Advanced surface-enhanced Raman gene probe systems and methods thereof

Claims

What is claimed is:

1. A method for using a Surface-Enhanced Raman (SER)-labeled Gene Probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide material comprising the steps of:

a) providing a support means;

b) preparing a solution of gene probe oligonucleotide strands of known sequence, said oligonucleotide strands being complementary to a target oligonucleotide strand;

c) disposing said gene probe oligonucleotide strands onto said support means;

d) incubating said support means having said disposed gene probe oligonucleotide strands for a period of time sufficient enough to immobilize said oligonucleotide strands on said support means;

e) synthesizing at least one SER-labeled oligonucleotide strand of unknown sequence taken from a target sample being suspect of containing target oligonucleotide strands, said SER-labeled oligonucleotide strand having at least one SER label;

f) preparing a SER-labeled oligonucleotide solution comprising at least one SER-labeled oligonucleotide strand wherein said SER label is unique for said target oligonucleotide strand of a particular sequence;

g) incubating said support means having said immobilized gene probe oligonucleotide strands thereon in an amount of said SER-labeled oligonucleotide solution sufficient enough to provide an amount of said SER-labeled target oligonucleotide strands that are complementary to said immobilized gene probe oligonucleotide strands to contact said immobilized gene probe oligonucleotide strands, incubating for a time period sufficient as for said SER-labeled target oligonucleotide strands to contact said immobilized gene probe oligonucleotide strands and sufficient enough as for hybridization to occur, thereby producing SER-labeled hybridized target oligonucleotide material;

h) removing said SER-labeled oligonucleotide strands that did not hybridize to said immobilized gene probe oligonucleotide strands;

i) activating said support means with a SER activating means, said support means having SER-labeled hybridized target oligonucleotide material; and

j) analyzing said SER-activated support means having SER-labeled hybridized target oligonucleotide material.

2. The method of claim 1 wherein said oligonucleotide strands comprise strands of Deoxyribonucleic acid, Ribonucleic acid or peptide nucleic acid.

3. The method of claim 1 wherein said support means is a substrate suitable for hybridization.

4. The method of claim 1 wherein said support means is a membrane suitable for hybridization.

5. The method of claim 1 wherein said support means is a blotting material.

6. The method of claim 1 wherein said support means is a polymer-based nanosphere.

7. The method of claim 1 wherein said support means is an alumina-based nanoparticle.

8. The method of claim 1 wherein said support means is a titanium oxide nanoparticle.

9. The method of claim 1 wherein said support means has a surface comprising cellulose, silica gel or polystyrene.

10. The method of claim 1 wherein said SER activating means comprises the step of coating said support means with metal-coated magnetic nanobeads.

11. The method of claim 1 wherein said SER activating means comprises inducing an electrochemical reaction to roughen the surface of said support means on which said hybridization occurs.

12. The method of claim 1 wherein said SER activating means comprises blotting said SER-labeled hybridized target oligonucleotide material directly onto a SER active support means using a blotting technique.

13. The method of claim 12, wherein said blotting technique comprises applying an electric field to adsorb said SER-labeled hybridized target oligonucleotide material onto a SERS active blot.

14. The method of claim 1 wherein said support means is disposed on a fiberoptic probe having a probe tip which supports said SER-activated support means, said fiberoptic probe having at least one optical fiber for transmitting exciting optical energy from an energy source to said SER-labeled gene probe on said SER-activated support means on said fiberoptic probe tip to generate a Raman optical signal and for collecting and transmitting said Raman optical signal to a signal analyzer.

15. The method of claim 1 wherein multiple SER-activated support means are disposed on an array of optical fibers for performing multiple assays, each said optical fiber having a fiberoptic tip which supports said SER-activated support means, said support means having uniquely labeled SER-labeled hybridized target oligonucleotide material wherein said SERS label is unique for a target oligonucleotide complementary to said gene probe oligonucleotide, said array further having an energy source for generating exciting optical energy and means for directing said exciting optical energy onto said optical fibers, and said optical fibers transmitting said exciting optical energy to said SER gene probe on said SER-activated support means on said fiberoptic probe tip to generate a Raman optical signal, said array further having means for collecting and transmitting said Raman optical signal to an array of signal analyzers.

16. The method of claim 1 wherein said SER-activated support means is disposed on a waveguide having a surface which supports said SER-activated support means, said waveguide further having means for transmitting exciting optical energy from an energy source to said surface and to said SER-labeled gene probe to generate a Raman optical signal and means for collecting and transmitting said Raman optical signal to a signal analyzer.

17. The method of claim 1 wherein, multiple SER-activated support means are disposed on a waveguide having a surface which supports said multiple SER-activated support means, each of said SER-activated support means having SER-labeled gene probes labeled uniquely for a particular target oligonucleotide complementary to said gene probe oligonucleotide strand, said waveguide further having means for transmitting exciting optical energy from an energy source to said surface and to said SER-labeled gene probe to generate a Raman optical signal and means for collecting and transmitting said Raman optical signal to a two dimensional charge-coupled device for analysis.

18. A method for using a Surface-Enhanced Raman (SER)-labeled Gene Probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide material comprising the steps of:

a) providing a support means;

b) exposing said support means to a target sample suspected of containing target oligonucleotide strands of unknown sequence;

c) incubating said support means with said target sample for a period of time sufficient enough to immobilize said oligonucleotide strands on said support means;

d) synthesizing SER-labeled gene probes wherein a SER-labeled gene probe comprises at least one oligonucleotide strand of known sequence labeled with at least one SER label unique for said target oligonucleotide strands of a particular sequence, said known oligonucleotide sequence being complementary to said target oligonucleotide strands;

e) preparing a SER-labeled gene probe solution comprising at least one SER-labeled gene probe;

f) incubating said support means having said immobilized oligonucleotide strands thereon in an amount of said SER-labeled gene probe solution sufficient enough to provide an amount of SER-labeled gene probes to contact said immobilized oligonucleotide strands, incubating for a time period sufficient as for said SER-labeled gene probes to contact said immobilized oligonucleotide strands and sufficient enough as for hybridization to occur between said SER-labeled gene probe and said immobilized target oligonucleotide, thereby producing SER-labeled hybridized target oligonucleotide material;

g) removing said oligonucleotide strands that did not hybridize to said immobilized oligonucleotide strands;

h) activating said support means with a SERS activating means, said support means having SER-labeled hybridized target oligonucleotide material; and

i) analyzing said SER-activated support means having SER-labeled hybridized target oligonucleotide material.

19. The method of claim 18 wherein said oligonucleotide strands comprise strands of Deoxyribonucleic acid, Ribonucleic acid or peptide nucleic acid.

20. The method of claim 18 wherein said support means is a substrate suitable for hybridization.

21. The method of claim 18 wherein said support means is a membrane suitable for hybridization.

22. The method of claim 18 wherein said support means is a blotting material.

23. The method of claim 18 wherein said support means is a polymer-based nanosphere.

24. The method of claim 18 wherein said support means is an alumina-based nanoparticle.

25. The method of claim 18 wherein said support means is a titanium oxide nanoparticle.

26. The method of claim 18 wherein said support means has a surface comprising cellulose, silica gel or polystyrene.

27. The method of claim 18 wherein said SER activating means comprises the step of coating said support means with a colloidal solution of metal hydrosols to produce metal nanoparticles.

28. The method of claim 27 wherein said metal is silver or gold.

29. The method of claim 18 wherein said SER activating means comprises the step of coating said support means with metal-coated magnetic nanobeads.

30. The method of claim 18 wherein said SER activating means comprises inducing an electrochemical reaction to roughen the surface of said support means on which said hybridization occurs.

31. The method of claim 18 wherein said SER activating means comprises blotting said SERS-labeled hybridized target oligonucleotide material directly onto a SER active support means using a blotting technique.

32. The method of claim 31 wherein said blotting technique comprises applying an electric field to adsorb said SER-labeled hybridized target oligonucleotide material onto a SERS active blot.

33. A method for using a Surface-Enhanced Raman (SER)-labeled Gene Probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide material comprising the steps of:

a) providing a support means;

b) preparing a solution of gene probe oligonucleotide strands of known sequence, said oligonucleotide strands being complementary to a target oligonucleotide strand;

c) disposing said gene probe oligonucleotide strands onto said support means;

d) incubating said support means having said disposed gene probe oligonucleotide strands for a period of time sufficient enough to immobilize said oligonucleotide strands on said support means;

e) preparing a solution comprising oligonucleotide strands of unknown sequence taken from a target sample containing suspect target oligonucleotide strands, said solution further comprising at least one SER label for a period of time sufficient enough for said target oligonucleotide strands to contact said immobilized gene probe oligonucleotide strands and sufficient enough for hybridization to occur between said immobilized gene probe oligonucleotide strands and said target oligonucleotide strands that are complementary to said immobilized gene probe oligonucleotide strands and sufficient enough as during hybridization, said SER label is intercalated between said hybridized oligonucleotide strands, thereby producing SER-labeled hybridized target oligonucleotide material;

f) removing said oligonucleotide strands that did not hybridize to said immobilized gene probe oligonucleotide strands;

g) activating said support means with a SERS activating means, said support means having SER-labeled hybridized target oligonucleotide material; and

h) analyzing said SERS activated support means having SER-labeled hybridized target oligonucleotide material.

34. The method of claim 33 wherein said oligonucleotide strands comprise strands of Deoxyribonucleic acid, Ribonucleic acid or peptide nucleic acid.

35. The method of claim 33 wherein said support means is a substrate suitable for hybridization.

36. The method of claim 33 wherein said support means is a membrane suitable for hybridization.

37. The method of claim 33 wherein said support means is a blotting material.

38. The method of claim 33 wherein said support means is a polymer-based nanosphere.

39. The method of claim 33 wherein said support means is an alumina-based nanoparticle.

40. The method of claim 33 wherein said support means is a titanium oxide nanoparticle.

41. The method of claim 33 wherein said support means has a surface comprising cellulose, silica gel or polystyrene.

42. The method of claim 33 wherein said SER activating means comprises the step of coating said support means with metal-coated magnetic nanobeads.

43. The method of claim 33 wherein said SER activating means comprises inducing an electrochemical reaction to roughen the surface of said support means on which said hybridization occurs.

44. The method of claim 33 wherein said SER activating means comprises blotting said SERS-labeled hybridized target oligonucleotide material directly onto a SER active support means using a blotting technique.

45. The method of claim 44 wherein said blotting technique comprises applying an electric field to adsorb said SER-labeled hybridized target oligonucleotide material onto a SERS active blot.

46. A method for using a Surface Enhanced Raman (SER)-labeled gene probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide material comprising the steps of:

a) providing a support means;

b) exposing said support means to a target sample suspected of containing target oligonucleotide strands of unknown sequence;

c) incubating said support means with said target sample for a period of time sufficient enough to immobilize said oligonucleotide strands on said support means;

d) preparing a gene probe solution comprising at least one gene probe oligonucleotide strand of known sequence complementary to said target oligonucleotide strands, said solution further comprising at least one SER label in a free state in said solution, said SER label unique for said target oligonucleotide strands of a particular sequence;

e) incubating said support means having said immobilized oligonucleotide strands thereon in an amount of said gene probe solution sufficient enough to provide enough gene probe oligonucleotide strands to contact said immobilized oligonucleotide strands, incubating for a period of time sufficient as for said gene probe oligonucleotide strands to contact said immobilized oligonucleotide strands and sufficient enough as for hybridization to occur between said gene probe oligonucleotide strands and said complementary immobilized target oligonucleotide strands, and sufficient enough as during hybridization, said SER label is intercalated between said hybridized oligonucleotide strands, thereby producing SER-labeled hybridized target oligonucleotide material;

f) removing said oligonucleotide strands that did not hybridize to said immobilized oligonucleotide strands;

g) activating said support means with a SERS activating means, said support means having SER-labeled hybridized target oligonucleotide material; and

h) analyzing said SER-activated support means having SER-labeled hybridized target oligonucleotide material.

47. The method of claim 46 wherein said oligonucleotide strands comprise strands of Deoxyribonucleic acid, Ribonucleic acid or peptide nucleic acid.

48. The method of claim 46 wherein said support means is a substrate suitable for hybridization.

49. The method of claim 46 wherein said support means is a membrane suitable for hybridization.

50. The method of claim 46 wherein said support means is a blotting material.

51. The method of claim 46 wherein said support means is a polymer-based nanosphere.

52. The method of claim 46 wherein said support means is an alumina-based nanoparticle.

53. The method of claim 46 wherein said support means is a titanium oxide nanoparticle.

54. The method of claim 46 wherein said support means has a surface comprising cellulose, silica gel or polystyrene.

55. The method of claim 46 wherein said SER activating means comprises the step of coating said support means with metal-coated magnetic nanobeads.

56. The method of claim 46 wherein said SER activating means comprises inducing an electrochemical reaction to roughen the surface of said support means on which said hybridization occurs.

57. The method of claim 46 wherein said SER activating means comprises blotting said SERS-labeled hybridized target oligonucleotide material directly onto a SER active support means using a blotting technique.

58. The method of claim 57 wherein said blotting technique comprises applying an electric field to adsorb said SER-labeled hybridized target oligonucleotide material onto a SERS active blot.

59. A method for using a Surface Enhanced Raman (SER)-labeled Gene Probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide strands comprising the steps of:

a) providing a support means;

b) synthesizing at least one SER-labeled oligonucleotide strand of unknown sequence taken from a target sample being suspect of containing target oligonucleotide strands, said SER-labeled oligonucleotide strand having at least one SER label;

c) preparing a solution comprising gene probe oligonucleotide strands of known sequence, said oligonucleotide strands being complementary to a target oligonucleotide strand, said solution further comprising at least one SER-labeled oligonucleotide strand wherein said SER label is unique for said target oligonucleotide strand of a particular sequence, and further comprising a SERS activating means;

d) incubating said support means with said solution for a period of time sufficient enough to immobilize said oligonucleotide strands on said support means while simultaneously allowing hybridization between said gene probe oligonucleotide strands of known sequence and said SER-labeled target oligonucleotide strands to occur, thereby producing SER-labeled hybridized target oligonucleotide material, and simultaneously SERS activating said support means with said SERS activating means; and

e) analyzing said SERS active support means having SER-labeled hybridized target oligonucleotide material.

60. The method of claim 59 wherein said oligonucleotide strands comprise strands of Deoxyribonucleic acid, Ribonucleic acid or peptide nucleic acid.

61. The method of claim 59 wherein said support means is a substrate suitable for hybridization.

62. The method of claim 59 wherein said support means is a membrane suitable for hybridization.

63. The method of claim 59 wherein said support means is a blotting material.

64. The method of claim 59 wherein said support means is a polymer-based nanosphere.

65. The method of claim 59 wherein said support means is an alumina-based nanoparticle.

66. The method of claim 59 wherein said support means is a titanium oxide nanoparticle.

67. The method of claim 59 wherein said support means has a surface comprising cellulose, silica gel or polystyrene.

68. The method of claim 59 wherein said SERS activating means comprises metal nanoparticles.

69. The method of claim 68 wherein said metal is silver or gold.

70. The method of claim 59 wherein said SERS activating means comprises metal-coated magnetic nanobeads.

71. A method for using a Surface Enhanced Raman (SER)-labeled Gene Probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide strands comprising the steps of:

a) providing a support means;

b) preparing a solution comprising SER-labeled hybridized target oligonucleotide material comprising at least one SER-labeled oligonucleotide strand of unknown sequence taken from a target sample being suspect of containing target oligonucleotide strands, said SERS label being unique for said target oligonucleotide strand of a particular sequence, and at least one gene probe oligonucleotide strand of known sequence complementary to said target oligonucleotide strand, said gene probe oligonucleotide strand is hybridized with said SER-labeled oligonucleotide strand, said solution further comprising a SERS activating means;

c) incubating said support means with said solution for a period of time sufficient enough to immobilize said SER-labeled hybridized target oligonucleotide material on said support means while simultaneously SERS activating said support means with said SERS activating means; and

d) analyzing said SERS active support means having SER-labeled hybridized target oligonucleotide material.

72. The method of claim 71 wherein said oligonucleotide strands comprise strands of Deoxyribonucleic acid, Ribonucleic acid or peptide nucleic acid.

73. The method of claim 71 wherein said support means is a substrate suitable for hybridization.

74. The method of claim 71 wherein said support means is a membrane suitable for hybridization.

75. The method of claim 71 wherein said support means is a blotting material.

76. The method of claim 71 wherein said support means is a polymer-based nanosphere.

77. The method of claim 71 wherein said support means is an alumina-based nanoparticle.

78. The method of claim 71 wherein said support means is a titanium oxide nanoparticle.

79. The method of claim 71 wherein said support means has a surface comprising cellulose, silica gel or polystyrene.

80. The method of claim 71 wherein said SERS activating means comprises metal nanoparticles.

81. The method of claim 80 wherein said metal is silver or gold.

82. The method of claim 71 wherein said SERS activating means comprises metal-coated magnetic nanobeads.

83. The method of claim 71 wherein said SERS activating means comprises delivering reagents onto said support means to form nanoparticles of metal sol directly on said support means.

84. A method for using a Surface-Enhanced Raman (SER)-labeled Gene Probe for hybridization, detection and identification of hybridized target oligonucleotide strands comprising the steps of:

a) providing a support means;

b) synthesizing SER-labeled gene probe oligonucleotide strands wherein a SER-labeled gene probe comprises at least one gene probe oligonucleotide strand of known sequence with at least one SER label unique for a target oligonucleotide strand of a particular sequence, said known gene probe oligonucleotide sequence being complementary to said target oligonucleotide strand;

c) preparing a solution comprising oligonucleotide strands of unknown sequence taken from a target sample being suspect of containing target oligonucleotide strands, said solution further comprising at least one SER-labeled gene probe oligonucleotide strand and a SERS activating means;

d) incubating said support means with said solution for a period of time sufficient enough to immobilize said oligonucleotide strands on said support means while simultaneously allowing hybridization between said target oligonucleotide strands and said SER-labeled gene probe oligonucleotide strand to occur, thereby producing SER-labeled hybridized target oligonucleotide material, and simultaneously SERS activating said support means with said SERS activating means; and

e) analyzing said SERS-active support means having SER-labeled hybridized target oligonucleotide material.

85. The method of claim 84 wherein said oligonucleotide strands comprise strands of Deoxyribonucleic acid, Ribonucleic acid or peptide nucleic acid.

86. The method of claim 84 wherein said support means is a substrate suitable for hybridization.

87. The method of claim 84 wherein said support means is a membrane suitable for hybridization.

88. The method of claim 84 wherein said support means is a blotting material.

89. The method of claim 84 wherein said support means is a polymer-based nanosphere.

90. The method of claim 84 wherein said support means is an alumina-based nanoparticle.

91. The method of claim 84 wherein said support means is a titanium oxide nanoparticle.

92. The method of claim 84 wherein said support means has a surface comprising cellulose, silica gel or polystyrene.

93. The method of claim 84 wherein said SERS activating means comprises metal nanoparticles.

94. The method of claim 93 wherein said metal is silver or gold.

95. The method of claim 84 wherein said SERS activating means comprises metal-coated magnetic nanobeads.

96. A method for using a Surface Enhanced Raman (SER)-labeled Gene Probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide strands comprising the steps of:

a) providing a support means;

b) synthesizing SER-labeled gene probe oligonucleotide strands comprising at least one gene probe oligonucleotide strand of known sequence with at least one SER label unique for a target oligonucleotide strand of a particular sequence, said known oligonucleotide sequence being complementary to said target oligonucleotide strand;

c) preparing a solution comprising SER-labeled hybridized oligonucleotide target material comprising oligonucleotide strands of unknown sequence taken from a target sample being suspect of containing target oligonucleotide strands, said oligonucleotide strands of unknown sequence hybridized with a SER-labeled gene probe oligonucleotide strand, said solution further comprising a SERS activating means;

d) incubating said support means with said solution for a period of time sufficient enough to immobilize said SER-labeled hybridized target oligonucleotide material on said support means while simultaneously SERS activating said support means with said SERS activating means; and

e) analyzing said SERS active support means having SER-labeled hybridized target oligonucleotide material.

97. The method of claim 96 wherein said oligonucleotide strands comprise strands of Deoxyribonucleic acid, Ribonucleic acid or peptide nucleic acid.

98. The method of claim 96 wherein said support means is a substrate suitable for hybridization.

99. The method of claim 96 wherein said support means is a membrane suitable for hybridization.

100. The method of claim 96 wherein said support means is a blotting material.

101. The method of claim 96 wherein said support means is a polymer-based nanosphere.

102. The method of claim 96 wherein said support means is an alumina-based nanoparticle.

103. The method of claim 96 wherein said support means is a titanium oxide nanoparticle.

104. The method of claim 96 wherein said support means has a surface comprising cellulose, silica gel or polystyrene.

105. The method of claim 96 wherein said SERS activating means comprises metal nanoparticles.

106. The method of claim 96 wherein said metal is silver or gold.

107. The method of claim 96 wherein said SERS activating means comprises metal-coated magnetic nanobeads.

FIELD OF THE INVENTION

The present invention relates to non-radioactive DNA gene probe systems, biosensors and methods for gene identification, particularly to non-radioactive gene probes, biosensors and methods for hybridization, detection and identification of hybridized oligonucleotides based on surface-enhanced Raman scattering (SERS) label detection, and more particularly to advanced SER gene probe systems, biosensors, detection systems and methods for detecting hybridized SER gene probes utilizing substrates and membranes suitable for hybridization or blotting materials.

BACKGROUND OF THE INVENTION

There is currently strong interest in the development of nonradioactive deoxyribonucleic acid (DNA) probes for use in a wide variety of applications, such as gene identification, gene mapping, DNA sequencing, medical diagnostics, and biotechnology. Among the various methods for gene identification, technologies using radioactive labels are currently the most widely used. Radioactive label techniques suffer from several disadvantages however. The principal isotope used, Phosphorus-32, has a limited shelflife because it has a 14-day half-life. Secondly, because there is one principal label for gene probes, DNA can only be probed for one sequence at a time. Due to material limitations, probing immobilized DNA with different .sup.3 P-labeled sequences can only be performed a few (3-4) times. Therefore, the researcher must have idea about the sequence prior to probing. In addition to these inconveniences, the potential safety hazard associated with use of radioactive materials makes the technology undesirable. Shipping, handling and waste disposal of radioactive materials are strictly regulated by federal and state guidelines.

Recently, luminescence labels such as fluorescent or chemiluminescent labels have been developed for gene detection. Although sensitivities achieved by luminescence techniques are adequate, alternative techniques with improved spectral selectivities must be developed to overcome the need for radioactive labels and the poor spectral specificity of luminescent labels.

Spectroscopy is an analytical technique concerned with the measurement of the interaction of radiant energy with matter and with the interpretation of the interaction both at the fundamental level and for practical analysis. Interpretation of the spectra produced by various spectroscopic instrumentation has been used to provide fundamental information on atomic and molecular energy levels, the distribution of species within those levels, the nature of processes involving change from one level to another, molecular geometries, chemical bonding, and interaction of molecules in solution. Comparisons of spectra have provided a basis for the determination of qualitative chemical composition and chemical structure, and for quantitative chemical analysis.

Vibrational spectroscopy is a useful technique for characterizing molecules and for determining their chemical structure. The vibrational spectrum of a molecule, based on the molecular structure of that molecule, is a series of sharp lines which constitutes a unique fingerprint of that specific molecular structure.

One particular spectroscopic technique, known as Raman spectroscopy, utilizes the Raman effect, which is a phenomenon observed in the scattering of light as it passes through a material medium, whereby the light suffers a change in frequency and a random alteration in phase. When exciting optical energy of a single wavelength interacts with a molecule, the optical energy scattered by the molecule contains small amounts of optical energy having wavelengths different from that of the incident exciting optical energy. The wavelengths present in the scattered optical energy are characteristic of the structure of the molecule, and the intensity of this optical energy is dependent on the concentration of these molecules.

Raman spectroscopy is a spectrochemical technique that is complementary to infrared spectroscopy, and has been an important analytical tool due to its excellent specificity for chemical group identification. Raman spectroscopy provides a means for obtaining similar molecular vibrational spectra over optical fibers using visible or near infrared light that is transmitted by the optical fibers without significant absorption losses. In Raman spectroscopy, monochromatic light is directed to a sample and the spectrum of the light scattered from the sample is determined.

Raman spectroscopy is a useful tool for chemical analysis due to its excellent capability of chemical group identification. One limitation of conventional Raman spectroscopy is its low sensitivity, often requiring the use of powerful and costly laser sources for excitation. However, a renewed interest has recently developed among Raman spectroscopists as a result of observation that Raman scattering efficiency can be enhanced by factors of up to 108 or more when a compound is adsorbed on or near special metal surfaces. Spectacular enhancement factors due to the microstructured metal surface scattering process is responsible for increasing the intrinsically weak normal Raman scattering (NRS). The technique associated with this phenomenon is known as surface-enhanced Raman scattering (SERS) effect which can increase the Raman signal as well as the resonance Raman signal significantly. When the laser excitation wavelength occurs in the ultraviolet absorption band, the Raman signal of the analyte is enhanced and often called resonance Raman scattering (RRS) signal. The Raman enhancement process is believed to result from a combination of several electromagnetic and chemical effects between the molecule and the metal surface.

Deoxyribonucleic acid is the main carrier of genetic information in most living organisms. DNA is essentially a complex molecule built up of deoxyribonucleotide repeating units. Each unit comprises a sugar, phosphate, and a purine or pyrimidine base. The deoxyribonucleotide units are linked together by the phosphate groups, joining the 3' position of one sugar to the 5' position of the next. The alternate sugar and phosphate residues form the backbone of the molecule, and the purine and pyrimidine bases are attached to the backbone via the 1' position of the deoxyribose. This sugar-phosphate backbone is the same in all DNA molecules. What gives each DNA its individuality is the sequence of the purine and pyrimidine bases. Peptide nucleic acid (PNA) is a DNA analog that combines sequence specific binding to genetic targets with biostability and synthetic versatility (M. Ericson, Nucleosides and Nucleotides, 16 (1997), p. 617). Molecular probes (DNA, RNA and PNA) having a SERS label provide an excellent combination of detection sensitivity and spectral selectivity, important for many bioassays.

OBJECTS OF THE INVENTION

It is an object of the subject invention to provide a SER gene probe detection system for the detection and identification of biotargets such as Deoxyribonucleic acid (DNA), Ribonucleic acid (RNA) and Peptide nucleic acid (PNA) for the detection of bacteria and viruses and for medical diagnostics.

It is another object of the invention to provide methods for using a SER gene probe for hybridization, detection and identification of hybridized target oligonucleotides such as DNA, RNA and PNA related to bacteria, viruses and genetic material in a variety of samples such as environmental, biological samples or clinical samples.

It is still yet another object of the invention to provide methods for using a SER gene probe for hybridization, detection and identification of hybridized target oligonucleotides such as DNA, RNA and PNA related to bacteria, viruses and genetic material in a variety of samples using substrates suitable for hybridization, membranes and blotting materials.

It is a further object of the invention to provide methods for using a SER gene probe for use in diagnostics of genetic disease, DNA polymorphism, drug screening and drug discovery.

It is still a further object of the invention to provide methods for using a SER gene probe for DNA mapping and sequencing using yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and P1 artificial chromosome (PAC) methods.

It is another object of the invention to provide a method for SER activating a support means such as a substrate or a membrane suitable for hybridization or a blotting material after hybridization has occurred.

It is yet another object of the invention to provide a method for SER activating a support means such as a substrate or a membrane suitable for hybridization or a blotting material simultaneously with the hybridization step.

Further and other objects of the present invention will become apparent from the description contained herein.

SUMMARY

The subject invention is a series of methods and systems using a SER gene probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide material. The SER-labeled gene probe is used for the detection and identification of biotargets, oligonucleotide material, such as DNA, RNA and PNA related to bacteria, viruses and genetic material in a variety of samples such as environmental, biological samples or clinical samples.

In acccordance with one object of the invention, a method for using a SER-labeled gene probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide material comprising the steps of disposing a gene probe oligonucleotide strand, being complementary to a target oligonucleotide strand, onto a support means and incubating for a period of time sufficient enough to immobilize the gene probe on the support means; then incubating the support means in an amount of solution comprising at least one SER-labeled oligonucleotide strand of unknown sequence taken from a target sample being suspect of containing target oligonucleotide strands, with the SER-labeled oligonucleotide strand having at least one SER label, incubated for a time sufficient as for the SER-labeled target oligonucleotide strands to hybridize with the immobilized gene probe oligonucleotide strands to produce SER-labeled hybridized target oligonucleotide material; then removing the SER-labeled oligonucleotide