Application of quantum dots for nuclear staining

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.

1 . A conjugate, comprising:
 a quantum dot; and   a DNA-binding moiety comprising a DNA-binding molecule, wherein the DNA-binding molecule is a major groove binder, a DNA intercalator, a DNA alkylating agent, or a combination thereof.   
     
     
         2 . The conjugate of  claim 1 , wherein the DNA-binding moiety comprises a DNA-binding molecule selected from 4′,6-diamidino-2-phenylindole (DAPI), bis-benzimide dyes, psoralen, and naphthalene diimide. 
     
     
         3 . The conjugate of  claim 1 , wherein the DNA-binding moiety further comprises a linker, and the conjugate has the structure
 quantum dot-linker-DNA-binding molecule.   
     
     
         4 . The conjugate of  claim 3 , wherein the linker comprises an aliphatic chain from 1 to 30 carbon atoms in length or (—OCH 2 CH 2 —) n  where n is from 2 to 15. 
     
     
         5 . The conjugate of  claim 3 , wherein the linker is a multi-functional linker and a plurality of DNA-binding molecules is bound to the multi-functional linker. 
     
     
         6 . The conjugate of  claim 3 , wherein the DNA binding moiety is 
       
         
           
           
               
               
           
         
       
     
     
         7 . A method for visualizing a nucleus, comprising:
 pretreating a tissue sample with a protease to form a pretreated tissue sample;   incubating the pretreated tissue sample with a conjugate comprising a) a nanoparticle and b) a DNA-binding moiety comprising a DNA-binding molecule 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.   
     
     
         8 . The method of  claim 7 , wherein the nanoparticle comprises a quantum dot, a metal nanoparticle, a metal oxide nanoparticle, or a transition metal complex nanoparticle. 
     
     
         9 . The method of  claim 7 , wherein the nanoparticle comprises a quantum dot and visualizing the nanoparticle comprises visualizing photostable fluorescence of the quantum dot. 
     
     
         10 . The method of  claim 7 , wherein the DNA-binding molecule is a minor groove binder, a major groove binder, a DNA intercalator, a DNA alkylating agent, or a combination thereof. 
     
     
         11 . The method of  claim 10 , wherein the DNA-binding molecule is 4′,6-diamidino-2-phenylindole (DAPI), a bis-benzimide dye, psoralen, or naphthalene diimide. 
     
     
         12 . The method of  claim 7 , wherein the DNA-binding moiety further comprises a linker, and the conjugate has the structure
 nanoparticle-linker-DNA-binding molecule.   
     
     
         13 . The method of  claim 12 , wherein the DNA-binding moiety is 
       
         
           
           
               
               
           
         
       
     
     
         14 . The method of  claim 7 , wherein the conjugate is incubated with the tissue sample at a concentration of at least 20 nM. 
     
     
         15 . The method of  claim 7 , further comprising using computer image analysis techniques to quantitatively measure nuclear features. 
     
     
         16 . The method of  claim 15 , wherein the nuclear features include chromosomal distribution, ploidy, shape, size, texture features, contextual features, or combinations thereof. 
     
     
         17 . The method of  claim 7 , wherein the tissue sample is pretreated with the protease for 4-8 minutes and the tissue sample is fixed before pretreating with the protease. 
     
     
         18 . The method of  claim 7 , 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.   
     
     
         19 . The method of  claim 18 , wherein detecting the probe comprises visualizing a quantum dot associated with the probe. 
     
     
         20 . The method of  claim 19 , 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. 
     
     
         21 . The method of  claim 7 , further comprising performing a fluorescence in situ hybridization procedure on the tissue sample. 
     
     
         22 . The method of  claim 21 , wherein the fluorescence in situ hybridization procedure comprises a HER2 assay, a TMPRSS2-ERG assay, a Chr17 assay, or a combination thereof. 
     
     
         23 . A kit for visualizing a nucleus, 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 comprising a) a nanoparticle and b) a DNA-binding moiety comprising a DNA-binding molecule; 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.   
     
     
         24 . The kit of  claim 23 , wherein the nanoparticle comprises a quantum dot, a metal nanoparticle, a metal oxide nanoparticle, or a transition metal complex nanoparticle. 
     
     
         25 . The kit of  claim 23 , wherein the DNA-binding molecule is a minor groove binder, a major groove binder, a DNA intercalator, a DNA alkylating agent, or a combination thereof.