of claim 43, wherein said complexes are illuminated by one or more lasers.

48. The method of claim 43, wherein said complexes are illuminated by one or more light-emitting diodes.

49. The method of claim 43, wherein said complexes are illuminated by a source of white-light.

50. The method of claim 43, wherein said complexes are illuminated using one or more optical filters adapted for illuminating said sample with light of a wavelength appropriate for detecting said complexes.

51. The method of claim 44, wherein said emitted, scattered, transmitted, or absorbed light is detected using optical filters adapted to detect the signals derived from the illumination of said complexes.

52. The method of claim 1, wherein said detecting does not employ illumination.

53. The method of claim 1, wherein said detecting detects thermal radiation.

54. The method of claim 1, wherein said detecting detects optical absorbance.

55. The method of claim 54, wherein said optical absorbance is in the infrared region.

56. The method of claim 1, wherein said detecting detects fluorescence polarization.

57. The method of claim 1, wherein said detecting detects optical reflectance.

58. The method of claim 1, wherein said detecting detects light scattering.

59. The method of claim 1, wherein said detecting detects Raman scattering.

60. The method of claim 1, wherein said labeling particles are less than 20 microns in size.

61. The method of claim 60, wherein said labeling particles are less than 10 microns in size.

62. The method of claim 61, wherein said labeling particles are less than 5 microns in size.

63. The method of claim 62, wherein said labeling particles are less than 1 micron in size.

64. The method of claim 63, wherein said labeling particles are less than 100 nm in size.

65. The method of claim 64, wherein said labeling particles are less than 10 nm in size.

66. The method of claim 1, wherein said labeling particles are latex particles, silica particles, quantum dots, resonance light scattering particles, up-converting phosphors, or particles composed chiefly of gold or silver.

67. The method of claim 1, wherein said labeling particles are coated with enzymatic signaling moieties.

68. The method of claim 67, wherein said labeling particles comprise enzymatic signaling moieties at an average density of greater than or equal to 2 enzymatic signaling moieties per cubic micron of particle volume.

69. The method of claim 1, wherein said labeling particles comprise signaling moieties are alkaline phosphatase or horseradish peroxidase enzymes.

70. The method of claim 1, wherein said labeling particles bind to category-binding molecules that have been previously contacted with the sample.

71. The method of claim 1, wherein said labeling particles comprise signaling moieties are selected from the group consisting of organic fluorophores, up-regulated phosphors, lanthanides, quantum dots, and enzymes that generate fluorescent product from non-fluorescent substrates.

72. The method of claim 1, wherein said labeling particles comprise signaling moieties are particles dyed with or conjugated to signaling moieties that have fluorescent signal character and that are selected from the group consisting of:

organic fluorophores, up-regulated phosphors, lanthanides, quantum dots, and enzymes that generate fluorescent product form non-fluorescent substrates.

73. The method of claim 1, wherein said labeling particles comprise signaling moieties with fluorescent signaling character.

74. The method of claim 1, wherein said labeling particles comprise signaling moieties with chemiluminescent signaling character.

75. The method of claim 74, wherein said signaling moieties are acridinium esters.

76. The method of claim 1, wherein said labeling particles comprise signaling moieties with chromogenic signaling character.

77. The method of claim 1, wherein said labeling particles comprise signaling moieties with light scattering character.

78. The method of claim 1, wherein step (a) comprises contacting said sample with labeling particles comprising category-binding molecules under conditions that allow the formation of complexes between said category-binding molecules and category-specific binding sites on said target molecules.

79. The method of claim 78, wherein said category-binding molecules comprise antibodies.

80. The method of claim 78, wherein said category-binding molecules comprise aptamers.

81. The method of claim 78, wherein said category-binding molecules comprise nucleic acids or peptide nucleic acids.

82. The method of claim 78, wherein said category-binding molecules comprise ligands.

83. The method of claim 78, wherein said category-binding molecules comprise molecules with molecular weights less than 100 kD.

84. The method of claim 83, wherein said category-binding molecules comprise molecules with molecular weights less than 10 kD.

85. The method of claim 84, wherein said category-binding molecules comprise molecules with molecular weights less than 1 kD.

86. The method of claim 1, wherein said labeling particles comprise different populations, wherein each population is conjugated to a different non-overlapping family of category-binding molecules.

87. The method of claim 86, wherein each of said populations binds specifically to a category of target molecules that also binds specifically to a corresponding, second family of category-binding molecules that is stably bound to a section of the detection zone, wherein each of said corresponding, second families is bound at a distinct site that can be distinguished by said detecting.

88. The method of claim 87, wherein each of said populations of labeling particles has the same signaling class and signature.

89. The method of claim 86, wherein each of said populations has a distinct signaling signature or signaling class.

90. The method of claim 89, wherein said method comprises optical filters adapted to discriminate between the signal signatures of said populations of labeling particles.

91. The method of claim 86, wherein said families of category-binding molecules have a family complexity that is greater than 1.

92. The method of claim 86, wherein said families of category-binding molecules have a family complexity≥

5.

93. The method of claim 86, wherein said families of category-binding molecules have a family complexity≥

10.

94. The method of claim 86, wherein said families of category-binding molecules have a family complexity≥

20.

95. The method of claim 1, wherein the container has a bar code or equivalent label for tracking the sample automatically.

96. The method of claim 1, wherein the detection surface has registration marks to facilitate alignment of multiple images of the same surface.

97. The method of claim 1, wherein said method detects control marks or control cells in a specified region of the detection zone.

98. The method of claim 1, wherein said detecting comprises use of a photoelectric detector.

99. The method of claim 1, wherein said detecting comprises use of a photoelectric array detector.

100. The method of claim 99, wherein said photoelectric detector comprises a CCD or CMOS detector.

101. The method of claim 44, wherein detecting of said emitted, scattered, or absorbed light does not employ an image intensifier.

102. The method of claim 1, wherein said detecting comprises use of a photomultiplier tube detector.

103. The method of claim 1, wherein said detecting comprises use of a photodiode detector.

104. The method of claim 1, wherein the detecting comprises use of photosensitive film.

105. The method of claim 1, wherein said detecting comprises direct visual detection.

106. The method of claim 1, wherein the number of target molecules is inferred from said detecting by analyzing images acquired by said detecting.

107. The method of claim 1, wherein the category of target molecules is inferred from said detecting using image analysis software.

108. The method of claim 107, wherein said image analysis software further comprises functions for discerning the signals generated by said complexes from other signals.

109. The method of claim 1, wherein said sample is obtained from raw, in-process, or finished material in the manufacture of medical or in vitro diagnostic devices;

industrial surfaces;

instrumentation;

or machinery.

110. The method of claim 1, wherein the liquid phase is aqueous.