Application of quantum dots for nuclear staining

Patent metadata

Field	Value
Publication number	US-9448231-B2
Application number	US-201213402567-A
Country	US
Kind code	B2
Filing date	Feb 22, 2012
Priority date	Feb 28, 2011
Publication date	Sep 20, 2016
Grant date	Sep 20, 2016

Abstract

Embodiments of a system, method, and kit for visualizing a nucleus are disclosed. A tissue sample is pretreated with a protease to permeabilize the nucleus, and then incubated with a nanoparticle/DNA-binding moiety conjugate. The DNA-binding moiety includes at least one DNA-binding molecule. The conjugate binds to DNA within the nucleus, and the nanoparticle is visualized, thereby visualizing the nucleus. Computer and image analysis techniques are used to evaluate nuclear features such as chromosomal distribution, ploidy, shape, size, texture features, and/or contextual features. The method may be used in combination with other multiplexed tests on the tissue sample, including fluorescence in situ hybridization. Kits for performing the method include a protease enzyme composition, a nanoparticle/DNA-binding moiety conjugate, and a reaction buffer.

First claim

We claim:

1. A method for visualizing a nucleus in a fixed tissue sample, comprising:
pretreating a tissue sample with a protease to permeabilize the nucleus, thereby forming a pretreated tissue sample;
incubating the pretreated tissue sample with a conjugate having a structure nanoparticle-linker-DNA-binding molecule, wherein the DNA binding molecule is selected from a minor groove binder, a major groove binder, a DNA intercalator, a DNA alkylating agent, or a combination thereof, under conditions sufficient to allow the conjugate to enter a nucleus within the pretreated tissue sample, wherein the conjugate binds to DNA in the nucleus; and
visualizing the nanoparticle, and thereby visualizing the nucleus.

2. The method of claim 1 , wherein the nanoparticle comprises a quantum dot, a metal nanoparticle, a metal oxide nanoparticle, or a transition metal complex nanoparticle.

3. The method of claim 1 , wherein the nanoparticle comprises a quantum dot and visualizing the nanoparticle comprises visualizing photostable fluorescence of the quantum dot.

4. The method of claim 1 , wherein the DNA-binding molecule is 4′,6-diamidino-2-phenylindole (DAPI), a bis-benzimide dye, psoralen, or naphthalene diimide.

5. The method of claim 1 , wherein the DNA-binding moiety is

6. The method of claim 1 , wherein the conjugate is incubated with the tissue sample at a concentration of at least 20 nM.

7. The method of claim 1 , further comprising using computer image analysis techniques to quantitatively measure nuclear features.

8. The method of claim 7 , wherein the nuclear features include chromosomal distribution, ploidy, shape, size, texture features, contextual features, or combinations thereof.

9. The method of claim 1 , wherein the tissue sample is pretreated with the protease for 4-8 minutes and the tissue sample is fixed before pretreating with the protease.

10. The method of claim 1 , further comprising:
providing a probe capable of hybridizing to a target within the tissue sample prior to incubating the pretreated tissue sample with the conjugate;
incubating the probe with the tissue sample under conditions sufficient to allow the probe to hybridize to the target within the tissue sample; and
detecting the probe.

11. The method of claim 10 , wherein detecting the probe comprises visualizing a quantum dot associated with the probe.

12. The method of claim 11 , wherein the nanoparticle of the conjugate comprises a quantum dot capable of emitting fluorescence at a different wavelength than the quantum dot associated with the probe.

13. The method of claim 1 , further comprising performing a fluorescence in situ hybridization procedure on the tissue sample.

14. The method of claim 13 , wherein the fluorescence in situ hybridization procedure comprises a HER2 assay, a TMPRSS2-ERG assay, a Chr17 assay, or a combination thereof.

15. A kit for visualizing a nucleus in a fixed tissue sample, comprising:
a protease enzyme composition comprising a protease enzyme and a protease buffer, wherein the protease buffer has a salt concentration and pH sufficient to allow the protease enzyme to exhibit proteolytic activity;
a conjugate having a structure nanoparticle-linker-DNA-binding molecule wherein the DNA-binding molecule is selected from a minor groove binder, a major groove binder, a DNA intercalator, a DNA alkylating agent, or a combination thereof; and
a reaction buffer, wherein the reaction buffer has a salt concentration and pH sufficient to enable the conjugate to enter a nucleus within a tissue sample pretreated with the protease enzyme composition.

16. The kit of claim 15 , wherein the nanoparticle comprises a quantum dot, a metal nanoparticle, a metal oxide nanoparticle, or a transition metal complex nanoparticle.

Assignees

• Yun Chol Steven
• Kelly Brian Daniel
• Ashworth-Sharpe Julia
• Bieniarz Christopher A
• Bamford Pascal
• Murillo Adrian E
• Ventana Med Syst Inc

Inventors

• Yun Chol Steven
• Kelly Brian Daniel
• Ashworth-Sharpe Julia
• Bieniarz Christopher A
• Bamford Pascal
• Murillo Adrian E

Classifications

• C07D487/04

  Ortho-condensed systems · CPC title

• G01N33/5308 Primary

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• C09B69/10

  Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q2600/158

  Expression markers · CPC title