Method of and system for multiplexed analysis by spectral imaging

• US 20030215791A1
• Filed: 10/23/2002
• Published: 11/20/2003
• Est. Priority Date: 05/20/2002
• Status: Abandoned Application

• Current Assignee: Applied Spectral Imaging Ltd.
• Original Assignee: Applied Spectral Imaging Ltd.
• Inventors: Cherepakhin, Vladimir, Hammill, Terry, Milman, Uri, Horn, Eli, Malinovich, Yacov, Garini, Yuval, Bar-Am, Irit, Katzir, Nir
• Application Number:
  US10/277,916
• Publication Number:
  US 20030215791A1
• Time in Patent Office: Days
• US Class Current: 435/5
• 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 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

A method of detecting the presence, absence and/or level of a plurality of analytes-of-interest in a sample, the method comprisES: (a) providing a plurality of objects, each of the plurality of objects having a predetermined, measurable and different imagery characteristic, and further having a predetermined and specific affinity to one analyte of the plurality of analytes-of-interest, each the imagery characteristic corresponding to one the predetermined specific affinity, hence each the imagery characteristic corresponds to one analyte of the plurality of analytes-of interest; (b) providing at least one affinity moiety having a predetermined and specific affinity or predetermined and specific affinities to the plurality of analytes-of-interest, each the affinity moiety having a predetermined, measurable response to light; (c) combining the objects, the at least one affinity moiety and the sample under conditions for affinity binding; and (d) simultaneously determining, for each object of the plurality of objects an imagery characteristic, and for at least a portion of the at least one affinity moiety a response to light, thereby detecting the presence, absence and/or level of the plurality of analytes-of-interest in the sample.

Claims

1. A method of detecting the presence, absence and/or level of a plurality of analytes-of-interest in a sample, the method comprising:

(a) providing a plurality of objects, each of said plurality of objects having a predetermined, measurable and different imagery characteristic, and further having a predetermined and specific affinity to one analyte of the plurality of analytes-of-interest, each said imagery characteristic corresponding to one said predetermined specific affinity, hence each said imagery characteristic corresponds to one analyte of the plurality of analytes-of interest;

(b) providing at least one affinity moiety having a predetermined and specific affinity or predetermined and specific affinities to the plurality of analytes-of-interest, each said affinity moiety having a predetermined, measurable response to light;

(c) combining said objects, said at least one affinity moiety and the sample under conditions for affinity binding; and

(d) simultaneously determining, for each object of said plurality of objects an imagery characteristic, and for at least a portion of said at least one affinity moiety a response to light, thereby detecting the presence, absence and/or level of the plurality of analytes-of-interest in the sample.

2. The method of claim 1, wherein said predetermined, measurable and different imagery characteristic is selected from the group consisting of a unique size, a unique geometrical shape and a unique response to light.

3. The method of claim 2, wherein said step (d) is by a spectral imaging device operable to construct a spectral image of the sample.

4. The method of claim 3, wherein said spectral image comprises at least two colors.

5. The method of claim 3, wherein said spectral image comprises at least three colors.

6. The method of claim 3, wherein said spectral image comprises at least four colors.

7. The method of claim 2, wherein said step (d) comprises determining, for each object, a wavelength value and an intensity value.

8. The method of claim 7, wherein said wavelength value is used to determine a presence of a particular analyte of said plurality of analytes-of-interest in the sample.

9. The method of claim 7, wherein said intensity value is used to determine a level of a particular analyte of said plurality of analytes-of-interest in the sample.

10. The method of claim 1, wherein the analytes-of-interest are dissolved, suspended or emulsed in a solution.

11. The method of claim 1, wherein the analytes-of-interest are selected from the group consisting of antigens, antibodies, receptors, haptens, enzymes, proteins, peptides, nucleic acids, drugs, hormones, chemicals, polymers, pathogens, toxins, and combination thereof.

12. The method of claim 1, wherein the analytes-of-interest are selected from the group consisting of viruses, bacteria, cells and combination thereof.

13. The method of claim 2, wherein said unique geometrical shape is selected from the group consisting of a spherical shape, a pyramidal shape, a flat shape and an irregular shape.

14. The method of claim 1, wherein a portion of said plurality of objects are beads.

15. The method of claim 1, wherein a portion of said plurality of objects are disks.

16. The method of claim 1, wherein said plurality of objects are predetermined spatial x-y locations on two-dimensional array.

17. The method of claim 16, wherein said two-dimensional array is a micro-array chip.

18. The method of claim 1, wherein said objects are of micrometer size.

19. The method of claim 1, wherein each of said plurality of objects comprises a predetermined combination of color-components, each color-component is selected from the group consisting of fluorochromes, chromogenes, quantum dots, nanocrystals, nanoprisms, nanobarcodes, scattering metallic objects, resonance light scattering objects and solid prisms.

20. The method of claim 19, wherein each of said color-components is characterized by a predetermined concentration level.

21. The method of claim 19, wherein each-of said fluorochromes is selected from the group consisting of Aqua, Texas-Red, FITC, rhodamine, rhodamine derivative, fluorescein, fluorescein derivative, cascade blue, Cyanine and Cyanine derivatives.

22. The method of claim 1, wherein said specific affinity of each of said plurality of objects and said specific affinity of each of said at least one affinity moiety are independently capable of binding to an analyte by means of an ionic linkage or a non-ionic linkage.

23. The method of claim 1, wherein said specific affinity of each of said plurality of objects and said specific affinity of each of said at least one affinity moiety are independently capable of binding to an analyte by means of covalent linkage or a non-covalent linkage.

24. The method of claim 1, wherein said specific affinity of each object of said plurality of objects is adsorbed onto a surface of said object.

25. The method of claim 1, wherein said specific affinity of each object of said plurality of objects is covalently linked to said object.

26. The method of claim 1, wherein said specific affinity of each of said plurality of objects and said specific affinity of each of said at least one affinity moiety are independently selected from the group consisting of a nucleic acid, an antibody, an antigen, a receptor, a ligand, an enzyme, a substrate and an inhibitor.

27. The method of claim 1, further comprising repeating said step (c) a plurality of times, each time on a different x-y location of a two-dimensional platform.

28. The method of claim 27, wherein said two-dimensional platform is a microtiter plate.

29. The method of claim 27, wherein said step (d) is performed for each x-y location separately.

30. The method of claim 27, wherein said step (d) is performed simultaneously for all x-y locations.

31. The method of claim 1, further comprising repeating said step (d) at least once, so as to optimize a signal-to-noise ratio.

32. The method of claim 3, further comprising performing at least one calibration spectral imaging measurement prior to said step (d).

33. The method of claim 2, wherein responses to light of said plurality of objects and responses to light of said at least one moiety are determined simultaneously.

34. The method of claim 2, wherein responses to light of said plurality of objects and responses to light of said at least one moiety are determined separately and independently.

35. The method of claim 1, wherein responses to light of said at least one moiety are determined by gray-level imaging.

36. The method of claim 3, further comprising subtracting background spectra from said spectral image, said background spectra are collected from a regions of said image which are characterized by absence of objects.

37. The method of claim 3, further comprising magnifying said spectral image by a magnification factor, said magnification factor is from 1 to 100.

38. The method of claim 2, further comprising selecting an optimal excitation and emission spectrum of each of said plurality of objects.

39. The method of claim 38, wherein said selecting an optimal excitation and emission spectrum is by an epi-fluorescent setup which comprises at least one spectral filter.

40. The method of claim 1, wherein said step (d) is effected by a procedure selected from a group consisting of a principle component analysis, a principle component regression and a spectral decomposition.

41. The method of claim 2, wherein said step (d) comprises using a library of reference spectra characterizing said plurality of objects.

42. The method of claim 3, wherein said spectral imaging device comprises a dispersion element and a detector.

43. The method of claim 42, wherein said dispersion element is an interferometer.

44. The method of claim 43, wherein said interferometer is selected from the group consisting of a moving type interferometer, a Michelson type interferometer and a Sagnac type interferometer.

45. The method of claim 42, wherein said dispersion element is at least one filter, selected so as to collect spectral data of intensity peaks characterizing a response to light of each of said plurality of objects.

46. The method of claim 45, wherein each of said at least one filter is independently selected from the group consisting of an acousto-optic tunable filter and a liquid-crystal tunable filter.

47. The method of claim 42, wherein said dispersion element is selected from the group consisting of a grating and a prism.

48. The method of claim 42, wherein said detector is selected from the group consisting of a CCD detector, a C-MOS detector, a line-scan array, an array of photo diodes and a photomultiplier.

49. The method of claim 42, wherein said spectral imaging device further comprises at least one light source.

50. The method of claim 49, wherein said at least one light source is selected from the group consisting of Mercury lamp, Xenon lamp, Tungsten lamp, Halogen lamp, laser light source, Metal-Halide lamp.

51. The method of claim 3, wherein said step (d) comprises:

(i) illuminating the sample with incident light; and

(ii) collecting exiting light from the sample so as to acquire a spectrum of each object of said plurality of objects.

52. The method of claim 51, wherein said exiting light is reflected from the sample.

53. The method of claim 51, wherein said exiting light is transmitted through the sample.

54. The method of claim 51, wherein said exiting light is emitted from the sample.

55. The method of claim 51, further comprising positioning at least a portion of said plurality of objects on a two-dimensional platform, prior to said step (i).

56. The method of claim 51, wherein said positioning is effected by a procedure selected from the group consisting of printing and gluing.

57. The method of claim 55, wherein said two-dimensional platform is a microtiter plate.

58. The method of claim 55, wherein said two-dimensional platform is a microscope slide.

59. The method of claim 51, further comprising using at least one filter to adjust a spectrum of said incident light.

60. The method of claim 51, further comprising substantially filtering out an exciting wavelength of said incident light while collecting said exiting light.

61. The method of claim 60, wherein said filtering out exciting wavelength is by an optical device selected from the group consisting of a dichroic mirror, a dark-field objective lens, a phase contrast device and a Numarski-prism.

62. The method of claim 51, further comprising acquiring an intensity value of each picture element of said at least a portion of the sample.

63. The method of claim 62, wherein said intensity value is used to determine a level of a particular analyte of said plurality of analytes-of-interest in the sample.

64. The method of claim 51, wherein said step (ii) is characterized by spectral resolution ranging between 1 nm and 50 nm and spatial resolution ranging between 0.1 mm and 1.0 mm.

65. The method of claim 51, further comprising generating individual spectra-images from spectra acquired in said step (ii).

66. The method of claim 42, wherein said illuminating is by at least one light source selected from the group consisting of Mercury lamp, Xenon lamp, Tungsten lamp, Halogen lamp, laser light source, Metal-Halide lamp.

67. The method of claim 3, wherein said spectral imaging device comprises an interferometer and a detector, said interferometer comprising two mirrors and one beam-splitter, and said detector comprising a two dimensional array of detector elements.

68. The method of claim 67, wherein said detector is a CCD detector.

69. The method of claim 67, wherein said step (d) comprises:

(i) collecting incident light simultaneously from said plurality of objects;

(ii) passing said incident light through said interferometer, so that said light is first split into two coherent beams having an optical path difference therebetween, and then said two coherent beams recombine to interfere with each other to form an exiting light;

(iii) focusing said exiting light on said detector, so that each of said detector elements produces a signal which is a particular linear combination of light intensity emitted by a respective object of said plurality of objects, said linear combination is a function of said optical path difference;

(iv) simultaneously scanning said optical path difference for said plurality of objects; and

(v) recording said signals of each of said detector elements as function of time.

70. The method of claim 69, further comprising passing said incident light through a collimator, prior said step (ii), said collimator designed and configured such that said light is simultaneously collected and collimated for each of said plurality of objects.

71. The method of claim 69, wherein said collimator is an afocal telescope.

72. The method of claim 69, wherein said collimator is a microscope.

73. The method of claim 69, wherein said simultaneously scanning said optical path difference is by rigidly rotating said beam-splitter and said two mirrors around an axis perpendicular to a plane formed by said two coherent beams.

74. The method of claim 69, wherein said interferometer further comprises a first periscope mirror, a second periscope mirror and a double sided mirror having a first side and a second side, and further wherein said simultaneously scanning said optical path difference is by rotating said double sided mirror around an axis perpendicular to a plane formed by said two coherent beams, in a manner that said incident light:

encounters said first side of said double sided mirror,

encounters said first periscope mirror,

splits and recombined in said beam-splitter and said two mirrors;

encounters said second periscope mirror, and

encounters said second side of said double sided mirror.

75. The method of claim 69, wherein said interferometer further comprises a single large mirror, and further wherein said simultaneously scanning said optical path difference is by rotating said large mirror around an axis perpendicular to a plane formed by said two coherent beams, in a manner that said incident light:

encounters said large mirror;

splits and recombined in said beam-splitter and said two mirrors; and

reflected by said large mirror.

76. The method of claim 69, wherein said beam-splitter and said two mirrors are combined in a single rigid element, shaped as a prism.

77. The method of claim 69, wherein said beam-splitter and said two mirrors are combined in a single rigid element, shaped as a grating.

78. The method of claim 69, wherein said beam-splitter and said two mirrors are combined in a single rigid element, shaped as a combination of a prism and a grating.

79. The method of claim 69, further comprising simultaneously transferring all data in real time from all said elements of said detector array to a computer, and displaying an image on an output device.

80. The method of claim 79, wherein said output device is a screen.

81. The method of claim 79, wherein said output device is a printed image.

82. A system for detecting the presence, absence and/or level of a plurality of analytes-of-interest in a sample, the system comprising:

(a) a plurality of objects, each of said plurality of objects having a predetermined, measurable and different imagery characteristic, and further having a predetermined and specific affinity to one analyte of the plurality of analytes-of-interest, each said predetermined imagery characteristic corresponding to one said predetermined specific affinity, hence each said imagery characteristic corresponds to one analyte of the plurality of analytes-of interest;

(b) at least one affinity moiety having a predetermined and specific affinity or predetermined and specific affinities to the plurality of analytes-of-interest, each said affinity moiety having a predetermined, measurable response to light;

(c) a container for combining said objects, said at least one affinity moiety and the sample under conditions for affinity binding; and

(d) a determinator for simultaneously determining, for each object of said plurality of objects an imagery characteristic, and for at least a portion of said at least one affinity moiety a response to light, thereby detecting the presence, absence and/or level of the plurality of analytes-of-interest in the sample.

83. The system of claim 82, wherein said predetermined, measurable and different imagery characteristic is selected from the group consisting of a unique size, a unique geometrical shape and a unique response to light.

84. The system of claim 83, wherein said unique geometrical shape is selected from the group consisting of a spherical shape, a pyramidal shape, a flat shape and an irregular shape.

85. The system of claim 83, wherein said determinator is a spectral imaging device operable to construct a spectral image of the sample.

86. The system of claim 85, wherein said spectral image comprises at least two colors.

87. The system of claim 85, wherein said spectral image comprises at least three colors.

88. The system of claim 85, wherein said spectral image comprises at least four colors.

89. The system of claim 83, wherein said determinator is operable to determine, for each object, a wavelength value and an intensity value.

90. The system of claim 89, wherein said determinator is operable to determine a presence of a particular analyte of said plurality of analytes-of-interest in the sample, based on said wavelength value.

91. The system of claim 89, wherein said determinator is operable to determine a level of a particular analyte of said plurality of analytes-of-interest in the sample, based on said intensity value.

92. The system of claim 82, wherein the analytes-of-interest are dissolved, suspended or emulsed in a solution.

93. The system of claim 82, wherein the analytes-of-interest are selected from the group consisting of antigens, antibodies, receptors, haptens, enzymes, proteins, peptides, nucleic acids, drugs, hormones, chemicals, polymers, pathogens, toxins, and combination thereof.

94. The system of claim 82, wherein the analytes-of-interest are selected from the group consisting of viruses, bacteria, cells and combination thereof.

95. The system of claim 83, wherein said unique geometrical shape is selected from the group consisting of a spherical shape, a pyramidal shape, a flat shape and an irregular shape.

96. The system of claim 82, wherein a portion of said plurality of objects are beads.

97. The system of claim 82, wherein a portion of said plurality of objects are disks.

98. The system of claim 82, wherein said plurality of objects are predetermined spatial x-y locations on two-dimensional array.

99. The system of claim 98, wherein said two-dimensional array is a micro-array chip.

100. The system of claim 82, wherein said objects are of micrometer size.

101. The system of claim 83, wherein each of said plurality of objects comprises a predetermined combination of color-components, each color-component is selected from the group consisting of fluorochromes, chromogenes, quantum dots, nanocrystals, nanoprisms, nanobarcodes, scattering metallic objects, resonance light scattering objects and solid prisms.

102. The system of claim 101, wherein each of said color-components is characterized by a predetermined concentration level.

103. The system of claim 101, wherein each of said fluorochromes is selected from the group consisting of Aqua, Texas-Red, FITC, rhodamine, rhodamine derivative, fluorescein, fluorescein derivative, cascade blue, Cyanine and Cyanine derivatives.

104. The system of claim 82, wherein said specific affinity of each of said plurality of objects and said specific affinity of each of said at least one affinity moiety are independently capable of binding to an analyte by means of an ionic linkage or a non-ionic linkage.

105. The system of claim 82, wherein said specific affinity of each of said plurality of objects and said specific affinity of each of said at least one affinity moiety are independently capable of binding to an analyte by means of covalent linkage or a non-covalent linkage.

106. The system of claim 82, wherein said specific affinity of each object of said plurality of objects is adsorbed onto a surface of said object.

107. The system of claim 82, wherein said specific affinity of each object of said plurality of objects is covalently linked to said object.

108. The system of claim 82, wherein said specific affinity of each of said plurality of objects and said specific affinity of each of said at least one affinity moiety are independently selected from the group consisting of a nucleic acid, an antibody, an antigen, a receptor, a ligand, an enzyme, a substrate and an inhibitor.

109. The system of claim 82, wherein said container comprises a plurality of x-y location on a two-dimensional platform.

110. The system of claim 109, wherein said two-dimensional platform is a microtiter plate.

111. The system of claim 109, wherein said determinator is operable to process each x-y location separately.

112. The system of claim 109, wherein said determinator is operable to process all x-y locations simultaneously.

113. The system of claim 83, wherein said determinator is operable to simultaneously determine responses to light of said plurality of objects and responses to light of said at least one moiety.

114. The system of claim 83, wherein said determinator is operable to simultaneously determine responses to light of said plurality of objects and responses to light of said at least one moiety one at a time.

115. The system of claim 82, wherein said determinator is operable to generate a gray-level image of responses to light of said at least one moiety.

116. The system of claim 85, further comprising a background subtractor for collecting and subtracting background spectra from said spectral image, said background spectra are collected from a regions of said image which are characterized by absence of objects.

117. The system of claim 85, further comprising a magnifier for magnifying said spectral image by a magnification factor, said magnification factor is from 1 to 100.

118. The system of claim 83, further comprising an epi-fluorescent setup which comprises at least one filter for selecting an optimal excitation and emission spectrum of each of said plurality of objects.

119. The system of claim 83, wherein said determinator comprises a spectral analyzer operable to perform a procedure selected from a group consisting of a principle component analysis, a principle component regression and a spectral decomposition.

120. The system of claim 83, wherein said determinator communicates with a library of reference spectra characterizing said plurality of objects.

121. The system of claim 85, wherein said spectral imaging device comprises a dispersion element and a detector.

122. The system of claim 121, wherein said dispersion element is an interferometer.

123. The system of claim 122, wherein said interferometer is selected from the group consisting of a moving type interferometer, a Michelson type interferometer and a Sagnac type interferometer.

124. The system of claim 121, wherein said dispersion element is at least one filter, selected so as to collect spectral data of intensity peaks characterizing a response to light of each of said plurality of objects.

125. The system of claim 124, wherein each of said at least one filter is independently selected from the group consisting of an acousto-optic tunable filter and a liquid-crystal tunable filter.

126. The system of claim 121, wherein said dispersion element is selected from the group consisting of a grating and a prism.

127. The system of claim 121, wherein said detector is selected from the group consisting of a CCD detector, a C-MOS detector, a line-scan array, an array of photo diode array and a photomultiplier.

128. The system of claim 121, wherein said spectral imaging device further comprises at least one light source.

129. The system of claim 128, wherein said at least one light source is selected from the group consisting of Mercury lamp, Xenon lamp, Tungsten lamp, Halogen lamp, laser light source, Metal-Halide lamp.

130. The system of claim 83, wherein said determinator comprises:

(i) at least one light source for illuminating the sample with incident light ; and

(ii) a collector for collecting exiting light from the sample so as to acquire a spectrum of each object of said plurality of objects.

131. The system of claim 130, wherein said exiting light is reflected from the sample.

132. The system of claim 130, wherein said exiting light is transmitted through the sample.

133. The system of claim 130, wherein said exiting light is emitted from the sample.

134. The system of claim 130, further comprising at least one filter for adjusting a spectrum of said incident light.

135. The system of claim 130, further comprising an optical device for substantially filtering out an exciting wavelength of said incident light while collecting said exiting light.

136. The system of claim 135, wherein said optical device is selected from the group consisting of a filter, a dichroic mirror, a dark-field objective lens, a phase contrast device and a Numarski-prism.

137. The system of claim 130, wherein said collector is characterized by spectral resolution ranging between 1 nm and 50 nm and spatial resolution ranging between 0.1 mm and 1.0 mm.

138. The system of claim 130, wherein said spectral imaging device is operable to generate individual spectra-images from spectra acquired by said collector.

139. The system of claim 121, wherein said at least one light source is selected from the group consisting of Mercury lamp, Xenon lamp, Tungsten lamp, Halogen lamp, laser light source, Metal-Halide lamp.

140. The system of claim 85, wherein said spectral imaging device comprises an interferometer and a detector, said interferometer comprising two mirrors and one beam-splitter, and said detector comprising a two dimensional array of detector elements.

141. The system of claim 140, wherein said detector is a CCD detector.

142. The system of claim 140, further comprising a collimator designed and configured such that light is simultaneously collected and collimated for each of said plurality of objects.

143. The system of claim 140, wherein said collimator is an afocal telescope.

144. The system of claim 140, wherein said collimator is a microscope.

145. The system of claim 140, wherein said beam-splitter and said two mirrors are operable to rotate rigidly about a predetermined axis.

146. The system of claim 140, wherein said interferometer further comprises a first periscope mirror, a second periscope mirror and a double sided mirror having a first side and a second side, and further wherein said double sided mirror is operable to rotate about a predetermined axis.

147. The system of claim 140, wherein said interferometer further comprises a single large mirror, operable to rotate about a predetermined axis.

148. The system of claim 140, wherein said beam-splitter and said two mirrors are combined in a single rigid element, shaped as a prism.

149. The system of claim 140, wherein said beam-splitter and said two mirrors are combined in a single rigid element, shaped as a grating.

150. The system of claim 140, wherein said beam-splitter and said two mirrors are combined in a single rigid element, shaped as a combination of a prism and a grating.

151. The system of claim 140, further comprising a transmitting unit for simultaneously transferring all data in real time from all said elements of said detector array to a computer, and displaying an image on an output device.

152. The system of claim 151, wherein said output device is a screen.

153. The system of claim 151 , wherein said output device is a printed image.