Modular probe for total internal reflection fluorescence spectroscopy

• US 6,300,638 B1
• Filed: 11/12/1998
• Issued: 10/09/2001
• Est. Priority Date: 11/12/1998
• Status: Expired due to Fees

• Current Assignee: American Research Corporation Of Virginia, Veridian Engineering Incorporated
• Original Assignee: American Research Corporation Of Virginia, Calspan Srl Corporation
• Inventors: Reynolds, Roger L., Raymonda, John, Sullivan, William F., Coolbaugh, Myron T., Lo, K. Peter, Groger, Howard P.
• Primary Examiner(s): Hannaher, Constantine
• Application Number:
  US09/189,841
• Time in Patent Office: 1,062 Days
• Field of Search: 250/458.1
• US Class Current: 250/458.1
• CPC Class Codes: G01N 21/552 Attenuated total reflection
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 21/7703 using reagent-clad optical fibres or optical waveguides using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B

Abstract

An instrument for total internal reflection fluorescence spectroscopy having modular probe that inexpensively monitors vapors and liquid-phase analytes under field conditions is described. The system is particularly helpful in evaluating multiple analytes using the fluorescence of a material immobilized in a thin sorbent polymer coating. At the same time, the system is capable of monitoring trace quantities of analyte using a fluorescence-read immunological reaction. The device includes a diode laser source, a shaped optical element (which may be as simple as a microscope slide), and one or more amplified photodiode detectors. The shaped optical element propagates light from the diode laser in a series of internally reflective bounces.

Claims

1. A detection device comprising a shaped optical element, a fluorophore deposited within an evanescent field of the shaped optical element, a diode laser operatively disposed relative to the shaped optical element and creating an evanescent field for excitation of the fluorophore and generation of fluorescence, and a photodetector positioned adjacent a surface of the optical element having an amplified diode capable of detecting the fluorescence at the surface of said optical element.

2. The device of claim 1, further comprising a focusing lens between the laser and the shaped optical element.

3. The device of claim 1, wherein the shaped optical element is a polymer material.

4. The device of claim 3, wherein the shaped optical element is formed by injection molding.

5. The device of claim 4, wherein the shaped optical element has a scratch/dig specification of 60/40 or better.

6. The device of claim 1, wherein the diode laser has an output wavelength of about 635 to about 1060 nm.

7. The device of claim 1, further comprising a flow cell, wherein the shaped optical element forms one side of the flow cell, wherein the fluorophore deposited on the shaped optical element as a fluorophore-polymerfilm, and wherein the flow cell has an inlet for allowing a material to be detected to interact with the fluorophore-polymer film, and an outlet.

8. The device of claim 7, further comprising a vaporization chamber connected to the inlet for vaporizing a liquid material before flowing the material as a vapor to the flow cell.

9. The device of claim 8, further comprising a thin film heater in the vaporization chamber for rapidly heating the liquid material to the vapor.

10. The device of claim 9, wherein the vaporization chamber further comprises an inlet valve connected to the inlet of the flow cell and an outlet valve capable of controlling expansion of the vapor into the flow cell for interacting with the fluorophore-polymerfilm.

11. The device of claim 8, further comprising sensors capable of monitoring the temperature and pressure within the vaporization chamber.

12. The device of claim 8, wherein the vaporization chamber is up to about 10 mm long, and up to about 500 microns wide and deep.

13. The device of claim 7, wherein at least two photodetectors are mounted on the flow cell.

14. The device of claim 13, wherein a reference segment of the fluorophore-polymer film has a coating effective to prohibit interaction with material to be detected.

15. The device of claim 14, wherein one photodetector monitors the fluorescence from the reference segment of the fluorophore-polymer film.

16. The device of claim 13, wherein the at least two photodetectors are capable of monitoring non-coated segments of the fluorophore-polymer film.

17. The device of claim 1, wherein the diode laser is modulated.

18. The device of claim 17, wherein the modulation frequency is about 1 kHz.

19. The device of claim 17, wherein the modulation frequency is between and including 500 Hz and 5000 Hz.

20. The device of claim 1, wherein the photodetector comprises a photodiode capable of collecting the fluorescence signal and an integrated amplifier capable of amplifying the fluorescence signal.

21. The device of claim 1, further comprising a lock-in amplifier connected to the amplified photodiode capable of further amplifying the fluorescence signal.

22. The device of claim 1, wherein the fluorophore is in a fluorophore-polymer film, which is drop cast on a surface of the shaped optical element.

23. The device of claim 22, wherein the volume drop cast is about 75 to about 200 μ

l.

24. The device of claim 1, further comprising an optical filter connected to the photodiode effective to restrict light at the excitation wavelength.

25. The device of claim 24, wherein the optical filter is a holographic filter.

26. A detection device comprising a shaped optical element, a fluorophore deposited within an evanescent field of the shaped optical element, a diode laser operatively disposed relative to the shaped optical element and creating an evanescent field for excitation of the fluorophore and generation of fluorescence, and a photodetector having an amplified diode capable of detecting the fluorescence from the surface of said optical element, wherein the shaped optical element is a microscope slide.

27. A detection device comprising a shaped optical element, a fluorophore deposited within an evanescent field of the shaped optical element, a diode laser operatively disposed relative to the shaped optical element and creating an evanescent field for excitation of the fluorophore and generation of fluorescence, and a photodetector having an amplified diode capable of detecting the fluorescence from the surface of a optical element, wherein the shaped optical element is polystyrene with a refractive index of about 1.59.

28. A detection device comprising a shaped optical element, a fluorophore deposited within an evanescent field of the shaped optical element, a diode laser operatively disposed relative to the shaped optical element and creating an evanescent field for excitation of the fluorophore and generation of fluorescence, and a photodetector having an amplified diode capable of detecting the fluorescence from a surface of said optical element, wherein the shaped optical element has a beveled edge and the laser output enters the shaped optical element through the beveled edge.

29. The device of claim 28, wherein an angle of the beveled edge is such that an angle between a normal to the beveled edge and the laser output is between about 10 and about 20 degrees.

30. The device of claim 28, wherein the beveled edge is formed by selecting from a group consisting of lithographic processing and injection molding.

31. The device of claim 28, wherein a plan area of the shaped optical element decreases as the distance from the beveled edge increases.

32. A detection device comprising a shaped optical element, a fluorophore deposited within an evanescent field of the shaped optical element, a diode laser operatively disposed relative to the shaped optical element and creating an evanescent field for excitation of the fluorophore and generation of fluorescence, and a photodetector having an amplified diode capable of detecting the fluorescence from a surface of said optical element, and a splitter attached to said laser.

33. The device of claim 32, wherein the splitter is selected from a group consisting of a beam-splitter, a line generator and a holographic beam splitter.

34. An analyte detector apparatus comprising a radiant energy transparent guide, a fluorophore that fluoresces in the presence of radiant energy and the analyte in a fluid, wherein the fluorophore is deposited in conjunction with a thin polymer layer on a surface of the guide, a flow cell partially surrounding the surface, fluid conduits having a fluid intake and a fluid exhaust connected to the fluid conduits capable of moving the fluid through the chamber, a mover connected to the fluid conduits capable of moving the fluid through the chamber, a radiant energy source connected to the guide capable of irradiating the guide and the fluorophore, and a detector positioned adjacent a surface of the optical element associated with the flow cell or the guide capable of detecting fluorescing of the fluorophore at a surface of said guide or said flow cell and thus detecting presence of the analyte.

35. The detector apparatus of claim 34, further comprising a vaporization chamber having an inlet valve and an outlet valve, wherein the inlet valve is connected to the intake and the outlet valve is connected to the sample chamber, a heater connected to the vaporization chamber for vaporizing fluid within the vaporization chamber, and wherein the mover further comprises a sequencer for opening the valves and subsequently closing the valves, energizing the heater and opening the outlet valve for transferring vapors from the vaporization chamber to the sample chamber.

36. The detector apparatus of claim 34, wherein the guide is an optically transparent plate having a bevel at one end, wherein the radiant energy source is a laser optically aligned with the bevel for directing laser energy into the transparent plate for reflecting the energy within the plate and leaking some of the energy through the surface and wherein the detector comprises a photodiode and an amplifier connected to the photodiode.

37. A method of detecting analytes comprising providing a transparent plate, providing on a surface of the plate a fluorophore that changes fluorescent reaction to photoexcitation in the presence of a target analyte, partially forming a flow cell with a surface of the plate, flowing a fluid into the flow cell, illuminating one end of the plate with a laser diode, internally reflecting light from the laser diode within the plate, contacting the fluorophore with the target analyte, changing fluorescent reaction of the fluorophore with the target analyte, exciting the fluorophore with light from the plate, and detecting, through a detector positioned adjacent a surface of the flow cell or the plate, fluorescence at the surface of the flow cell or the plate.

38. The method of claim 37, further comprising providing multiple distinct fluorophores in parallel stripes along the surface of the plate, splitting illumination from the laser diode into parallel beams, directing the parallel beams into the plate, and internally reflecting the parallel beams in the plate.

39. The method of claim 37, wherein the detecting comprises detecting the fluorescence with an amplified photodiode and further amplifying output of the photodiode with an integral lock-in amplifier.

40. The method of claim 37, further comprising providing a bevel on the one end of the plate and wherein the illuminating comprises directing laser light through the bevel into the plate.

41. The method of claim 37, wherein the providing a fluorophore comprises depositing a fluorophore in conjunction with a thin polymer layer on the surface.

42. The method of claim 37, further comprising introducing a liquid into an evaporation chamber, closing valves to and from the evaporation chamber, heating the evaporation chamber, vaporizing liquid therein, opening a valve from the evaporation chamber, and flowing vapor to the flow cell.

46. The device of claim 42, wherein the shaped optical element is a polymer material.

47. The device of claim 46, wherein the shaped optical element is formed by injection molding.

48. The device of claim 47, wherein the shaped optical element has a scratch/dig specification of 60/40 or better.

43. A detection device comprising a shaped optical element having a beveled edge, a fluorophore deposited within an evanescent field of said shaped optical element, a diode laser capable of directing radiant energy onto the beveled edge of the shaped optical element and a photodetector having an amplified diode capable of detecting fluorescence of the fluorophore.

44. The device of claim 43, further comprising a focusing lens between the laser and the shaped optical element.

45. The device of claim 43, wherein the shaped optical element is a microscope slide.

49. The device of claim 43, wherein the shaped optical element is polystyrene with a refractive index of about 1.59.

50. The device of claim 43, wherein the diode laser has an output wavelength of about 635 to about 1060 nm.

51. The device of claim 43, wherein an angle of the beveled edge is such that an angle between a normal to the beveled edge and the laser output is between about 10 and about 20 degrees.

52. The device of claim 43, wherein the beveled edge is formed by selecting from a group consisting of lithographic processing and injection molding.

53. The device of claim 43, wherein a plan area of the shaped optical element decreases as the distance from the beveled edge increases.

54. The device of claim 43, further comprising of flow cell, wherein the shaped optical element forms one side of the flow cell, wherein the fluorophore is deposited on the shaped optical element as a fluorophore-polymer film, and wherein the flow cell has an inlet capable of allowing a material to be detected to interact with the fluorophore-polymer film, and an outlet.

55. The device of claim 54, further comprising a vaporization chamber connected to the inlet capable of vaporizing a liquid material before flowing the material as a vapor to the flow cell.

56. The device of claim 55, further comprising a thin film heater in the vaporization chamber capable of rapidly heating the liquid material to the vapor.

57. The device of claim 56, wherein the vaporization chamber further comprises an inlet valve connected to the inlet of the flow cell and an outlet valve capable of controlling expansion of the vapor into the flow cell for interacting with the fluorophore-polymer film.

58. The device of claim 55, further comprising sensors capable of monitoring the temperature and pressure within the vaporization chamber.

59. The device of claim 55, wherein the vaporization chamber is up to about 10 mm long, and up to about 500 microns wide and deep.

60. The device of claim 54, wherein at least two photodetectors are mounted on the flow cell.

61. The device of claim 60, wherein a reference segment of the fluorophore-polymer film has a coating effective to prohibit interaction with material to be detected.

62. The device of claim 61, wherein one of the at least two photodetectors is capable of monitoring the fluorescence from the reference segment of the fluorophore-polymer film.

63. The device of claim 60, wherein the at least two photodetectors are capable of monitoring non-coated segments of the fluorophore-polymer film.

64. The device of claim 43, wherein the diode laser has a modulation frequency.

65. The device of claim 64, wherein the modulation frequency is about 1 kHz.

66. The device of claim 64, wherein the modulation frequency is between and including about 500 Hz and about 5000 Hz.

67. The device of claim 43, further comprising a splitter attached to said laser.

68. The device of claim 67, wherein the splitter is selected from a group consisting of a beam-splitter, a line generator and a holographic beam splitter.

69. The device of claim 43, wherein the photodetector comprises a photodiode capable of collecting the fluorescence signal and an integrated amplifier capable of ambling the fluorescence signal.

70. The device of claim 43, further comprising a lock-in amplifier connected to the amplified photodiode capable of further amplifying the fluorescence signal.

71. The device of claim 43, wherein the fluorophore is in a fluorophore-polymer film, drop cast on a surface of the shaped optical element.

72. The device of claim 71, wherein the volume drop cast on a surface is about 75 to about 200 μ

l.

73. The device of claim 43, further comprising an optical filter connected to the photodiode effective to restrict light to the excitation wavelength.

74. The device of claim 73, wherein the optical filter is a holographic filter.

75. An analyte detector apparatus comprising a radiant energy transparent guide having beveled edge, a fluorophore capable of fluorescing in the presence of radiant energy and the analyte in a fluid, the fluorophore being deposited in conjunction with a thin polymer layer on a surface of the guide, a flow cell partially surrounding the surface, fluid conduits having a fluid intake and a fluid exhaust connected to the fluid conduits capable of moving the fluid through the chamber, a mover connected to the fluid conduits capable of moving the fluid through the chamber, a radiant energy source capable of emitting radiant energy onto the beveled edge of the guide and a detector associated with the flow cell of the guide capable of detecting fluorescing of the fluorophore emitted from a surface of said guide or said flow cell in response to presence of the analyte and the radiant energy.

76. The detector apparatus of claim 75, further comprising a vaporization chamber having an inlet valve and an outlet valve, wherein the inlet valve is connected to the intake and the outlet valve is connected to the sample chamber, a heater connect ed to the vaporization chamber capable of vaporizing fluid within the vaporization chamber, and wherein the mover further comprises a sequencer capable of opening the valves and subsequently closing the valves, energizing the heater and opening the outlet valve to transfer vapors from the vaporization chamber to the sample chamber.

77. The detector apparatus of claim 75, wherein the radiant energy source is a laser, wherein radiant energy is reflected within the plate.

78. The detector apparatus of claim 75, wherein the detector comprises a photodiode and an amplifier connected to said photodiode.

79. A method of detecting analytes comprising providing a transparent plate with a beveled edge comprising, providing on a surface of a plate a fluorophore that undergoes a fluorescent reaction to photoexcitation in the presence of a target analyte, partially forming a flow cell within the surface of the plate, flowing a liquid into the flow cell, illuminating the plate through the beveled edge with a laser diode, internally reflecting light from the laser diode within the plate, contacting the fluorophore with the target analyte, exciting the fluorophore, and detecting fluorescence emitted from the surface of one of the flow cell or the plate.

80. The method of claim 79, further comprising providing multiple distinct fluorophores in parallel stripes along the surface of the plate, splitting illumination from the laser diode into parallel beams, directing the parallel beams into the plate, and internally reflecting the parallel beams in the plate.

81. The method of claim 79, wherein the detecting comprises detecting the fluorescence with an amplified photodiode and amplifying output of the photodiode with an integral lock-in amplifier.

82. The method of claim 79, wherein the providing a fluorophore comprises depositing a fluorophore in conjunction with a thin polymer layer on the surface.

83. The method of claim 79, further comprising introducing a liquid into an evaporation chamber, closing valves to and from the evaporation chamber, vaporizing the liquid inside the evaporation chamber, opening a valve from the evaporation chamber, and flowing vapor to the flow cell.

84. A detection device comprising a shaped optical element having a beveled edge, a fluorophore deposited within the shaped optical element, a diode laser capable of directing radiant energy onto the beveled edge of the shaped optical element and a photodetector having an amplified diode capable of detecting the fluorescence emitted from the surface of said optical element.

85. The device of claim 84, further comprising a focusing lens between the laser and the shaped optical element.

86. The device of claim 84, wherein the shaped optical element is a microscope slide.

87. The device of claim 84, wherein the shaped optical element is a polymer material.

88. The device of claim 87, wherein the shaped optical element is formed by injection molding.

89. The device of claim 88, wherein the shaped optical element has a scratch/dig specification of 60/40 or better.

90. The device of claim 84, wherein the shaped optical element is polystyrene with a refractive index of about 1.59.

91. The device of claim 84, wherein the diode laser has an output wavelength of about 635 to about 1060 nm.

92. The device of claim 84, wherein an angle of the beveled edge is such that an angle between a normal to the beveled edge and the laser output is between about 10 and about 20 degrees.

93. The device of claim 84, wherein the beveled edge is formed by selecting from a group consisting of lithographic processing and injection molding.

94. The device of claim 84, wherein a plan area of the shaped optical element decreases as the distance from the beveled edge increases.

95. The device of claim 84, further comprising a flow cell, wherein the shaped optical element forms one side of the flow cell, wherein the fluorophore deposited on the shaped optical element as a fluorophore-polymer film, and wherein the flow cell has an inlet capable of allowing a material to be detected to interact with the fluorophore-polymer film, and an outlet.

96. The device of claim 95, further comprising a vaporization chamber connected to the inlet capable of vaporizing a liquid material before flowing the material as a vapor to the flow cell.

97. The device of claim 96, further comprising a thin film heater in the vaporization chamber capable of rapidly heating the liquid material to the vapor.

98. The device of claim 97, wherein the vaporization chamber further comprises an inlet valve connected to the inlet of the flow cell and an outlet valve capable of controlling expansion of the vapor into the flow cell to interact with the fluorophore-polymer.

99. The device of claim 96, further comprising sensors capable of monitoring the temperature and pressure within the vaporization chamber.

100. The device of claim 96, wherein the vaporization chamber is up to about 10 mm long, and up to about 500 microns wide and deep.

101. The device of claim 95, wherein at least two photodetectors are mounted on the flow cell.

102. The device of claim 101, wherein a reference segment of the fluorophore-polymer film has a coating effective to prohibit interaction with material to be detected.

103. The device of claim 102, wherein one of the two photodetectors is capable of monitoring the fluorescence from the reference segment of the fluorophore-polymer film.

104. The device of claim 101, wherein the at least two photodetectors monitor non-coated segments of the fluorophore-polymer film.

105. The device of claim 84, wherein the diode laser has a modulation frequency.

106. The device of claim 105, wherein the modulation frequency is about 1 kHz.

107. The device of claim 105, wherein the modulation frequency is between and including about 500 Hz and about 5000 Hz.

108. The device of claim 84, further comprising a splitter attached to said laser.

109. The device of claim 108, wherein the splitter is selected from a group consisting of a beam-splitter, a line generator and a holographic beam splitter.

110. The device of claim 84, wherein the photodetector comprises a photodiode capable of collecting the fluorescence signal, and an integrated amplifier capable of amplifying the fluorescence signal.

111. The device of claim 84, further comprising a lock-in amplifier connected to the amplified photodiode capable of further amplifying the fluorescence signal.

112. The device of claim 84, wherein the fluorophore is in a fluorophore-polymer film, drop cast on a surface of the shaped optical element.

113. The device of claim 112, wherein the volume drop cast is about 75 to about 200 μ

l.

114. The device of claim 84, further comprising an optical filter connected to the photodiode effective to restrict light to the excitation wavelength.

115. The device of claim 114, wherein the optical filter is a holographic filter.

116. An analyte detector apparatus comprising a radiant energy transparent guide having a beveled edge, a fluorophore that fluoresces in the presence of radiant energy and the analyte in a fluid, the fluorophore being deposited in conjunction with a thin polymer layer on a surface of the guide, a flow cell partially surrounding the surface, fluid conduits having a fluid intake and a fluid exhaust connected to the fluid conduits capable of moving the fluid through the chamber, a mover connected to the fluid conduits capable of moving the fluid through the chamber, a radiant energy source capable of emitting radiant energy onto the beveled edge of the guide, and a detector associated with the guide or the flow cell capable of detecting fluorescing of the fluorophore emitted from a surface of said guide or said flow cell.

117. The detector apparatus of claim 116, further comprising a vaporization chamber having an inlet valve and an outlet valve, wherein the inlet valve is connected to the intake and the outlet valve is connected to the sample chamber, a heater connected to the vaporization chamber capable of vaporizing fluid within the vaporization chamber, and wherein the mover further comprises a sequencer capable of opening the valves and subsequently closing the valves, energizing the heater and opening the outlet valve to transfer vapors from the vaporization chamber to the sample chamber.

118. The detector apparatus of claim 116, wherein the radiant energy source is a laser, wherein radiant energy is reflected with the plate.

119. The detector apparatus of claim 116, wherein the detector comprises a photodiode and an amplifier connected to said photodiode.