Assessing blood brain barrier dynamics or identifying or measuring selected substances, including ethanol or toxins, in a subject by analyzing Raman spectrum signals

• US 20040019283A1
• Filed: 03/25/2003
• Published: 01/29/2004
• Est. Priority Date: 07/13/1998
• Status: Active Grant

• Current Assignee: California Institute of Technology, Childrens Hospital Los Angeles
• Original Assignee: California Institute of Technology, Childrens Hospital Los Angeles
• Inventors: Borchert, Mark S., Lambert, James L.
• Granted Patent: US 7,398,119 B2
• Time in Patent Office: Days
• US Class Current: 600/476
• CPC Class Codes: A61B 5/14532 for measuring glucose, e.g. by tissue impedance measurement
• CPC Class Codes: A61B 5/14546 for measuring analytes not otherwise provided for, e.g. ions, cytochromes
• CPC Class Codes: A61B 5/1455 using optical sensors, e.g. spectral photometrical oximeters
• CPC Class Codes: A61B 5/4064 Evaluating the brain A61B5/031, A61B5/369, A61B5/14553 take precedence
• CPC Class Codes: A61B 5/411 Detecting or monitoring allergy or intolerance reactions to an allergenic agent or substance
• CPC Class Codes: G01N 21/65 Raman scattering

Abstract

A non-invasive method for analyzing the blood-brain barrier includes obtaining a Raman spectrum of a selected portion of the eye and monitoring the Raman spectrum to ascertain a change to the dynamics of the blood brain barrier.

Also, non-invasive methods for determining the brain or blood level of an analyte of interest, such as glucose, drugs, alcohol, poisons, and the like, comprises: generating an excitation laser beam at a selected wavelength (e.g., at a wavelength of about 400 to 900 nanometers); focusing the excitation laser beam into the anterior chamber of an eye of the subject so that aqueous humor, vitreous humor, or one or more conjunctiva vessels in the eye is illuminated; detecting (preferably confocally detecting) a Raman spectrum from the illuminated portion of the eye; and then determining the blood level or brain level (intracranial or cerebral spinal fluid level) of an analyte of interest for the subject from the Raman spectrum. In certain embodiments, the detecting step may be followed by the step of subtracting a confounding fluorescence spectrum from the Raman spectrum to produce a difference spectrum; and determining the blood level and/or brain level of the analyte of interest for the subject from that difference spectrum, preferably using linear or nonlinear multivariate analysis such as partial least squares analysis. Apparatus for carrying out the foregoing methods are also disclosed.

Claims

1. A method for evaluating in vivo the blood brain barrier of a subject and/or determining the presence and/or quantity of a selected substance in the brain of a subject, comprising:

monitoring the blood brain barrier of a subject using a Raman spectral response of a selected region of the eye of the subject.

2. A method according to claim 1, further comprising evaluating the Raman spectrum to determine the presence and/or quantity of an analyte in the intracranial and/or cerebral spinal fluid.

3. A method according to claim 1, further comprising administering a non-specific marker to the subject selected for its normal reluctance to cross the blood brain barrier under a normal condition, and wherein said monitoring step comprises detecting the presence of the non-specific marker in the selected region of the eye of the subject.

4. A method according to claim 1, further comprising:

intentionally altering the dynamics of the blood brain barrier of the subject from a normal condition; and

then

administering a quantity of a selected therapeutic agent to a subject after said altering step.

5. A method according to claim 4, further comprising returning the blood brain barrier to a substantially normal state after a sufficient quantity of the therapeutic agent has been delivered to the brain.

6. A method according to claim 5, wherein said method further comprises assessing the dose of the therapeutic agent delivered to the brain.

7. A method according to claim 1, wherein the selected region of the eye comprises the aqueous humor.

8. A method according to claim 1, wherein the selected region of the eye comprises the vitreous humor.

9. A method according to claim 1, wherein the selected region of the eye comprises a conjunctive vessel.

10. A method according to claim 4, wherein the therapeutic agent is a cytotoxic agent suitable for treating a cancer in the brain.

11. A method according to claim 4, wherein said altering step is carried out by introducing a chemical to the subject to temporarily open the blood brain barrier which allows larger molecules to pass therethrough.

12. A method according to claim 4, wherein said altering step is carried out by increasing the intracranial pressure of the subject.

13. A method according to claim 4, wherein the therapeutic agent is directed to treating brain cancer.

14. A method of non-invasively detecting the presence or absence of an analyte in the cerebral spinal fluid or brain and/or monitoring the blood brain barrier of a subject, comprising the steps of:

generating an excitation beam at a wavelength of between about 400 to 900 nanometers;

focusing the excitation beam of said generating step into the eye of the subject so that a selected region is illuminated;

obtaining a Raman spectrum corresponding to the illuminated region; and

detecting the presence or absence of an analyte in the brain or cerebral spinal fluid and/or monitoring the blood brain barrier dynamics based on said detecting step.

15. A method according to claim 14, wherein said detecting step comprises assessing whether the dynamics of the blood brain barrier allow at least one selected analyte, which would normally be inhibited from traveling through the blood brain barrier, to pass into the intracranial spinal fluid through the blood brain barrier.

16. A method according to claim 15, wherein said method further comprises the steps of:

administering a non-specific marker to the subject, the non-specific marker being selected for its normal reluctance to pass through the blood brain barrier;

comparing the Raman spectrum from said obtaining step to predetermined reference spectra corresponding to the non-specific marker; and

identifying the presence of the non-specific marker in the obtained Raman spectrum thereby indicating that the dynamics of the blood brain barrier have changed.

17. A method according to claim 14, wherein said detecting step is carried out by illuminating the aqueous humor of the eye.

18. A method according to claim 14, wherein said detecting step is carried out by illuminating the vitreous humor of the eye.

19. A method according to claim 14, wherein said detecting step is carried out when the subject is exposed to elevated ambient pressures.

20. A method according to claim 16, further comprising the step of introducing a cytotoxic agent to the subject after said identifying step.

21. A method according to claim 14, wherein said monitoring step is carried out when the subject is exposed to gravity deficient environments.

22. A method according to claim 14, wherein said method is carried out during drug trials to assess the impact of the drug on the dynamics of the blood brain barrier of the subject.

23. A method according to claim 14, wherein said method is carried out when the brain of the subject has been exposed to a trauma injury to determine whether there is an alteration of the dynamics of the blood brain barrier.

24. A method according to claim 14, wherein said detecting step is carried out by obtaining a first in vivo Raman spectrum of the aqueous humor of the subject then obtaining a second in vivo Raman spectrum of the aqueous humor of the subject and comparing the first and second Raman spectrums to perform at least one of:

(a) detecting an alteration in the function of the blood brain barrier;

(b) identifying the presence or absence of a selected substance in the brain or cerebral spinal fluid; and

(c) determining a brain or cerebral spinal fluid concentration level of a selected substance.

25. A computer program product for monitoring the blood brain barrier and/or determining the presence and/or quantity of a selected substance in the brain of a subject, comprising:

a computer readable storage medium having computer readable program code embodied in said medium, said computer-readable program code comprising;

computer readable program code for analyzing a Raman spectral response of a selected region of the eye of a subject to monitor the blood brain barrier and/or determine the presence and/or quantity of a selected substance in the intracranial and/or cerebral spinal fluid of a subject.

26. A computer program product according to claim 25, further comprising computer program code for comparing the Raman spectral response to a priori data using a hybrid linear analysis technique.

27. A computer program product according to claim 25, further comprising computer program code for comparing the Raman spectral response to a priori data using a partial least squares technique.

28. A computer program product according to claim 25, wherein the program code is configured to analyze a selected one or more peaks of interest in the Raman spectral response to identify the presence and/or quantity of the substance in the subject.

29. A computer program product according to claim 28, wherein the computer program product is configured to analyze a chemical shift peak associated with a carbon-carbon double bond on the molecular structure of the analyte of interest to identify the concentration of the substance in the subject.

30. A method of monitoring ethanol and/or its metabolites or constituents in a subject using Raman spectroscopy, comprising:

obtaining a plurality of reference Raman spectra of varying concentrations of ethanol and a plurality of metabolites thereof;

transmitting an excitation beam at a wavelength to an eye of a subject;

obtaining a Raman spectrum of the eye of the subject generated in response to the transmitted excitation beam;

comparing the Raman spectrum from said obtaining step to at least one of the reference spectra; and

determining the presence and/or level of ethanol and/or its metabolites in the subject.

31. A method according to claim 30, wherein the determining step determines the concentration of ethanol, acetaldehyde and acetic acid based on the obtaining and comparing steps.

32. A method according to claim 30, wherein the excitation beam is focused on the aqueous humor, said method further comprising obtaining biometric data of the eye of the subject to confirm the identity of the subject proximate in time to the transmitting and obtaining steps.

33. A method according to claim 30, further comprising subtracting spectral contributions from predetermined constituents of the aqueous humor from the obtained Raman spectrum using data from an a priori baseline Raman spectrum of the aqueous humor without ethanol and ethanol metabolites.

34. A method according to claim 30, wherein the determining step is capable of determining in vivo ethanol values of between about 5-500 mg/dL.

35. A method according to claim 30, wherein the excitation beam has a wavelength of between about 400 to 900 nanometers, wherein the obtained Raman spectrum includes at least a portion of the spectrum in the range of about 700-1700 cm⁻

1, and wherein the determining step is carried out by selectively excluding regions of the spectra in a 700-1700 cm<sup>−

1</sup> response region in the Raman response signal at which spectral shifts can occur at increased concentrations of ethanol.

36. A method according to claim 30, further comprising providing a calibration model that analyzes a complex signal of mixed constituents for a plurality of separate constituent spectra comprising at least ethanol, acetaldehyde and acetate.

37. A method according to claim 36, wherein the calibration model is a linear model.

38. A method according to claim 36, wherein the calibration model is a piecewise linear calibration model.

39. A method according to claim 37, wherein the calibration model is an implicit model.

40. A method according to claim 37, wherein the calibration model employs a partial least squares statistical evaluation technique and is configured so that the mathematical addition of the spectra of the separate constituent spectra results in a Raman response curve that is substantially identical to the measured Raman spectra of the mixed sample in the eye.

41. A method according to claim 37, wherein the calibration model employs a hybrid linear analysis statistical evaluation technique and is configured so that the mathematical addition of the spectra of the separate constituents results in a Raman response curve that is substantially identical to the measured Raman spectra of the mixed sample in the eye.

42. A method according to claim 30, wherein the determining step determines the cerebral spinal fluid or intracranial ethanol level.

43. A method according to claim 30, further comprising dividing by the area of the Raman spectrum associated with a hydrogen-bonding region in the 400-600 cm⁻

1 range in the spectrum to normalize the obtained Raman spectrum data.

44. A method according to claim 30, wherein the determining step comprises subtracting the obtained Raman spectrum from a baseline Raman spectrum taken of an artificial aqueous humor with 0% ethanol.

45. A method according to claim 30, wherein the transmitting, obtaining and determining steps are carried out intermittently over a monitoring period to thereby provide data regarding the metabolism of ethanol.

46. A method according to claim 30, wherein the determining step is carried out using a plurality of predetermined narrow segments having a width that is less than about 200 cm⁻

1 and residing in the Raman spectrum within a 700-1700 cm<sup>−

1</sup> range.

47. A method according to claim 30, wherein the transmitting step is carried out using low energy.

48. A method according to claim 30, wherein the transmitting step is carried out using an excitation signal having an energy of less than about 100 mJ.

49. A method according to claim 30, wherein the transmitting step is carried out using an excitation signal having a length of between 5-10 seconds and a power exposure level of less than about 75 mJ.

50. A method according to claim 49, wherein the transmitting step is carried out with an energy of about 2 mW for between about 5-10 seconds.

51. A method according to claim 30, wherein the step of transmitting is carried out by confocally delivering the excitation beam to the aqueous humor of the eye of the subject.

52. A method according to claim 31, said method further comprising sampling selected wave numbers of the obtained Raman spectrum using a partial least squares statistical model to determine the concentration of ethanol in the subject.

53. A method according to claim 31, further comprising sampling selected wave numbers of the obtained Raman spectrum using a hybrid linear analysis statistical model to determine the concentration of ethanol in the subject.

54. A method according to claim 30, wherein the step of identifying is carried out to be able to determine the concentration level in the subject in the nano-micromolar range.

55. A method according to claim 30, further comprising accepting user input to define an adjustable test threshold level that corresponds to a concentration of ethanol that is illegal in a particular testing jurisdiction.

56. A method according to claim 55, further comprising automatically generating a report record of subjects when the determining step finds that the subject has a concentration level that is above the threshold level and fails the test.

57. An apparatus for in vivo determination of the presence and/or level of ethanol and/or its metabolites or constituents in a subject using Raman spectroscopy, comprising:

computer program code representing a plurality of reference Raman spectra of varying concentrations of ethanol and a plurality of metabolites thereof;

means for illuminating an eye of a subject with an excitation beam at a wavelength between about 400 to 1000 nanometers;

means for obtaining a Raman spectrum of the eye of the subject generated in response to the excitation beam;

computer program code for comparing the Raman spectrum from said obtaining step to at least one of the reference spectra; and

computer program code for determining the presence and/or level of ethanol and/or its metabolites in the subject.

58. An apparatus according to claim 57, further comprising means for obtaining biometric data of the eye of the subject to confirm the identity of the subject.

59. An apparatus according to claim 58, wherein the means for obtaining biometric data is configured to perform an iris-image analysis.

60. An apparatus according to claim 57, wherein the computer program code for determining is configured to determine the concentration of ethanol, acetaldehyde and acetic acid based on data in the Raman spectrum.

61. An apparatus according to claim 57, wherein the means for illuminating is configured to generate an excitation beam with a wavelength between about 400 to 900 nanometers to the aqueous humor, wherein the obtained Raman spectrum includes at least a portion of a response in the range of about 700-1700 cm⁻

1, and wherein the determining step is carried out by selectively excluding regions of the spectra in a 700-1700 cm<sup>−

1</sup> response region in the Raman response signal at which spectral shifts can occur at increased concentrations of ethanol.

62. An apparatus according to claim 57, wherein the computer program code adjusts the obtained Raman spectrum based on baseline Raman spectra data taken of an artificial aqueous humor with 0% ethanol and the normal baseline constituents of the aqueous humor of the eye.

63. An apparatus according to claim 57, wherein the computer program code includes a calibration model that analyzes a complex signal of mixed constituents for a plurality of separate constituent spectra comprising at least ethanol, acetaldehyde and acetate.

64. An apparatus according to claim 63, wherein the calibration model employs a partial least squares technique and is configured so that the mathematical addition of the spectra of the separate constituent spectra results in a Raman response curve that is substantially identical to the measured Raman spectra of the mixed sample in the eye.

65. An apparatus according to claim 63, wherein the calibration model employs a hybrid linear analysis technique and is configured so that the mathematical addition of the spectra of the separate constituents results in a Raman response curve that is substantially identical to the measured Raman spectra of the mixed sample in the eye.

66. An apparatus according to claim 63, further comprising computer program code for dividing the area of the spectrum associated with a hydrogen-bonding region in the 400-600 cm⁻

1 range in the obtained Raman spectrum to normalize the obtained Raman spectrum data.

67. An apparatus according to claim 57, wherein the computer program code includes program code for subtracting the obtained Raman spectrum from a baseline Raman spectrum taken of an artificial aqueous humor with 0% ethanol.

68. An apparatus according to claim 57, wherein the apparatus is configured to intermittently operate to obtain a plurality of readings over a monitoring period to thereby provide data regarding the metabolism of ethanol.

69. An apparatus according to claim 57, wherein the computer program code for determining the presence and/or concentration includes computer program code that selectively analyzes a plurality of predetermined narrow segments having a width that is less than about 200 cm⁻

1 that reside in the Raman spectrum within a 700-1700 cm<sup>−

1</sup> range.

70. An apparatus according to claim 57, wherein the means for illuminating step operates with low energy.

71. An apparatus according to claim 57, wherein the means for illuminating comprises a laser, and said apparatus further comprises a feedback circuit that controls the current or power delivered to the laser.

72. An apparatus according to claim 57, further comprising an electronic monitoring circuit that dynamically measures the intensity, energy or power of the excitation beam output to the subject.

73. An apparatus according to claim 57, wherein the means for illuminating comprises a laser, said apparatus further comprising an electronic monitoring circuit that dynamically monitors the load or power of the laser and dynamically adjusts the current, voltage or power input to the laser.

74. An apparatus according to claim 72, wherein said apparatus further comprises computer program code that adjusts the obtained Raman spectrum based on the measured power used to generate the Raman spectrum.

75. A non-invasive method for identifying the presence and/or concentration of a substance in a subject, comprising the steps of:

providing a library of reference Raman spectra of a plurality of different substances at different concentrations from in vitro artificial aqueous humor samples;

generating an excitation beam at a wavelength of from 400 to 900 nanometers;

focusing the excitation beam of said generating step into the aqueous humor of the eye of a subject so that the selected region is illuminated;

detecting a Raman spectrum corresponding to the illuminated selected region;

comparing the detected Raman spectrum to the library of reference spectra corresponding to different concentrations of the plurality of different substances; and

identifying the substance in the subject based on said detecting and comparing steps.

76. A method according to claim 75, further comprising determining the concentration of the substance in the subject.

77. A method according to claim 75, further comprising providing a calibration model that analyzes a complex signal of mixed constituents.

78. A method according to claim 77, wherein the calibration model is a linear model.

79. A method according to claim 78, wherein the calibration model is a piecewise linear calibration model.

80. A method according to claim 77, wherein the calibration model is an implicit model.

81. A method according to claim 77, wherein the calibration model employs a partial least squares technique and is configured so that the mathematical addition of the spectra of the separate constituent spectra results in a Raman spectrum that is substantially similar to the obtained Raman spectrum of the mixed sample in the eye.

82. A method according to claim 77, wherein the calibration model employs a hybrid linear analysis technique and is configured so that the mathematical addition of the spectra of the separate constituents results in a Raman spectrum that is substantially similar to the measured Raman spectra of the mixed sample in the eye.

83. A method according to claim 75, further comprising subtracting a baseline Raman spectrum of the aqueous humor with its normal constituents of lactate, urea, glucose, and ascorbate.

84. A method according to claim 75, wherein the excitation beam is generated by a laser, said method further comprising:

dynamically monitoring the power or current load to the laser to adjust for temperature, drift or power fluctuation; and

adjusting the input to the laser so that it is able to output the excitation beam to within at least 98% of the target output strength based on the monitoring step.

85. A method according to claim 75, wherein the excitation beam is generated by a laser, said method further comprising:

dynamically measuring the excitation light beam output to the subject; and

adjusting the obtained Raman spectrum to account for the actual excitation signal transmitted to the subject.

86. A method according to claim 75, further comprising obtaining biometric data of the eye of the subject to confirm the identity of the subject proximate in time to said detecting step.

87. A method according to claim 86, wherein the means for obtaining biometric data is configured to use iris-image analysis.

88. A method according to claim 75, wherein said excitation beam is low energy.

89. A method according to claim 75, wherein, for a single detecting step, the identifying step has the capability to identify the presence of a plurality of substances in the subject.

90. A method according to claim 75, further comprising the step of subtracting a fluorescence spectrum from said vessel Raman spectrum to produce a difference spectrum.

91. A method according to claim 75, wherein said identifying step identifies an elevated antibody level in the subject.

92. A method according to claim 75, wherein said identifying step detects the presence of a poison in the subject.

93. A method according to claim 92, wherein the reference spectra of selected analytes of interest are based on selected household chemicals.

94. A method according to claim 75, wherein the reference library of different substances comprises a plurality of different illegal narcotics.

95. A method according to claim 75, wherein said reference library comprises spectra generated from a Raman spectra training set comprising at least 10 in vitro aqueous humor samples for each substance of interest with each of the at least 10 samples including different amounts of the substance therein, and a substance level corresponding to each of the samples.

96. A computer program product for determining the identity of an unknown substance in a subject, the computer program product comprising:

a computer readable storage medium having computer readable program code embodied in said medium, said computer-readable program code comprising;

computer readable program code for defining at least one signature reference spectrum for at least one selected substance of interest; and

computer readable program code for analyzing an in vivo obtained Raman spectrum of the aqueous humor of the subject; and

computer readable program code for comparing selected characteristics between the reference spectrum and the in vivo spectrum to evaluate whether the in vivo Raman spectrum corresponds to at least one of the at least one signature reference spectrum, wherein said computer code for comparing comprises a predetermined calibration model that employs hybrid linear analysis and/or partial least squares techniques.

97. A computer program product according to claim 96, wherein the program code is configured to analyze Raman spectrum obtained by exciting a subject with a wavelength of between about 400-900 nm.

98. A computer program product according to claim 96, wherein the computer readable program code for defining the different reference spectrums is for a plurality of different selected substances.

99. A computer program product according to claim 96, wherein the different selected substances comprise banned substances for athletes in competition.

100. A computer program product according to claim 96, wherein the different selected substances comprise a plurality of illegal narcotic substances.

101. A computer program product according to claim 96, wherein the plurality of different selected substances comprise a plurality of household products which are potentially poisonous to a subject when ingested.

102. A computer program product according to claim 96, further comprising computer program code for evaluating the identity of the subject using biometric data.

103. An apparatus for the non-invasive determination of a level of an analyte of interest in a subject, comprising:

a laser source for generating an excitation beam;

an optical system operatively associated with said laser for directing said excitation beam into the eye of said subject so that a selected region in the eye is sufficiently illuminated to generate a detectable Raman spectrum thereof;

a detector operatively associated with said optical system and configured to detect a Raman spectrum from the selected region in the eye;

a system processor operably associated with said detector and including computer programs with computer program code for comparing the detected Raman spectrum signal to reference spectra associated with a plurality of analytes of interest to identify the presence or absence and/or concentration of at least one analyte of interest in the subject, said system processor comprising computer program code of a calibration model that evaluates Raman spectra of complex mixtures using partial least squares and/or hybrid linear analysis techniques;

a housing component configured to overlie at least one eye of a subject during use and to house said optical system and said detector therein, so that, in position, the optical system is spaced apart from the eye of the subject; and

a focus adjustment unit including a visual display adapted to display visual indicia thereon, mounted in said housing component so that, in operation, the visual indicia on said display is visible to a user to allow a user to look at and focus on a visual reference presented thereon so that the excitation beam can be directed to the appropriate location in the eye of the subject and so that said detector can detect the generated Raman spectrum.

104. An apparatus according to claim 103, wherein said signal processor further comprises a biometric assessment computer program module.

105. An apparatus according to claim 104, wherein said housing is a portable housing.

106. An apparatus according to claim 105, wherein said system processor comprises computer program code for determining in vivo the blood level of at least one analyte of interest for the subject based on the detected Raman spectrum.

107. An apparatus according to claim 106, wherein said system processor comprises means for subtracting a fluorescence spectrum for said Raman spectrum to produce a difference spectrum.

108. An apparatus according to claim 106, wherein said optical system is configured to illuminate the aqueous humor of the eye.

109. An apparatus according to claim 103, wherein said excitation beam is low energy.

110. An apparatus according to claim 103, wherein the computer program reference spectra are produced with a training set of Raman spectra corresponding to at least 20 aqueous humor samples of varying concentrations of analytes therein for each of a plurality of different selected analytes and an identified blood level of said analyte of interest for each of said samples.

111. An apparatus according to claim 110, wherein said reference spectra of selected analytes comprise reference spectra of narcotics.

112. An apparatus according to claim 111, wherein said reference spectrums comprise reference spectra of poisons including at least one of a household chemical, at least one of a pesticide or herbicide, and at least one of a petroleum product.

113. An apparatus according to claim 103, wherein said reference spectra include at least one of an analyte associated with a poisonous plant.

114. An apparatus according to claim 103, wherein said reference spectra include at least one spectrum of a pharmaceutical drug.

115. An apparatus according to claim 103, wherein said housing is configured and sized to fit onto and rest against the head of the subject during operation.

116. An apparatus according to claim 103, wherein said housing is configured and sized to be a hand-held device.

117. An apparatus according to claim 103, wherein said apparatus is configured to obtain a plurality of intermittent readings over a desired monitoring period.