Reagentless analysis of biological samples

• US 20030048432A1
• Filed: 07/09/2002
• Published: 03/13/2003
• Est. Priority Date: 08/27/1998
• Status: Active Grant

• Current Assignee: Eric B. Shain, Gary M. Oosta, Joseph Larry Pezzaniti, Larry L. Mcdowell, Tzyy-Wen Jeng
• Original Assignee: Eric B. Shain, Gary M. Oosta, Joseph Larry Pezzaniti, Larry L. Mcdowell, Tzyy-Wen Jeng
• Inventors: Shain, Eric B., Jeng, Tzyy-Wen, McDowell, Larry L., Oosta, Gary M., Pezzaniti, Joseph Larry
• Granted Patent: US 6,773,922 B2
• Time in Patent Office: Days
• US Class Current: 356/39
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/274 Calibration, base line adjustment, drift correction
• CPC Class Codes: G01N 21/4133 Refractometers, e.g. differential
• CPC Class Codes: Y10T 436/144444 Glucose
• CPC Class Codes: Y10T 436/146666 Bile pigment
• CPC Class Codes: Y10T 436/147777 Plural nitrogen in the same ring [e.g., barbituates, creatinine, etc.]
• CPC Class Codes: Y10T 436/171538 Urea or blood urea nitrogen
• CPC Class Codes: Y10T 436/200833 Carbonyl, ether, aldehyde or ketone containing

Abstract

Apparatus and method for determining at least one parameter, e.g., concentration, of at least one analyte, e.g., urea, of a biological sample, e.g., urine. A biological sample particularly suitable for the apparatus and method of this invention is urine. In general, spectroscopic measurements can be used to quantify the concentrations of one or more analytes in a biological sample. In order to obtain concentration values of certain analytes, such as hemoglobin and bilirubin, visible light absorption spectroscopy can be used. In order to obtain concentration values of other analytes, such as urea, creatinine, glucose, ketones, and protein, infrared light absorption spectroscopy can be used. The apparatus and method of this invention utilize one or more mathematical techniques to improve the accuracy of measurement of parameters of analytes in a biological sample. The invention also provides an apparatus and method for measuring the refractive index of a sample of biological fluid while making spectroscopic measurements substantially simultaneously.

Claims

1. A method for determining concentration of at least one analyte of interest in a biological sample from an individual by means of spectroscopic analysis, said method comprising the steps of:

(a) identifying at least one analyte that is a major component of said biological sample, said at least one analyte accounting for significant variations with respect to a plurality of spectra of biological samples from a plurality of donors of said biological samples;

(b) measuring a spectrum for each of said plurality of biological samples from said plurality of donors of said biological samples;

(c) calculating a model spectrum for each of said plurality of biological samples from said plurality of donors of said biological samples by mathematically fitting spectra of said analytes of said at least one identified analyte to each spectrum of each of said biological samples from said plurality of donors of said biological samples;

(d) calculating a residual spectrum for each spectrum of each of said biological samples from said plurality of donors of said biological samples by subtracting each value of said model spectrum from each value of the spectrum of said biological samples from said plurality of donors of said biological samples that corresponds to said model spectrum;

(e) repeating steps (a), (b), (c), and (d) at least one time by introducing at least one additional analyte to said model spectrum until said calculated residual spectra are substantially constant from one biological sample to another biological sample of said plurality of biological samples from said plurality of donors of said biological samples;

(f) determining a set of calibration parameters from said model spectra, said set of calibration parameters accounting for effects of said substantially constant residual spectra; and

(g) using said calibration parameters to determine concentration of an analyte of interest in said sample of biological fluid of said individual.

2. The method claim 1, wherein said biological sample is a fluid.

3. The method of claim 1, wherein said biological sample is selected from the group consisting of urine, whole blood, plasma, serum, sputum, saliva, sweat, interstitial fluid, cerebral spinal fluid, and dialysate obtained in kidney dialysis.

4. The method claim 1, wherein said biological sample is urine.

5. The method of claim 4, wherein said at least one analyte of interest is selected from the group consisting of urea, creatinine, protein, glucose, and ketones.

6. The method of claim 4, wherein said at least one analyte of interest is selected from the group consisting of hemoglobin and bilirubin.

7. The method of claim 1, wherein said spectroscopic analysis is selected from the group consisting of ultraviolet absorption spectroscopy, visible absorption spectroscopy, infrared absorption spectroscopy, ultraviolet scattering spectroscopy, visible scattering spectroscopy, infrared scattering spectroscopy, fluorescence spectroscopy, and Raman spectroscopy.

8. The method of claim 1, wherein said spectroscopic analysis is filter photometry.

9. The method of claim 1, wherein said spectroscopic analysis is derivative spectroscopy.

10. The method of claim 1, wherein said spectroscopic analysis utilize measurement of transmitted light.

11. The method of claim 1, wherein said spectroscopic analysis utilizes measurement of reflected light.

12. The method of claim 1, further including the step of employing at least one measurement selected from the following additional measurements:

light scattering, impedance, pH, ion, temperature, and refractive index.

13. A method for (a) determining concentration of at least one analyte of interest in a biological sample by means of a spectroscopic analysis and (b)measuring refractive index of said biological sample, said method comprising the steps of:

(a) providing a beam of light for a spectroscopic measurement;

(b) providing a beam of light for a refractive index measurement, said beam of light for said spectroscopic measurement and said beam of light for said refractive index measurement beam not being co-linear;

(c) introducing said beam of light for said spectroscopic measurement into said sample;

(d) introducing said beam of light for said refractive index measurement into said sample;

(e) detecting said beam of light for said spectroscopic measurement emerging from said sample;

(f) detecting said beam of light for said refractive index measurement emerging from said sample;

(g) determining concentration of said analyte from said detected beam of light for said spectroscopic measurement;

(h) determining said refractive index of said sample from said detected beam of light for said refractive index measurement.

14. The method claim 13, wherein said biological sample is a fluid.

15. The method of claim 14, wherein said biological sample is selected from the group consisting of urine, whole blood, plasma, serum, sputum, saliva, sweat, interstitial fluid, cerebral spinal fluid, and dialysate obtained in kidney dialysis.

16. The method claim 13, wherein said biological sample is urine.

17. The method of claim 16, wherein said at least one analyte of interest is selected from the group consisting of urea, creatinine, protein, glucose, and ketones.

18. The method of claim 16, wherein said at least one analyte of interest is selected from the group consisting of hemoglobin and bilirubin.

19. The method of claim 13, wherein said spectroscopic analysis is selected from the group consisting of ultraviolet absorption spectroscopy, visible absorption spectroscopy, infrared absorption spectroscopy, ultraviolet scattering spectroscopy, visible scattering spectroscopy, infrared scattering spectroscopy, fluorescence spectroscopy, and Raman spectroscopy.

20. The method of claim 13, wherein said spectroscopic analysis is filter photometry.

21. The method of claim 13, wherein said spectroscopic analysis is derivative spectroscopy.

22. The method of claim 13, wherein said spectroscopic analysis utilizes measurement of transmitted light.

23. The method of claim 13, wherein said spectroscopic analysis utilizes measurement of reflected light.

24. The method of claim 13, wherein said beam of light for said refractive index measurement is perpendicular to said beam of light for said spectroscopic measurement.

25. The method of claim 13, wherein the wavelength of said beam of light for said refractive index measurement ranges from about 500 nm to about 1000 nm.

26. The method of claim 13, wherein the wavelength of said beam of light for said spectroscopic measurement ranges from about 300 nm to about 2500 nm.

27. The method of claim 13, further including the step of employing at least one measurement selected from the following additional measurements:

light scattering, impedance, pH, ion, and temperature.

28. An apparatus for measuring refractive index of a biological sample comprising:

(a) a source of light capable of transmitting a beam of light through said biological sample;

(b) a sample cell assembly having a chamber for containing said biological sample, said chamber having a first window and a second window; and

(c) a detector, said source of light, said first window, and said second window positioned such that a beam of light from said source of light is capable of entering said first window of said chamber, is capable of being transmitted through said sample, is capable of emerging from said second window of said chamber, wherein said beam of light entering said first window is not parallel to said beam of light emerging from said second window and is not perpendicular to said first window.

29. The apparatus of claim 28, wherein said source of light is selected from the group consisting of a laser diode, light emitting diode, and incandescent lamp.

30. The apparatus of claim 28, wherein said sample cell assembly has a third window and a fourth window, said third window and said fourth window perpendicular to said first window and said second window, said third window parallel to said fourth window.

31. An apparatus for measuring refractive index of a biological sample comprising:

(a) a source of light capable of transmitting a beam of light through said biological sample;

(b) a sample cell assembly having a chamber for containing said biological sample, said chamber having a first window and a second window, at least one window of said chamber being curved, whereby a beam of light entering said first window and exiting said second window is caused to focus at a focal point; and

(c) a detector, said detector positioned such that said beam of light forms a spot on said detector.

32. The apparatus of claim 31, wherein said source of light is selected from the group consisting of a laser diode, light emitting diode, and incandescent lamp.

33. The apparatus of claim 31, wherein said sample cell assembly has a third window and a fourth window, said third window and said fourth window perpendicular to said first window and said second window, said third window parallel to said fourth window.

34. An apparatus for measuring refractive index of a biological sample comprising:

(a) a source of light capable of transmitting a beam of light through said biological sample;

(b) a sample cell assembly having a chamber containing said biological sample, said chamber having a first window and a second window, said first window parallel to said second window; and

(c) a detector substantially adjacent to said second window, said beam of light entering said first window not perpendicular to said first window.

35. The apparatus of claim 34, wherein said source of light is selected from the group consisting of a laser diode, light emitting diode, and incandescent lamp.

36. The apparatus of claim 34, wherein said sample cell assembly has a third window and a fourth window, said third window and said fourth window perpendicular to said first window and said second window, said third window parallel to said fourth window.

37. A sample cell assembly for containing a biological sample to be used in determining concentration at least one analyte in said biological sample comprising:

(a) a port into which said biological sample can be introduced into the sample cell assembly;

(b) said port of (a) communicating with an expansion zone;

(c) said expansion zone communicating with a read zone;

(d) said read zone communicating with a contraction zone;

(e) said contraction zone communicating with a port from which said biological sample can be removed from said sample cell assembly;

(f) a first pair of windows; and

(g) a second pair of windows.

38. The sample cell assembly of claim 37, wherein said read zone has at least one dimension that is of sufficient size that a beam of light entering said read zone for making a spectroscopic measurement will not be truncated.

39. The sample cell assembly of claim 37, wherein said expansion zone has a first dimension where it communicates with said inlet port and a second dimension where it communicates with said read zone, said second dimension being greater than said first dimension.

40. The sample cell assembly of claim 37, wherein said contraction zone has a first dimension where it communicates with said port from which said biological sample can be removed from said sample cell assembly and a second dimension where it communicates with said read zone, said second dimension being greater than said first dimension.

41. The sample cell assembly of claim 37, wherein said first pair of windows are parallel to one another and are aligned with said read zone such that a beam of light for a spectroscopic measurement can be transmitted through said pair of windows and said sample.

42. The sample cell assembly of claim 37, wherein said second pair of windows defines a second read zone for a refractive index measurement.

43. The sample cell assembly of claim 42, wherein a beam of light entering said read zone for said spectroscopic measurement is not co-linear with a beam of light entering said second read zone for said refractive index measurement, wherein said second pair of windows are positioned relative to one another such that a beam of light is capable of entering said first window of said second pair of windows, is capable of being transmitted through said read zone for refractive index measurement, is capable of emerging from said second window of said second pair of windows, wherein said beam of light entering said first window of said second pair of windows is not parallel to said beam of light emerging from said second window of said second pair of windows, and said beam of light capable of entering said first window of said second pair of windows is not capable of being oriented in a perpendicular direction to said first window of said second pair of windows.

44. The sample cell assembly of claim 37, including a first port into which said biological sample can be introduced into the sample cell assembly and a second port from which said biological sample can be removed from said sample cell assembly.

45. The sample cell assembly of claim 37, wherein said expansion zone and said contraction zone are the same.

46. The sample cell assembly of claim 37, wherein said expansion zone and said contraction zone are different.

47. A method for reducing noise in a determination of concentration of at least one analyte of interest in a biological sample by means of spectroscopic analysis, said method comprising the steps of:

(a) identifying a mathematical function that is substantially similar to a region of a non-smoothed spectrum of said sample over a selected range of the non-smoothed spectrum;

(b) selecting a portion of said region of said non-smoothed spectrum such that noise in said selected portion is substantially random;

(c) determining coefficients of said mathematical function that result in a close fit of the function to said selected portion of said non-smoothed spectrum;

(d) calculating at least one value of said non-smoothed spectrum at at least one wavelength of said non-smoothed spectrum by means of said coefficients and said mathematical function of step (c), wherein said at least one wavelength includes the center of said region of said non-smoothed spectrum;

(e) assigning said at least one calculated value of said non-smoothed spectrum to a wavelength including the center of said selected portion of said region of said non-smoothed spectrum to form at least one point of a smoothed spectrum;

(f) shifting a selected distance in said non-smoothed spectrum and repeating steps (c), (d), and (e) until a desired amount of said smoothed spectrum is formed;

(g) forming a residual spectrum by subtracting each point of said desired amount of said smoothed spectrum at a given wavelength from each point of said non-smoothed spectrum at said given wavelength;

(h) inspecting said residual spectrum to determine if it is random; and

(i) if said residual spectrum is not random, repeating steps (b), (c), (d), (e), (e (g), and (h) to achieve a smoothing, wherein said residual spectrum is random.

48. The method claim 47, wherein said biological sample is a fluid.

49. The method of claim 47, wherein said biological sample is selected from the group consisting of urine, whole blood, plasma, serum, sputum, saliva, sweat, interstitial fluid, cerebral spinal fluid, and dialysate obtained in kidney dialysis.

50. The method claim 47, wherein said biological sample is urine.

51. The method of claim 50, wherein said at least one analyte of interest is selected from the group consisting of urea, creatinine, protein, glucose, and ketones.

52. The method of claim 50, wherein said at least one analyte of interest is selected from the group consisting of hemoglobin and bilirubin.

53. The method of claim 47, wherein said spectroscopic analysis is selected from the group consisting of ultraviolet absorption spectroscopy, visible absorption spectroscopy, infrared absorption spectroscopy, ultraviolet scattering spectroscopy, visible scattering spectroscopy, infrared scattering spectroscopy, fluorescence spectroscopy, and Raman spectroscopy.

54. The method of claim 47, wherein said spectroscopic analysis is filter photometry.

55. The method of claim 47, wherein said spectroscopic analysis is derivative spectroscopy.

56. The method of claim 47, wherein said spectroscopic analysis utilizes measurement of transmitted light.

57. The method of claim 47, wherein said spectroscopic analysis utilizes measurement of reflected light.

58. The method of claim 47, wherein said mathematical function is a polynomial function.

59. The method of claim 47, wherein said spectral range selected has a width of from about 190 nm to about 40,000 nm.

60. The method of claim 47, wherein said portion has a width of from 1 nm to about 100 nm.

61. The method of claim 47, wherein said mathematical function is selected from the group consisting of polynomial functions, Taylor series functions, Fourier series functions, and Gaussian functions.

62. The method of claim 47, wherein randomness is inspected by means of an auto-correlation function.

63. The method of claim 47, wherein said mathematical function is fitted by a least squares technique.

64. The method of claim 47, wherein said mathematical function is fitted by a sum and difference method.

65. The method of claim 47, wherein the width of the spectral portions can be varied from spectral region to spectral region across the spectrum.

66. The method of claim 47, wherein the order of the polynomial function can be varied from spectral region to spectral region across the spectrum.

67. The method of claim 47, further including the step of employing at least one measurement selected from the following additional measurements:

light scattering, impedance, pH, ion, temperature, and refractive index.

68. A container for containing at least one reagent and for conducting an assay with said at least one reagent comprising:

(a) a receptacle wherein a chemical reaction can take place; and

(b) a cap for said receptacle, said cap containing said at least one reagent, said cap capable of sealing said receptacle after a biological sample has been introduced into said receptacle, said cap separating said receptacle from said at least one reagent after said receptacle has been sealed by said cap, wherein said at least one reagent is capable of being released into said biological sample in said receptacle by means of rupturing said sealed cap.

69. The container of claim 68, wherein said at least one reagent is capable of performing colorimetric reactions for materials that do not have a significant spectral signature.

70. The container of claim 68, wherein said at least one reagent is a liquid.

71. The container of claim 68, wherein said at least one reagent is a solid.