1. A nanoparticle probe for detecting for the presence of a target analyte in a sample, wherein the target analyte has at least two binding sites, the probe comprising a nanoparticle having bound thereto:
(i) a first member of a first specific binding pair;
(ii) a capture probe comprising a specific binding complement of the target analyte labeled with a second member of the first specific binding pair; and
(iii) a reporter comprising a non-nucleic acid linker having two ends, a second member of a first specific binding pair bound to the first end of the linker and a first member of a second specific binding pair bound to the second end of the linker, wherein the reporter and capture probe are bound to the first member of the first specific binding pair.
2. The probe of claim 1, wherein the first specific binding pair comprises DNPanti-DNP antibody or DIGanti-DIG antibody.
3. The probe of claim 1, wherein the non-nucleic acid linker comprises a polymer,
wherein:
R1 has the formula X (CH2)m;
X is \u2014CH3, \u2014CHCH3, \u2014COOH, \u2014CO2(CH2)mCH3, \u2014OH, \u2014CH2OH, ethylene glycol, hexa(ethylene glycol), \u2014O(CH2)mCH3, \u2014NH2, \u2014NH(CH2)mNH2, halogen, glucose, maltose, fullerene C60, or a cyclic olefin; and
m is 0-30.
4. The probe of claim 1, wherein the second specific binding pair is biotinstreptavidin or biotinavidin.
5. The probe of claim 1, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles.
6. The probe of claim 5, wherein the nanoparticles are gold nanoparticles.
7. The probe of claim 1, wherein the first and second specific binding pairs are independently an antibody and an antigen, a receptor and a ligand, an enzyme and a substrate, a drug and a target molecule, or two strands of at least partially complementary oligonucleotides.
8. The probe of claim 1, wherein the target has more than two binding sites.
9. The probe of claim 1, wherein at least two types of probes are provided, the first type of probe having a specific binding complement to a first binding site on the target analyte and the second type of probe having a specific binding complement to a second binding site on the target analyte.
10. The probe of claim 8, wherein a plurality of types of probes are provided, each type of probe having a specific binding complement to different binding sites on the target analyte.
11. The probe of claim 1, wherein the specific binding complement and the target analyte are members of a specific binding pair.
12. The probe of claim 11, wherein members of the specific binding pair comprise nucleic acid, oligonucleotide, peptide nucleic acid, polypeptide, antibody, antigen, carbohydrate, protein, peptide, amino acid, hormone, steroid, vitamin, drug, virus, polysaccharides, lipids, lipopolysaccharides, glycoproteins, lipoproteins, nucleoproteins, oligonucleotides, antibodies, immunoglobulins, albumin, hemoglobin, coagulation factors, peptide and protein hormones, non-peptide hormones, interleukins, interferons, cytokines, peptides comprising a tumor-specific epitope, cells, cell-surface molecules, microorganisms, fragments, portions, components or products of microorganisms, small organic molecules, nucleic acids and oligonucleotides, or metabolites of or antibodies to any of the above substances.
13. The probe of claim 12 wherein nucleic acid and oligonucleotide comprise genes, viral RNA and DNA, bacterial DNA, fungal DNA, mammalian DNA, cDNA, mRNA, RNA and DNA fragments, oligonucleotides, synthetic oligonucleotides, modified oligonucleotides, single-stranded and double-stranded nucleic acids, or natural and synthetic nucleic acids.
14. The probe according to claim 1, wherein the target analyte is a nucleic acid and the specific binding complement is an oligonucleotide.
15. The probe according to claim 1, wherein the target analyte is a protein or hapten and the specific binding complement is an antibody comprising a monoclonal or polyclonal antibody.
16. The probe according to claim 1, wherein the target analyte is a sequence from a genomic DNA sample and the specific binding complements are oligonucleotides, the oligonucleotides having a sequence that is complementary to at least a portion of the genomic sequence.
17. The probe of claim 16, wherein the genomic DNA is eukaryotic, bacterial, fungal or viral DNA.
18. The probe according to claim 1, wherein the specific binding complement and the target analyte are members of an antibody-ligand pair.
19. The probe according to claim 1, wherein in addition to its first binding site, the target analyte has been modified to include a second binding site.
20. A method for detecting for the presence or absence of a target analyte in a sample, wherein the target analyte has at least two binding sites, the method comprising:
(a) providing a first substrate and a nanoparticle probe comprising a nanoparticle having bound thereto: (i) a first member of a first specific binding pair;
(ii) a capture probe comprising a specific binding complement of the target analyte labeled with a second member of the first specific binding pair; and (iii) a reporter comprising a non-nucleic acid linker having two ends, a second member of a first specific binding pair bound to the first end of the linker and a first member of a second specific binding pair bound to the second end of the linker, wherein the reporter and capture probe are bound to the first member of the first specific binding pair;
(b) immobilizing the target analyte onto the first substrate;
(c) contacting the immobilized target analyte with the nanoparticle probe under conditions effective to allow for binding between the target analyte and the nanoparticle probe to form a complex on the substrate;
(d) washing the substrate to remove unbound nanoparticle probes; and
(e) detecting for the presence or absence of the reporter, wherein the presence or absence of the reporter is indicative of the presence or absence of the target analyte in the sample.
21. The method of claim 20, wherein subsequent to step (d) and prior to step (e), further comprising step (d1) subjecting the complex to conditions effective to release the reporter.
22. The method of claim 20, wherein prior to step (e), further comprising steps (d2) capturing the reporter onto a second substrate; (d2) contacting the immobilized reporter with a second nanoparticle probe, the second nanoparticle probe having a specific binding complement to the reporter, under conditions effective to allow binding between the reporter and the second nanoparticle probe to form a complex on the second substrate; and (d3) washing the second substrate to remove any unbound second nanoparticle probe.
23. The method of claim 22, wherein step (e) detecting comprises contacting the washed second substrate with a stain.
24. The method of claim 20, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles.
25. The method of claim 23, wherein the second nanoparticle probe is a gold nanoparticle probe.
26. The method of claim 22, wherein the second substrate is a wave guide and step (e) comprises illuminating the substrate subsequent to step (d3) and observing for any changes in the intensity of light scattered.
27. The method of claim 20, wherein the specific binding pair is an antibody and an antigen, a receptor and a ligand, an enzyme and a substrate, a drug and a target molecule, or two strands of at least partially complementary oligonucleotides.
28. The method of claim 20, wherein the target has more than two binding sites.
29. The method of claim 20, wherein at least two types of probes are provided, the first type of probe having a specific binding complement to a first binding site on the target analyte and the second type of probe having a specific binding complement to a second binding site on the target analyte.
30. The method of claim 28, wherein a plurality of types of probes are provided, each type of probe having a specific binding complement to different binding sites on the target analyte.
31. The method of claim 20, wherein the specific binding complement and the target analyte are members of a specific binding pair.
32. The method of claim 20, wherein members of a specific binding pair comprise nucleic acid, oligonucleotide, peptide nucleic acid, polypeptide, antibody, antigen, carbohydrate, protein, peptide, amino acid, hormone, steroid, vitamin, drug, virus, polysaccharides, lipids, lipopolysaccharides, glycoproteins, lipoproteins, nucleoproteins, oligonucleotides, antibodies, immunoglobulins, albumin, hemoglobin, coagulation factors, peptide and protein hormones, non-peptide hormones, interleukins, interferons, cytokines, peptides comprising a tumor-specific epitope, cells, cell-surface molecules, microorganisms, fragments, portions, components or products of microorganisms, small organic molecules, nucleic acids and oligonucleotides, or metabolites of or antibodies to any of the above substances.
33. The method of claim 32 wherein nucleic acid and oligonucleotide comprise genes, viral RNA and DNA, bacterial DNA, fungal DNA, mammalian DNA, cDNA, mRNA, RNA and DNA fragments, oligonucleotides, synthetic oligonucleotides, modified oligonucleotides, single-stranded and double-stranded nucleic acids, or natural and synthetic nucleic acids.
34. The method according to claim 20, wherein the target analyte is a nucleic acid and the specific binding complement is an oligonucleotide.
35. The method according to claim 20, wherein the target analyte is a protein or hapten and the specific binding complement is an antibody comprising a monoclonal or polyclonal antibody.
36. The method according to claim 20, wherein the target analyte is a sequence from a genomic DNA sample and the specific binding complements are oligonucleotides, the oligonucleotides having a sequence that is complementary to at least a portion of the genomic sequence.
37. The method of claim 20, wherein the genomic DNA is eukaryotic, bacterial, fungal or viral DNA.
38. The method according to claim 20, wherein the specific binding complement and the target analyte are members of an antibody-ligand pair.
39. The method according to claim 20, wherein in addition to its first binding site, the target analyte has been modified to include a second binding site.
40. A nanoparticle probe for detecting for the presence of a target analyte, wherein the target analyte is a first member of a first specific binding pair and wherein the target analyte has at least two binding sites, the probe comprising a nanoparticle having bound thereto:
(i) a first member of a second specific binding pair;
(ii) a capture probe comprising a second member of the first specific binding pair labeled with a second member of the second specific binding pair;
(iii) a reporter comprising a non-nucleic acid linker having two ends, a second member of a second specific binding pair bound to the first and second ends of the linker, wherein the reporter and capture probe are bound to the first member of the second specific binding pair.
41. The probe of claim 40, wherein the second specific binding pair is biotinstreptavidin or biotinavidin.
42. The probe of claim 41, wherein the second member of the second specific binding pair is biotin.
43. A method for detecting for the presence or absence of a target analyte in a sample, wherein the target analyte has at least two binding sites, the method comprising:
(a) providing a first substrate and a nanoparticle probe comprising a nanoparticle having bound thereto: (i) a first member of a second specific binding pair; (ii) a capture probe comprising a second member of the first specific binding pair labeled with a second member of the second specific binding pair; (iii) a reporter comprising a non-nucleic acid linker having two ends, a second member of a second specific binding pair bound to the first and second ends of the linker, wherein the reporter and capture probe are bound to the first member of the second specific binding pair;
(b) immobilizing the target analyte onto the first substrate;
(c) contacting the immobilized target analyte with the probe under conditions effective to allow for binding interactions between the target analyte and the nanoparticle probe to form a complex on the substrate in the presence of the target analyte;
(d) washing the substrate to remove unbound nanoparticle probes; and
(e) detecting for the presence or absence of the reporter, wherein the presence or absence of the reporter is indicative of the presence or absence of the target analyte in the sample.
44. The method of claim 43, wherein subsequent to step (d) and prior to step (e), further comprising step (d1) subjecting the complex to conditions effective to release the reporter.
45. The method of claim 43, wherein prior to step (e), further comprising steps (d2) capturing the reporter onto a second substrate; (d3) contacting the immobilized marker with a second nanoparticle probe, the second nanoparticle probe having a specific binding complement to the reporter, under conditions effective to allow binding between the reporter and the second nanoparticle probe to form a complex on the second substrate; and (d4) washing the second substrate to remove any unbound second nanoparticle probe.
46. The method of claim 45, wherein step (e) detecting comprises contacting the washed second substrate with a stain.
47. The method of claim 46, wherein the second nanoparticle probe is a gold nanoparticle probe.
48. The method of claim 45, wherein the second substrate is a waveguide and step (e) comprises illuminating the substrate subsequent to step (d4) and observing for any changes in the intensity of light scattered.
49. A method for detecting for the presence or absence of a target analyte in a sample, wherein the target analyte has at least two binding sites, the method comprising:
(a) providing a first substrate;
(b) providing a first nanoparticle probe comprising a nanoparticle having (i) a first member of a first specific binding pair bound thereto and (ii) a releasable specific binding complement to the target analyte, the specific binding complement labeled with a second member of the first specific binding pair;
(c) immobilizing the target analyte onto the first substrate;
(d) contacting the immobilized target analyte with the nanoparticle probe under conditions effective to allow for binding interactions between the target analyte and the first nanoparticle probe to form a complex on the substrate in the presence of the target analyte;
(e) washing the substrate to remove unbound first nanoparticle probes;
(f) releasing the specific binding complement from the first nanoparticle probe to form a second nanoparticle probe having the first member of the first specific binding pair; and
(g) detecting for the presence or absence of the second nanoparticle probe, wherein the presence or absence of the second nanoparticle probe is indicative of the presence or absence of the target analyte in the sample.
50. The method of claim 49, wherein subsequent to step (f) and prior to step (g), further comprising steps (f1) capturing the second nanoparticle probe onto a second substrate having a second member of the first specific binding pair under conditions effective to allow binding interactions between the second nanoparticle probe and the second member of the first specific binding pair to form a complex on the second substrate in the presence of the second nanoparticle probe; and (f2) washing the second substrate to remove any unbound second nanoparticle probe.
51. The method of claim 50, wherein step (g) detecting comprises contacting the washed second substrate with a stain.
52. The method of claim 51, wherein the second nanoparticle probe is a gold nanoparticle probe.
53. The method of claim 50, wherein the second substrate is a waveguide and step (g) comprises illuminating the substrate subsequent to step (f2) and observing for any changes in the intensity of light scattered.
54. A nanoparticle probe comprising:
(i) a first nanoparticle having a first member of a non-nucleic acid specific binding pair bound thereto; and
(ii) a second nanoparticle having a non-nucleic acid linker bound thereto, the linker having a first end and a second end, wherein a the first end of the linker is bound to the second nanoparticle and the second end of the linker is bound to a second member of the specific binding pair, wherein the first and second nanoparticles are bound to each other by specific binding pair interactions.
55. The probe of claim 54, wherein the specific binding pair comprises is biotinstreptavidin or biotinavidin.
56. The probe of claim 55, wherein the second member of the specific binding pair is biotin.
57. A method for detecting for the presence or absence of a target analyte in a sample, wherein the target analyte has at least two binding sites, the method comprising:
(a) providing a first substrate;
(b) providing a first nanoparticle probe comprising a nanoparticle having (i) a first member of a first specific binding pair bound thereto and (ii) a releasable specific binding complement to the target analyte, the specific binding complement labeled with a second member of the first specific binding pair;
(c) immobilizing the target analyte onto the first substrate;
(d) contacting the immobilized target analyte with the nanoparticle probe under conditions effective to allow for binding between the target analyte and the first nanoparticle probe to form a complex on the substrate in the presence of the target analyte;
(e) washing the first substrate to remove unbound first nanoparticle probes;
(f) releasing the specific binding complement from the first nanoparticle probe to form a second nanoparticle probe having the first member of the first specific binding pair;
(g) contacting the second nanoparticle probe with one or more third nanoparticle probes to form an aggregate probe in the presence of the second nanoparticle probe, the third nanoparticle probes comprising a nanoparticle having a non-nucleic acid linker molecule bound thereto, wherein a first end of the linker is bound to the third nanoparticle and a second end of the linker is bound to a second member of the first specific binding pair; and
(h) detecting for the presence or absence of the third nanoparticle probe, wherein the presence or absence of the third nanoparticle probe is indicative of the presence or absence of the target analyte in the sample.
58. The method of claim 57, wherein subsequent to step (g) but prior to step (h), further comprising step (g1) isolating the aggregate probe; (g2) releasing the third nanoparticle probe from the aggregate probe; (g3) capturing the third nanoparticle probe onto a second substrate having the first member of the first specific binding pair; and (g4) washing the second substrate to remove any unbound third nanoparticle probe.
59. The method of claim 58, wherein step (h) detecting comprises contacting the washed second substrate with a stain.
60. The method of claim 57, wherein the second nanoparticle probe is a gold nanoparticle probe.
61. The method of claim 58, wherein the second substrate is a waveguide and further comprising subsequent to step (g4), step (g5) illuminating the substrate and observing for any changes in the intensity of light scattered.
62. A nanoparticle probe for detecting for the presence of a target analyte, wherein the target analyte is a first member of a first specific binding pair, the probe comprising a nanoparticle having bound thereto a second member of the first specific binding pair, the second member of the first specific binding pair labeled with a first member of a second specific binding pair.
63. The probe of claim 62, wherein the second specific binding pair comprises is biotinstreptavidin or biotinavidin.
64. The probe of claim 62, wherein the second member of the first specific binding pair is a target specific antibody and the first member of the second specific binding pair is biotin.
65. A method for detecting for the presence or absence of a target analyte in a sample, wherein the target analyte is a first member of a first specific binding pair, the method comprising:
(a) providing a first substrate;
(b) providing a first nanoparticle probe comprising a nanoparticle having bound thereto a second member of the first specific binding pair, the second member of the first specific binding pair labeled with a first member of a second specific binding pair;
(c) immobilizing the target analyte onto the first substrate;
(d) contacting the immobilized target analyte with the first nanoparticle probe under conditions effective to allow for binding interactions between the target analyte and the first nanoparticle probe to form a complex on the substrate in the presence of the target analyte;
(e) washing the substrate to remove unbound first nanoparticle probes;
(f) releasing the first nanoparticle probe; and
(g) detecting for the presence or absence of the first nanoparticle probe, wherein the presence or absence of the first nanoparticle probe is indicative of the presence or absence of the target analyte in the sample.
66. The method of claim 65, wherein subsequent to step (f) and prior to step (g), further comprising steps (f1) capturing the first nanoparticle probe onto a second substrate having a second member of the first specific binding pair under conditions effective to allow binding interactions between the first nanoparticle probe and the second member of the first specific binding pair to form a complex on the second substrate in the presence of a second nanoparticle probe; and (f2) washing the second substrate to remove any unbound second nanoparticle probe.
67. The method of claim 66, wherein step (g) detecting comprises contacting the washed second substrate with a stain.
68. The method of claim 67, wherein the second nanoparticle probe is a gold nanoparticle probe.
69. The method of claim 66, wherein the second substrate is a waveguide and step (g) comprises illuminating the substrate subsequent to step (f2) and observing for any changes in the intensity of light scattered.
70. A nanoparticle probe for detecting for the presence of a target analyte, the probe comprising a nanoparticle having bound thereto (i) a specific binding complement of a target analyte; and (ii) a first member of a first specific binding pair.
71. The probe of claim 70, wherein the first specific binding pair comprises is biotinstreptavidin or biotinavidin.
72. The probe of claim 70, wherein the specific binding complement of the target analyte is a target specific antibody and the first member of the first specific binding pair is streptavidin.
73. A method for detecting for the presence or absence of a target analyte in a sample, wherein the target analyte has at least two binding sites, the method comprising:
(a) providing a first substrate;
(b) providing a first nanoparticle probe comprising a nanoparticle having bound thereto (i) a specific binding complement of a target analyte; and (ii) a first member of a first specific binding pair;
(c) immobilizing the target analyte onto the first substrate;
(d) contacting the immobilized target analyte with the first nanoparticle probe under conditions effective to allow for binding interactions between the target analyte and the first nanoparticle probe to form a complex on the substrate in the presence of the target analyte;
(e) washing the substrate to remove unbound first nanoparticle probes;
(f) releasing the first nanoparticle probe; and
(g) detecting for the presence or absence of the first nanoparticle probe, wherein the presence or absence of the first nanoparticle probe is indicative of the presence or absence of the target analyte in the sample.
74. The method of claim 73, wherein subsequent to step (f) and prior to step (g), further comprising steps (f1) capturing the first nanoparticle probe onto a second substrate having a second member of the first specific binding pair under conditions effective to allow binding interactions between the first nanoparticle probe and the second member of the first specific binding pair to form a complex on the second substrate in the presence of the first nanoparticle probe; and (f2) washing the second substrate to remove any unbound first nanoparticle probe.
75. The method of claim 74, wherein step (g) detecting comprises contacting the washed second substrate with a stain.
76. The method of claim 75, wherein the second nanoparticle probe is a gold nanoparticle probe.
77. The method of claim 74, wherein the second substrate is a waveguide and step (g) comprises illuminating the substrate subsequent to step (f2) and observing for any changes in the intensity of light scattered.
78. A method for detecting for the presence or absence of a target analyte in a sample, wherein the target analyte has at least two binding sites, the method comprising:
(a) providing a first substrate and a second substrate;
(b) labeling a sample believed to have the target analyte with a first member of a first specific binding pair;
(c) immobilizing labeled target analyte onto the first substrate;
(d) washing the first substrate to remove unbound labeled target analyte;
(e) releasing the labeled target analyte;
(f) recapturing the labeled target analyte onto the second substrate;
(g) contacting the recaptured target analyte with the a nanoparticle probe comprising a nanoparticle having a second member of the first specific binding pair under conditions effective to allow for binding between the recaptured labeled target analyte and the first nanoparticle probe to form a complex on the substrate in the presence of the labeled target analyte;
(h) washing the substrate to remove unbound nanoparticle probes; and
(i) detecting for the presence or absence of the nanoparticle probe, wherein the presence or absence of the nanoparticle probe is indicative of the presence or absence of the target analyte in the sample.
79. The method of claim 78, wherein step (i) detecting comprises contacting the washed second substrate with a stain.
80. The method of claim 79, wherein the nanoparticle probe is a gold nanoparticle probe.
81. The method of claim 78, wherein the second substrate is a waveguide and step (i) comprises illuminating the substrate subsequent to step (h) and observing for any changes in the intensity of light scattered.
82. A method for detecting for the presence or absence of a target analyte in a sample, wherein the target analyte has at least two binding sites, the method comprising:
(a) providing a first substrate and a second substrate;
(b) providing a first nanoparticle probe comprising a nanoparticle having (i) a first member of a first specific binding pair bound thereto and (ii) a specific binding complement to the target analyte, the specific binding complement labeled with a second member of the first specific binding pair;
(c) providing a second nanoparticle probe comprising a nanoparticle having a non-nucleic acid linker molecule bound thereto, wherein a first end of the linker is bound to the second nanoparticle and a second end of the linker is bound to a second member of the first specific binding pair;
(d) immobilizing the target analyte onto the first substrate;
(e) contacting the immobilized target analyte with the nanoparticle probe under conditions effective to allow for binding interactions between the target analyte and the first nanoparticle probe to form a complex on the substrate in the presence of the target analyte;
(f) washing the first substrate to remove unbound first nanoparticle probes;
(g) releasing the first member of the first specific binding pair from the first nanoparticle probe;
(h) immobilizing the first member of the first specific binding pair onto the second substrate;
(i) washing the second substrate to remove unbound first member of the specific binding pair;
(j) contacting the captured first member of the first specific binding pair on the second substrate with the second nanoparticle probe under conditions effective to allow for binding between the captured first member of the first specific binding pair and the second nanoparticle probe to form a complex in the presence of the first member;
(k) washing the second substrate so as to remove unbound second nanoparticle probes; and
(l) detecting for the presence or absence of the second nanoparticle probe, wherein the presence or absence of the second nanoparticle probe is indicative of the presence or absence of the target analyte in the sample.
83. The method of claim 82, wherein step (l) detecting comprises contacting the washed second substrate with a stain.
84. The method of claim 83, wherein the second nanoparticle probe is a gold nanoparticle probe.
85. The method of claim 82, wherein the second substrate is a waveguide and step (l) comprises illuminating the substrate and observing for any changes in the intensity of light scattered.
86. A method for detecting for the presence or absence of a target analyte in a sample, wherein the target analyte has at least two binding sites, the method comprising:
(a) providing a first substrate;
(b) providing a first particle probe comprising a polyacrylic acid polymer having bound thereto (i) a specific binding complement of the target analyte; and (ii) a first member of a first specific binding pair;
(c) immobilizing the target analyte onto the first substrate;
(d) contacting the immobilized target analyte with the first particle probe under conditions effective to allow for binding interactions between the target analyte and the first particle probe to form a complex on the first substrate in the presence of the target analyte;
(e) washing the first substrate to remove unbound first particle probes;
(f) denaturing the first particle probe to form fragments; and
(g) detecting for the presence or absence of the fragments, wherein the presence or absence of the fragments is indicative of the presence or absence of the target analyte in the sample.
87. The method of claim 86, wherein subsequent to step (f) and prior to step (g), further comprising steps (f1) capturing the fragments onto a second substrate having a second member of the first specific binding pair under conditions effective to allow binding interactions between the fragments and the second member of the first specific binding pair and form a complex on the second substrate in the presence of the fragments; (f2) washing the second substrate to remove any unbound fragments; and (f3) contacting the fragments bound to the second substrate with a nanoparticle probe comprising a nanoparticle having bound thereto the second member of the first specific binding pair.
88. The method of claim 87, wherein step (g) detecting comprises contacting the washed second substrate with a stain.
89. The method of claim 88, wherein the nanoparticle probe is a gold nanoparticle probe.
90. The method of claim 87, wherein the second substrate is a waveguide and step (g) comprises illuminating the substrate subsequent to step (f3) and observing for any changes in the intensity of light scattered.
91. A method for detecting for the presence or absence of a target analyte in a sample, wherein the target analyte has at least two binding sites, the method comprising:
(a) providing a first substrate and a second substrate having a first member of a first specific binding pair bound thereto;
(b) providing a specific binding complement to the target analyte, the specific binding complement labeled with a second member of the first specific binding pair;
(c) providing a nanoparticle probe comprising a nanoparticle having the second member of the first specific binding pair bound thereto;
(d) immobilizing the target analyte onto the first substrate;
(e) contacting the immobilized target analyte with the specific binding complement under conditions effective to allow for binding between the target analyte and the specific binding complement to form a complex on the first substrate in the presence of the target analyte;
(f) washing the first substrate to remove unbound specific binding complement;
(g) releasing specific binding complement;
(h) capturing the released specific binding complement onto the second substrate;
(i) washing the second substrate to remove unbound specific binding complements;
(j) contacting the captured specific binding complement on the second substrate with the nanoparticle probe under conditions effective to allow for binding between the captured specific binding complement and the nanoparticle probe to form a complex in the presence of captured specific binding complement;
(k) washing the second substrate so as to remove unbound nanoparticle probe; and
(l) detecting for the presence or absence of the nanoparticle probe, wherein the presence or absence of the nanoparticle probe is indicative of the presence or absence of the target analyte in the sample.
92. The method of claim 91, wherein step (l) detecting comprises contacting the washed second substrate with a stain.
93. The method of claim 92, wherein the nanoparticle probe is a gold nanoparticle probe.
94. The method of claim 91, wherein the second substrate is a wave guide and step (l) comprises illuminating the substrate and observing for any changes in the intensity of light scattered.
95. A kit for detecting for one or more target analytes in a sample, the kit comprising the nanoparticle probe of any one of claims 1, 40, 54, 62 and 70 and an optional substrate.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. In an electronic vacuum pulsating machine for milking domestic animals, such as cows and goats, an elongated teat cup liner located inside said teat cup; said teat cup liner is having a proximal portion in communication to the milking vacuum system and the milk pail and an distal portion in communication with the animal teat; said teat cup liner comprising:
a) a plurality of segments barriered by a means of one or more rings, said ring are elastomeric rubber-like sleeve structure preventing the liner from collapsing at the milk extracting pulls; said inflatabledeflatable ring is adapted to contract andor expand in a manner selected from a ring after a ring andor rings after rings; and,
b) a vacuum duct comprising a plurality of vacuum valves, said inlet opening of the duct is in communication with an effective vacuum source; each of said valve is in communication with one or more rings and is adapted to provide a reversible inflationdeflation of said elastomeric rubber-like sleeve according to a predetermined pulse control provided by a pulsator;
wherein vacuum or air is flow from the vacuumair-pressure source, via the vacuum duct and vacuum valve to the segments in a distal-to-proximal direction, such that the ring is inflated and milk is filling segment which is vacuumed in its turn, from the udder, then the vacuum pulse is terminated in the manner that the ring is deflated, and a beginning of a new cycle is provided until the animal’s udder is empty.
2. The teat cup liner according to claim 1, wherein the rings andor segments are selected from compartments, chambers, inflatable members and the like, adapted to reversibly inflate and deflate to a predetermined initial and terminal diameter and size.
3. The teat cup liner according to claim 1, wherein a plurality of N segments andor ris used, wherein N is any integer number higher or equal 2, said plurality of segments are arranged in any 2D or 3D array of segments, sub-segments, coupled segments or any combination thereof.
4-5. (canceled)
6. An automated milking robot comprising the teat cup liner as defined in claim 1.
7. A method for milking domestic animals by means of the teat cup liner as defined in claim 1 comprising applying vacuum pulses to a plurality of elastomeric rubber-like sleeve segments such that vacuum or air is flow from the vacuumair-pressure source, via the vacuum duct and vacuum valve to the segments in a distal-to-proximal direction, such that the ring is inflated and milk is filling segment which is vacuumed in its turn, from the udder, then the vacuum pulse is terminated in the manner that the ring is deflated, and a beginning of a new cycle is provided until the animal’s udder is empty.