Practical method and apparatus for analyte detection with colloidal particles

• US 6,770,488 B1
• Filed: 03/16/2000
• Issued: 08/03/2004
• Est. Priority Date: 03/19/1999
• Status: Expired due to Term

• Current Assignee: University of Wyoming
• Original Assignee: University of Wyoming
• Inventors: Corcoran, Robert C., Carron, Keith T., Sulk, Roberta Ann
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US09/527,226
• Time in Patent Office: 1,601 Days
• Field of Search: 435/7.1, 435/968, 435/973, 436/164, 436/166, 436/172, 436/518, 436/523, 436/524, 436/525, 436/536, 436/539, 436/540, 436/541, 436/805, 356/301, 356/317, 356/318, 356/337
• US Class Current: 436/525
• CPC Class Codes: G01N 2021/651 Cuvettes therefore
• CPC Class Codes: G01N 21/658 enhancement Raman, e.g. surface plasmons
• CPC Class Codes: G01N 2201/101 Scanning measuring head
• CPC Class Codes: G01N 2201/102 Video camera
• CPC Class Codes: G01N 2201/129 Using chemometrical methods
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/805 Optical property

Abstract

A colloidal system for detection of a variety of analytes involves techniques which permit reconstitution of a desiccated substance such as for surface enhanced Raman spectroscopic analysis and multiple sensors at once, each having different spectra through the use of markers or the like. Competitive assay techniques and a variety of substances are explained to permit a practical an versatile system which can also be used for immunological assays and can include antibodies tagged to provide spectroscopic indicia.

Claims

1. A method of analyte detection, comprising the steps of:

a. establishing a colloidal substance;

b. providing said colloidal substance in a liquid;

c. desiccating said colloidal substance in said liquid to establish a residue of said colloidal substance;

d. reconstituting said residue of said colloidal substance to generate a colloidal sensor solution containing said colloidal substance;

e. interacting an analyte with a portion of said colloidal substance contained in said colloidal sensor solution;

f. subjecting said colloidal sensor solution to an illumination;

g. affecting said illumination with said portion of said colloidal substance contained in said colloidal sensor solution interacting with said analyte to generate an affected emission; and

h. spectroscopically analyzing said affected emission to detect said analyte.

2. A method of analyte detection as described in claim 1 wherein said step of establishing a colloidal substance comprises the step of establishing a surface enhanced Raman spectroscopic colloidal substance, and wherein said step of affecting said illumination with said portion of said colloidal substance contained in said colloidal sensor solution interacting with said analyte to generate an affected emission comprises the step of generating a Raman spectral signal, and wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the step of analyzing a surface enhanced Raman spectrum.

3. A method of analyte detection as described in claim 2 wherein said step of establishing a surface enhanced Raman spectroscopic substance comprises the step of establishing a surface enhanced Raman spectroscopic substance selected from a group consisting of silver colloids, gold colloids, colloids formed from the chemical reduction of silver salts, colloids formed from the chemical reduction of gold salts, metal colloids, colloids formed from the chemical reduction of metal salts, colloids formed from the electrochemical reduction of metal salts, colloids formed from the electrochemical reduction of gold salts, colloids formed from the electrochemical reduction of silver salts, colloids formed from the ablation of metals, colloids formed from the ablation of silver, and colloids formed from the ablation of gold.

4. A method of analyte detection as described in claim 1 wherein said step of affecting said illumination with said portion of said colloidal substance contained in said colloidal sensor solution interacting with said analyte to generate an affected emission comprises the step of generating a fluorescence spectral signal, and wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the step of analyzing said fluorescence spectral signal.

5. A method of analyte detection as described in claim 4 wherein said step of generating a fluorescence spectral signal comprises the step of generating a surface enhanced fluorescence spectral signal.

6. A method of analyte detection as described in claim 1 further comprising the step of coating said colloidal substance with an analyte responsive coating to generate a coated colloidal substance.

7. A method of analyte detection as described in claim 6 wherein said step of coating a portion of said colloidal substance with an analyte responsive coating to generate a coated colloidal substance comprises the step of coating a portion of said colloidal substance with a first analyte responsive coating.

8. A method of analyte detection as described in claim 7 wherein said step of coating a portion of said colloidal substance with an analyte responsive coating to generate a coated colloidal substance comprises the step of coating a second portion of said colloidal substance with a second analyte responsive coating.

9. A method of analyte detection as described in claim 8 wherein said step of reconstituting said residue of said colloidal substance to generate a colloidal sensor solution comprises the step of reconstituting said residue of said coated colloidal substance to generate a colloidal sensor solution containing said coated colloidal substance having said first analyte responsive coating and having said second analyte responsive coating, and wherein said step of interacting an analyte with a portion of said colloidal substance contained in said colloidal sensor solution comprises the step of interacting a first analyte with said first analyte responsive coating.

10. A method of analyte detection as described in claim 9 wherein said step of desiccating said colloidal substance in said liquid to establish a residue of said colloidal substance comprises the step of desiccating said coated colloidal substance having said first analyte responsive coating and said second analyte responsive coating.

11. A method of analyte detection as described in claim 9 wherein said step of interacting an analyte with a portion of said colloidal substance contained in said colloidal sensor solution further comprises the step of interacting a second analyte with said second analyte responsive coating.

12. A method of analyte detection as described in claim 11 wherein said step of interacting a second analyte with said second analyte responsive coating occurs substantially simultaneously with said step of interacting a first analyte with a first analyte responsive coating.

13. A method of analyte detection as described in claim 12 wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the step of spectroscopically analyzing said affected emission to detect said first analyte.

14. A method of analyte detection as described in claim 13 wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the step of spectroscopically analyzing said affected emission to detect said second analyte.

15. A method of analyte detection as described in claim 6 further comprising the step of selectively interacting an illumination affecting substance with said analyte as said analyte interacts with said portion of said coated colloidal substance contained in said colloidal sensor solution, and wherein said step of affecting said illumination with said portion of said coated colloidal substance contained in said colloidal sensor solution interacting with said analyte to generate an affected emission comprises the step of altering said affected emission by the presence of said illumination affecting substance interacting with said analyte.

16. A method of analyte detection as described in claim 1 further comprising the step of coating said colloidal substance with an analyte-responsive coating to create a coated sensor colloid.

17. A method of analyte detection as described in claim 15 wherein said step of coating said colloidal substance with an analyte responsive coating to generate a coated colloidal substance comprises the step of coating a portion of said colloidal substance with a first analyte responsive coating.

18. A method of analyte detection as described in claim 17 wherein said step of coating said colloidal substance with an analyte responsive coating to generate a coated colloidal substance further comprises the step of coating a second portion of said colloidal substance with a second analyte responsive coating.

19. A method of analyte detection as described in claim 18 wherein said step of interacting an analyte with a portion of said analyte responsive colloidal substance contained in said colloidal sensor solution comprises the step of interacting a first analyte with said first analyte responsive coating, and wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the step of spectroscopically analyzing said affected emission to detect said first analyte.

20. A method of analyte detection as described in claim 19 wherein said step of interacting an analyte with a portion of said analyte responsive colloidal substance contained in said colloidal sensor solution comprises the step of interacting a second analyte with said second analyte responsive coating and wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the steps of:

i. spectroscopically analyzing said affected emission to detect said first analyte; and

ii. spectroscopically analyzing said affected emission to detect said second analyte.

21. A method of analyte detection as described in claim 20 wherein said step of interacting a second analyte with said second analyte reactive coating occurs substantially simultaneously with said step of interacting said first analyte with said first analyte reactive coating.

22. A method of analyte detection as described in claim 21 wherein said step of spectroscopically analyzing said affected emission to detect said first analyte and said step of spectroscopically analyzing said affected emission to detect said second analyte occur substantially simultaneously.

23. A method of analyte detection as described in claim 22 wherein said step of spectroscopically analyzing said affected emission to detect said first analyte and said step of spectroscopically analyzing said affected emission to detect said second analyte comprise the step of analyzing a single spectrum to detect both said first analyte and said second analyte.

24. A method of analyte detection as described in claim 23 wherein said step of coating said colloidal substance with an analyte responsive coating to generate a coated colloidal substance comprises the step of establishing a surface enhanced Raman spectroscopic substance, and wherein said step of affecting said illumination with said portion of said coated colloidal substance contained in said colloidal sensor solution interacting with said analyte to generate an affected emission comprises the step of generating a surface enhanced Raman spectral signal, and wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the step of analyzing a surface enhanced Raman spectrum.

25. A method of analyte detection as described in claim 6 wherein said analyte comprises an analyte analog and wherein said step of affecting said illumination with said portion of said coated colloidal substance contained in said colloidal sensor solution interacting with analyte comprises the step of affecting said illumination with said portion of said coated colloidal substance contained in said colloidal sensor solution interacting with said analyte analog.

26. A method of analyte detection as described in claim 6 wherein said step of interacting an analyte with a portion of said coated colloidal substance in said colloidal sensor solution comprises the step of interacting said analyte and interacting an analyte analog with said coated colloidal substance in said sensor colloid solution substantially simultaneously and wherein said step of affecting said illumination with said portion of said coated colloidal substance contained-in said colloidal sensor solution interacting with said analyte to generate an affected emission comprises the step of affecting said illumination with said analyte responsive colloidal substance contained in said colloidal sensor solution interacting with said analyte analog and said analyte.

27. A method of analyte detection as described in claim 6 wherein said step of interacting an analyte with a portion of said coated colloidal substance contained in said colloidal sensor solution comprises the step of interacting said analyte with said portion of said coated colloidal substance contained in said sensor colloidal solution prior to interaction of an analyte analog with said coated colloidal substance contained in said sensor colloid solution and wherein said step of affecting said illumination with said portion of said coated colloidal substance contained in said colloidal sensor solution interacting with said analyte to generate an affected emission comprises the step of affecting said illumination with said analyte responsive colloidal substance contained in said colloidal sensor solution interacting with said analyte and said analyte analog.

28. A method of analyte detection as described in claim 6 wherein said step of interacting an analyte with a portion of said coated colloidal substance contained in said sensor colloidal solution comprises the step of interacting said analyte with said portion of said coated colloidal substance contained in said sensor colloidal solution after interaction of said analyte analog with said portion of said coated colloidal substance contained in said sensor colloid solution and wherein said step of affecting said illumination with said portion of said coated colloidal substance contained in said colloidal sensor solution interacting with said analyte to generate an affected emission comprises the step of affecting said illumination with said analyte responsive colloidal substance contained in said colloidal sensor solution interacting with said analyte and said analyte analog.

29. A method of analyte detection as described in claim 26, 27 or 28 wherein said step of interacting an analyte analog with a portion of said coated colloidal substance contained in said colloidal sensor solution generates an intermediate solution, and further comprising the steps of:

a. subjecting said intermediate solution to an illumination;

b. affecting said illumination with said portion of said colloidal substance contained in said intermediate solution interacting with said analyte and said analyte analog to generate an intermediate emission; and

c. spectroscopically analyzing said intermediate emission, and wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the step of comparing said affected emission to said intermediate emission.

30. A method of analyte detection as described in claim 6 further comprising the step of interacting an analyte analog with a portion of said coated colloidal substance in said liquid prior to said step of desiccating said coated colloidal substance in said liquid to establish a residue of said colloidal substance.

31. A method of analyte detection as described in claim 30 wherein said step of interacting an analyte with a portion of said coated colloidal substance contained in said colloidal sensor solution generates an intermediate solution, and further comprising the steps of:

a. subjecting said intermediate solution to an illumination;

b. affecting said illumination with said analyte interacting with said portion of said coated colloidal substance within said intermediate solution to generate an intermediate emission; and

c. spectroscopically analyzing said intermediate emission, and wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the step of comparing said affected emission to said intermediate emission.

32. A method of analyte detection as described in claim 28 wherein said step of interacting said analyte with said portion of said coated colloidal substance contained in said sensor colloidal solution after interaction of said analyte analog with said portion of said coated colloidal substance contained in said sensor colloid solution comprises the steps of:

a. binding said analyte analog with said coated colloidal substance contained in said colloidal sensor solution;

b. displacing said analyte analog from said coated colloidal substance with said analyte; and

c. replacing said analyte analog with said analyte on said coated colloidal substance.

33. A method of analyte detection as described in claim 25, 26, 27, 28, or 30 wherein said analyte analog comprises a tagged analyte analog.

34. A method of analyte detection as described in claim 33 wherein said step of binding a tagged analyte analog with at least some of said sensor colloid complex comprises the step of binding a tagged analyte with at least some of said sensor colloid complex.

35. A method of analyte detection as described in claim 28 wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the step of analyzing to observe a decrease in said affected emission.

36. A method of analyte detection as described in claim 6 wherein said step of coating a portion of said colloidal substance with an analyte responsive coating to generate an coated colloidal substance comprises the steps of:

i. coating a first portion of said colloidal substance with a first analyte responsive substance to generate a first coated colloidal substance having a first analyte responsive coating, and

ii. coating a second portion of said colloidal substance with a second analyte responsive substance to generate a second coated colloidal substance having a second analyte responsive coating, wherein said step of reconstituting said residue of said coated colloidal substance to generate a colloidal sensor solution containing said coated colloidal substance comprises the step of reconstituting a residue mixture of said first coated colloidal substance having said first analyte responsive coating and said second coated colloidal substance having said second analyte responsive coating to generate a colloidal sensor solution containing said first coated colloidal substance and said second coated colloidal substance, and wherein said step of interacting an analyte with a portion of said analyte responsive colloidal substance contained in said colloidal sensor solution comprises the step of interacting a first analyte with said first coated colloidal substance contained in said colloidal sensor solution.

37. A method of analyte detection as described in claim 36 and further comprising the step of mixing said first sensor colloid complex and said second sensor colloid complex together.

38. A method of analyte detection as described in claim 36 and wherein said step of interacting an analyte with a portion of said coated colloidal substance contained in said colloidal sensor solution further comprises the step of interacting a second analyte with said second coated colloidal substance contained in said colloidal sensor solution, and wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the steps of:

i. spectroscopically analyzing said affected emission to detect said first analyte; and

ii. spectroscopically analyzing said affected emission to detect said second analyte.

39. A method of analyte detection as described in claim 38 wherein said step of interacting a second analyte with said second coated colloidal substance contained in said colloidal sensor solution occurs substantially simultaneously with said step of interacting a first analyte with said first coated colloidal substance contained in said colloidal sensor solution.

40. A method of analyte detection as described in claim 39 wherein said step of spectroscopically analyzing said affected emission to detect said first analyte and said step of spectroscopically analyzing said affected emission to detect said second analyte occur substantially simultaneously.

41. A method of analyte detection as described in claim 40 wherein said step of spectroscopically analyzing said affected emission to detect said first analyte and said step of spectroscopically analyzing said affected emission to detect said second analyte comprise the step of analyzing a single spectra to detect both said first analyte and said second analyte.

42. A method of analyte detection as described in claim 41 wherein said step of establishing a coated colloidal substance responsive to at least one analyte comprises the step of establishing a surface enhanced Raman spectroscopic substance, wherein said step of affecting said illumination with said portion of said coated colloidal substance contained in said sensor colloidal solution interacting with said analyte comprises the step of producing a Raman spectral signal, and wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the step of analyzing a surface enhanced Raman spectrum.

43. A method of analyte detection as described in claim 6 further comprising the step of providing a spectroscopic marker retained by said coated colloidal substance.

44. A method of analyte detection as described in claim 6 further comprising the step of providing a spectroscopic marker retained by said analyte.

45. A method of analyte detection as described in claim 6 further comprising the step of providing a spectroscopic marker retained by said analyte analog.

46. A method of analyte detection as described in claim 43 wherein said step of providing a spectroscopic marker retained by said coated colloidal substance further comprises the step of introducing said spectroscopic marker during said step of reconstituting said residue of said coated colloidal substance to generate a colloidal sensor solution.

47. A method of analyte detection as described in claim 43, 44, 45, or 46 wherein said spectroscopic marker comprises a dye.

48. A method of analyte detection as described in claim 47 wherein said step of providing a spectroscopic marker retained by said analyte comprises the steps of:

i. providing a first dye retained by a first analyte; and

ii. providing a second dye retained by a second analyte, and wherein said first dye and said second dye affect said illumination differently.

49. A method of analyte detection as described in claim 1 or 6 further comprising the step of including a reconstitution substance with said colloidal substance when desiccating said colloidal substance to establish a residue of said colloidal substance.

50. A method of analyte detection as described in claim 6 further comprising the step of including a reconstitution substance with said colloidal substance when desiccating said colloidal substance to establish a residue of said colloidal substance.

51. A method of analyte detection as described in claim 49 further comprising the step of coordinating a container with said colloidal substance prior to desiccation to permit reconstitution of said residue of said analyte responsive colloidal substance in said container.

52. A method of analyte detection as described in claim 50 further comprising the step of coordinating a container with said coated colloidal substance prior to desiccation to permit reconstitution of said residue of said analyte responsive colloidal substance in said container.

53. A method of analyte detection as described in claim 15 wherein said step of selectively interacting an illumination affecting substance with said analyte as said analyte interacts with said coated colloidal substance contained in said colloidal sensor solution comprises binding a reagent to said analyte while said analyte interacts with said coated colloidal substance contained in said colloidal sensor solution.

54. A method of analyte detection as described in claim 6 wherein said step of coating said colloidal substance with an analyte responsive coating to generate an coated colloidal substance comprises a step selected from the group consisting of binding an antibody to said colloidal substance, binding polyclonal antibodies to said colloidal substance, binding monoclonal antibodies to said colloidal substance, binding polyclonal antibodies to a first antigen and binding polyclonal antibodies to a second antigen to said colloidal substance, binding monoclonal antibodies to a first antigen and binding polyclonal antibodies to a second antigent to said colloidal substance, and binding at least one reagent to said colloidal substance.

55. A method of analyte detection as described in claim 54 wherein said step of coating said colloidal substance with an analyte responsive coating to generate an coated colloidal substance comprises the steps of:

i. coating a portion of said colloidal substance with a first analyte responsive coating to a first analyte responsive colloidal substance having a first analyte responsive coating, and

ii. coating another portion of said colloidal substance with a second analyte responsive coating to generate a second analyte responsive colloidal substance having a second analyte responsive coating, and wherein said step of reconstituting said residue of said coated colloidal substance to generate a colloidal sensor solution containing said coated colloidal substance comprises the step of reconstituting said residue of said coated colloidal substance to generate a colloidal sensor solution containing said first coated colloidal substance and said second coated colloidal substance, and wherein said step of interacting an analyte with said coated colloidal substance contained in said colloidal sensor solution comprises the step of interacting a first analyte with said first analyte responsive colloidal substance contained in said colloidal sensor solution.

56. A method of analyte detection as described in claim 6 wherein said step of coating said colloidal substance with an analyte responsive coating to generate an coated colloidal substance comprises the step of coating said colloidal substance with an analyte responsive coating selected from a group consisting of a virus binding substance, a bacteria binding substance, a fungal spore binding substance, an anthrax binding substance, a coating reactive to heroin, a coating reactive to THC, a coating reactive to morphine, a coating reactive to codeine, a coating reactive to mescaline, a coating reactive to alcohol, a coating reactive to amphetamine, a coating reactive to methamphetamine, a coating reactive to polychlorinated biphenyl (PCB), a coating reactive to phenolic compound, a coating reactive to furan, a coating reactive to pyrrole, a coating reactive to a substance in blood, a coating reactive to a substance in urine, a coating reactive to reactive to bilirubin, a coating reactive to salicylic compound, a coating reactive to neurotransmitter, a coating reactive to human growth hormone, a coating reactive to thyroid stimulating hormone, antibodies to a hormone, antibodies to a protein, antibodies to a bacteria, antibodies to a virus, antibodies to a prion, antibodies to human growth hormone, antibodies to thyroid stimulating hormone, antibodies to prostate specific antigen, antibodies to h. Pylori, antibodies to giardia, antibodies to a prion protein, antibodies to trinitrotoluene, and antibodies to polychlorinated biphenyls.

57. A method of analyte detection as described in claim 15 wherein said step of selectively interacting an illumination affecting substance with said analyte as said analyte interacts with said coated colloidal substance contained in said colloidal sensor solution comprises the step of selectively interacting an illumination affecting substance selected from the group consisting of a spectroscopically marked antibody, spectroscopically marked polyclonal antibodies, spectroscopically marked monoclonal antibodies, spectroscopically marked antibodies to a hormone, spectroscopically marked antibodies to a protein, spectroscopically marked antibodies to a bacteria, spectroscopically marked antibodies to a virus, spectroscopically marked antibodies to a prion, spectroscopically marked antibodies to human growth hormone, spectroscopically marked antibodies to thyroid stimulating hormone, spectroscopically marked antibodies to prostate specific antigen, spectroscopically marked antibodies to h. Pylori, spectroscopically marked antibodies to giardia, spectroscopically marked antibodies to a prion protein, spectroscopically marked antibodies to trinitrotoluene, and spectroscopically marked antibodies to polychlorinated biphenyls.

58. A method of analyte detection as described in claim 26, 27, 28, or 30 wherein said analyte analog is selected from a group consisting of a morphine analog, a morphine analog with a phthalocyanine dye, a methamphetamine analog, amphetamine analog with an azo dye, tetrahydrocannibinol analog, tetrahydrocannibinol with an aminotriarylmethane dye, a 2,4-dichlorophenoxyacetic acid analog, a 2,4-dichlorophenoxyacetic acid analog with an azo dye, a carbofuran analog, and a carbofuran analog with an aminotriarylmethane dye.

59. A method of analyte detection as described in claim 43 wherein said step of providing a spectroscopic marker retained by said coated colloidal substance comprises the step of providing a spectroscopic marker selected from the group consisting of a SERS marker, a phthalocyanine, a aminotriarylmethane, an oxazine, an anthraquinone, a xanthene, an indigoid, a carbocyanine, an azine, a thiazine, a coumarin, and an azo dye.

60. A method of analyte detection as described in claim 49 wherein said step of including a reconstitution substance with said colloidal substance when desiccating said colloidal substance to establish a residue of said colloidal substance comprises the step of including a reconstitution substance selected from the group consisting of a hydrocarbon, a paraffin, an organosilicon material, an oxygenated derivative of an organosilicon material, a fluorocarbon, a teflon material, a polyether, an oxygenated analog of a hydrocarbon, a perfluorocarbon, a hydrophobic substance, a halogenated hydrocarbon, an agent having a similar charge to that on the analyte responsive colloidal substance, a polyether, polyethylene glycol, a salt of inorganic derivatives of sulphuric acid, a salt of organic derivatives of sulphuric acid, a sulphonic acid, a phosphoric acid, a phosphonic acid, a carboxylic acid, a substance containing a free acid of a sulphate monoester, a substance containing a free acid of a sulfonic acid, a substance containing a free acid of a phosophate monoester, a substance containing a free acid of a phosophate diester, a substance containing a free acid of a phosphonic acid, a substance containing a free acid of a phosophate monoester, a substance containing a free acid of a carboxylic acid, a substance containing a salt of a sulphate monoester, a substance containing a salt of a sulfonic acid, a substance containing a salt of a phosophate monoester, a substance containing a salt of a phosophate diester, a substance containing a salt of a phosphonic acid, a substance containing a salt of a phosophate monoester, and a substance containing a salt of a carboxylic acid.

61. A method of analyte detection as described in claim 50 wherein said step of including a reconstitution substance with said coated colloidal substance when desiccating said colloidal substance to establish a residue of said coated colloidal substance comprises the step of including a reconstitution substance selected from the group consisting of a hydrocarbon, a paraffin, an organosilicon material, an oxygenated derivative of an organosilicon material, a fluorocarbon, a teflon material, a polyether, an oxygenated analog of a hydrocarbon, a perfluorocarbon, a hydrophobic substance, a halogenated hydrocarbon, an agent having a similar charge to that on the analyte responsive colloidal substance, a polyether, polyethylene glycol, a salt of inorganic derivatives of sulphuric acid, a salt of organic derivatives of sulphuric acid, a sulphonic acid, a phosphoric acid, a phosphonic acid, a carboxylic acid, a substance containing a free acid of a sulphate monoester, a substance containing a free acid of a sulfonic acid, a substance containing a free acid of a phosophate monoester, a substance containing a free acid of a phosophate diester, a substance containing a free acid of a phosphonic acid, a substance containing a free acid of a phosophate monoester, a substance containing a free acid of a carboxylic acid, a substance containing a salt of a sulphate monoester, a substance containing a salt of a sulfonic acid, a substance containing a salt of a phosophate monoester, a substance containing a salt of a phosophate diester, a substance containing a salt of a phosphonic acid, a substance containing a salt of a phosophate monoester, and a substance containing a salt of a carboxylic acid.

62. A method of analyte detection as described in claim 14 wherein said step of spectroscopically analyzing said affected emission to detect said analyte comprises the step of spectroscopically analyzing said affected emission to detect said first analyte and to detect said second analyte simultaneously from a single spectrum.

63. A method of analyte detection as described in claim 44 further comprising the step of displacing said spectroscopic marker retained by said analyte.

64. A method of analyte detection as described in claim 45, further comprising the step of displacing said spectroscopic marker retained by said analyte analog.

65. A method of analyte detection as described in claim 63, wherein said step of displacing said spectroscopic marker retained by said analyte responsive colloidal substance decreases said affected emission.

66. A method of analyte detection as described in claim 64, wherein said step of displacing said spectroscopic marker retained by said analyte decreases said affected illumination.