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

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 complementary 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.

1 . A composition comprising: 
 fluorescent core/shell nanocrystal functionalized with a coating that comprises amino acid molecules; the functionalized fluorescent nanocrystal operably bonded to a molecular probe and a plurality of polynucleotide strands of predetermined sequence, the polynucleotide strands of predetermined sequence capable of hybridizing under suitable conditions with complementary polynucleotide strands operably bonded to fluorescent core/shell nanocrystals, a terminal portion of each of the plurality of polynucleotide strands of predetermined sequence being operably linked to the functionalized fluorescent nanocrystal, and wherein the opposite terminus of each of the plurality polynucleotide strands of predetermined sequence is unbound to the functionalized nanocrystal and extends outwardly therefrom.    
     
     
         2 - 16 . (canceled)  
     
     
         17 . The composition according to  claim 1 , where the amino acid molecules operably link the plurality of polynucleotide strands of predetermined sequence to the coating.  
     
     
         18 . The composition of  claim 1  where the molecular probe is a polynucleotide which has binding specificity for a target molecule.  
     
     
         19 . The composition of  claim 1 , where the polynucleotide strands of predetermined sequence of the plurality of polynucleotide strand comprises a reactive functionality selected from the group consisting of an amino-reactive group, a carboxyl-reactive group, and a thiol group.  
     
     
         20 . The composition of  claim 1 , wherein a linker operably links a polynucleotide strand of predetermined sequence to the functionalized fluorescent nanocrystal.  
     
     
         21 . The composition of  claim 1  where the molecular probe is a monoclonal antibody operably linked to the functionalized fluorescent nanocrystal.  
     
     
         22 . The composition of  claim 1  where the molecular probe is avidin operably linked to the functionalized fluorescent nanocrystal.  
     
     
         23 . The composition of  claim 1  where a linker operably links a polynucleotide strand of the functionalized fluorescent nanocrystal to the molecular probe.  
     
     
         24 . The composition of  claim 1  where the coating further includes a diaminocarboxylic acid.  
     
     
         25 . The composition according to  claim 1 , wherein the functionalized fluorescent nanocrystal comprises a core of CdX wherein X is Se, Te, or S.  
     
     
         26 . The composition of  claim 1  where the molecular probe and the functionalized fluorescent nanocrystal are operably linked through a bond between: a reactive functionality on one or more of the plurality of polynucleotide strands of predetermined sequence and a reactive functionality associated with the molecular probe, a reactive functionality of the amino acid coating and a reactive functionality associate with the molecular probe, a linker which has one portion that binds to a reactive functionality on one or more polynucleotide strands of predetermined sequence and another portion which binds to a reactive functionality associated with the molecular probe, a linker which has one portion that hybridizes to one or more polynucleotide strands of predetermined sequence and another portion which hybridizes to the molecular probe comprising a nucleic acid molecule, or synthesizing the molecular probe as part of one or more polynucleotide strands of predetermined sequence.  
     
     
         27 . The composition of  claim 1  where the molecular probe is operably bonded to biotin, a terminal portion of the molecular probe operably linked to the functionalized fluorescent nanocrystal, the opposite terminus of the molecular probe is unbound to the functionalized nanocrystal, extends outwardly and is bonded to biotin.  
     
     
         28 . A composition comprising: 
 a primary fluorescent core/shell nanocrystal functionalized with a coating that comprises amino acid molecules; the functionalized fluorescent nanocrystal operably bonded to a molecular probe and a plurality of polynucleotide strands of predetermined sequence, the polynucleotide strands of predetermined sequence able to hybridize under suitable conditions with complementary polynucleotide strands operably bonded to fluorescent core/shell nanocrystals, a terminal portion of each of the plurality of polynucleotide strands of predetermined sequence is operably linked to the functionalized fluorescent nanocrystal, and the opposite terminus of each of the plurality polynucleotide strands of predetermined sequence is unbound to the functionalized fluorescent nanocrystal and extends outwardly therefrom, the primary functionalized fluorescent nanocrystal operably bonded to a target molecule.    
     
     
         29 . The composition of  claim 28  further comprising: a plurality of secondary fluorescent core/shell nanocrystals functionalized with a plurality of polynucleotide strands having a sequence complementary to the sequence of the plurality of polynucleotide strands of predetermined sequence of the primary functionalized fluorescent nanocrystal, the polynucleotide strands of predetermined sequence hybridizing with the complementary polynucleotide strands.  
     
     
         30 . The composition of  claim 28 , where the intensity of fluorescent emission from the primary nanocrystal hybridized to the secondary nanocrystals is greater than the fluorescent emission of the primary nanocrystal operably bonded to the target molecule.  
     
     
         31 . The composition of  claim 28 , wherein the primary fluorescent core/shell nanocrystal functionalized to be water soluble comprises a core of CdX wherein X is Se, Te, or S.  
     
     
         32 . A method of detecting a target molecule comprising: 
 contacting a sample with primary functionalized fluorescent nanocrystals, the primary functionalized fluorescent nanocrystals having a coating that comprises amino acid molecules; the primary functionalized fluorescent nanocrystal operably bonded to a molecular probe the operably bonds with a target molecule if present in the sample and a plurality of polynucleotide strands of predetermined sequence, the polynucleotide strands of predetermined sequence able to hybridize under suitable conditions with complementary polynucleotide strands operably bonded to secondary fluorescent core/shell nanocrystals, a terminal portion of each of the plurality of polynucleotide strands of predetermined sequence is operably linked to the primary functionalized fluorescent nanocrystal, and the opposite terminus of each of the plurality polynucleotide strands of predetermined sequence is unbound to the primary functionalized fluorescent nanocrystal and extends outwardly therefrom;    contacting the sample with successive additions of secondary fluorescent core/shell nanocrystal and primary functionalized fluorescent nanocrystals, the secondary functionalized fluorescent nanocrystals operably bonded to a plurality of polynucleotide strands of complementary sequence able to hybridize with primary functionalized fluorescent nanocrystals under suitable conditions for hybridization; and    detecting the fluorescence from the sample exposed to an excitation light source.    
     
     
         33 . The method of  claim 32  where the molecular probe is a polynucleotide that has binding specificity for a target molecule.  
     
     
         34 . The method of  claim 32  where the molecular probe is a monoclonal antibody.  
     
     
         35 . The method of  claim 32  where the molecular probe is avidin operably linked to the functionalized fluorescent nanocrystal.  
     
     
         36 . The method of  claim 32  where the molecular probe is operably bonded to biotin, a terminal portion of the molecular probe operably linked to the functionalized fluorescent nanocrystal, the opposite terminus of the molecular probe is unbound to the functionalized nanocrystal, extends outwardly from the functionalized fluorescent nanocrystal and is bonded to biotin.