Nanocrystals having polynucleotide strands and their use to form dendrimers in a signal amplification system

• US 6,261,779 B1
• Filed: 11/09/1999
• Issued: 07/17/2001
• Est. Priority Date: 11/10/1998
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Bio-Pixels, Ltd.
• Inventors: Barbera-Guillem, Emilio, Nelson, M. Bud, Castro, Stephanie L.
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): Lundgren, Jeffrey S.
• Application Number:
  US09/437,076
• Time in Patent Office: 616 Days
• Field of Search: 435/6
• US Class Current: 435/6.11
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/588 with semiconductor nanocrystal label, e.g. quantum dots
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Provided are compositions and assay kits comprising functionalized nanocrystals having extending therefrom a plurality of polynucleotide strands of known sequence; wherein primary dots are used to operably link to a molecular probe, and secondary dots comprise a plurality of polynucleotide strands which are complemetary to the plurality of polynucleotide strands of the primary dots. Also provided is a method for detecting the presence or absence of target molecule in a sample comprising operably linking primary dots to molecular probe, contacting the complex formed with the sample, contacting the sample with successive additions of secondary dots and primary dots. If target molecule is present in the sample, the primary dots and secondary dots will form dendrimers that can be detected by fluorescence emission.

Claims

1. An assay kit comprising:

(a) primary dots, each of which comprises a functionalized nanocrystal comprising a plurality of polynucleotide strands of a predetermined sequence, wherein a terminal portion of each of the plurality of polynucleotide strands is operably linked to the functionalized nanocrystal, wherein the opposite terminus of each of the plurality polynucleotide strand is unbound to the functionalized nanocrystal and extends outwardly from the functionalized nanocrystal, and wherein the primary dots have one or more reactive functionalities; and

(b) secondary dots each of which comprises a functionalized nanocrystal comprising a plurality of polynucleotide strands of a predetermined sequence which is complementary to and can under suitable conditions hybridize to the sequence of the polynucleotide strands of the primary dots, wherein a terminal portion of each of the plurality of polynucleotide strands is operably linked to the functionalized nanocrystal, and wherein the opposite terminus of each of the plurality polynucleotide strand is not bound to the functionalized nanocrystal and extends outwardly from the functionalized nanocrystal.

2. The assay kit according to claim 1, wherein the primary dots and secondary dots comprise a uniform size.

3. The assay kit according to claim 1, further comprising a linker for linking the primary dots to molecular probe.

4. The assay kit according to claim 1, further comprising a molecular probe.

5. The assay kit according to claim 4, wherein the molecular probe is operably linked to the primary dots.

6. The assay kit according to claim 1, further comprising a linker, and molecular probe.

7. The assay kit according to claim 1, further comprising primary dots which lack reactive functionalities.

8. The assay kit according to claim 1, wherein the primary dots and secondary dots comprise a first set;

and wherein the assay kit further comprises a second set of primary dots and secondary dots, wherein the primary dots and secondary dots of the second set comprise a uniform size which is different than the uniform size of the primary dots and secondary dots of the first set, wherein the primary dots of the second set comprise a plurality of polynucleotide strands which are complementary in sequence to the plurality of polynucleotide strands of the secondary dots of the second set, and wherein the plurality of polynucleotide strands of the primary dots of the second set lack sufficient complementarity to specifically hybridize to the plurality of polynucleotide strands of the primary dots of the first set.

9. A method of using functionalized nanocrystals comprising primary dots and secondary dots for detecting the presence or absence of a target molecule in a sample, the method comprising:

(a) operably linking primary dots to molecular probe in forming primary dot-molecular probe complexes;

(b) contacting an effective amount of primary dot-molecule probe complexes to the sample under suitable conditions for molecular probe to specifically bind to target molecule, if present in the sample;

(c) contacting an effective amount of secondary dots with the sample under suitable conditions for promoting hybridization of the plurality of polynucleotide strands of the second dots to the polynucleotide strands of the primary dots, if present in the sample;

(d) contacting the sample with successive additions of an effective amount of primary dots and an effective amount of secondary dots under suitable conditions for promoting hybridization;

(e) exposing the sample to an excitation light source that is suitable for exciting the functionalized nanocrystals to emit a fluorescence emission;

wherein detection by detection means of a signal comprising peak fluorescence emission, generated by excited functionalized nanocrystals, and amplified by dendrimer formation, is indicative of the presence of target molecule in the sample; and

absence of the signal is indicative of the absence of target molecule in the sample.

10. The method according to claim 9, further comprising a wash step after each addition of functionalized nanocrystals to the sample to remove unbound or non-specifically bound functionalized nanocrystals from the sample.

11. The method according to claim 9, wherein the primary dots and secondary dots comprise a uniform size.

12. The method according to claim 9, further comprising quantitating the amount of target molecule present in a sample by measuring the fluorescence intensity of the signal generated, and relating an amount of fluorescence intensity to an amount of target molecule.

13. The method according to claim 9, wherein the excitation light source comprises a spectral range of from about 200 nanometers to about 500 nanometers.

14. The method according to claim 9, wherein the excitation light source comprises a spectral range of from about 300 nanometers to about 400 nanometers.

15. The method according to claim 9, wherein the fluorescence emission is in the spectral range of about 400 nanometers to about 750 nanometers.

16. A method of using functionalized nanocrystals comprising primary dots and secondary dots for detecting the presence or absence of a target molecule in a sample, the method comprising:

(a) contacting an effective amount of molecule probe to the sample under suitable conditions for molecular probe to specifically bind to target molecule, if present in the sample;

(b) contacting an effective amount of primary dots to the sample under suitable conditions for the primary dots to operably link to molecular probe, if present in the sample;

(c) contacting an effective amount of secondary dots with the sample under suitable conditions for promoting hybridization of the plurality of polynucleotide strands of the second dots to the polynucleotide strands of the primary dots, if present in the sample;

(d) contacting the sample with successive additions of an effective amount of primary dots and an effective amount of secondary dots under suitable conditions for promoting hybridization;

(e) exposing the sample to an excitation light source that is suitable for exciting the functionalized nanocrystals to emit a fluorescence emission;

wherein detection by detection means of a signal comprising peak fluorescence emission, generated by excited functionalized nanocrystals, and amplified by dendrimer formation, is indicative of the presence of target molecule in the sample; and

absence of the signal is indicative of the absence of target molecule in the sample.

17. The method according to claim 16, further comprising a wash step after each addition of functionalized nanocrystals to the sample to remove unbound or non-specifically bound functionalized nanocrystals from the sample.

18. The method according to claim 16, wherein the primary dots and secondary dots comprise a uniform size.

19. The method according to claim 16, further comprising quantitating the amount of target molecule present in a sample by measuring the fluorescence intensity of the signal generated, and relating an amount of fluorescence intensity to an amount of target molecule.

20. The method according to claim 16, wherein the excitation light source comprises a spectral range of from about 200 nanometers to about 500 nanometers.

21. The method according to claim 16, wherein the excitation light source comprises a spectral range of from about 300 nanometers to about 400 nanometers.

22. The method according to claim 16, wherein the fluorescence emission is in the spectral range of about 400 nanometers to about 750 nanometers.