Method of detecting an analyte in a sample using semiconductor nanocrystals as a detectable label

Abstract

The use of semiconductor nanocrystals as detectable labels in various chemical and biological applications is disclosed. The methods find use for detecting a single analyte, as well as multiple analytes by using more than one semiconductor nanocrystal as a detectable label, each of which emits at a distinct wavelength.

We claim:  
     
         1 . A method of detecting one or more target analytes in a sample containing or suspected of containing the one or more analytes, comprising the steps of: 
 (a) providing the sample on a solid support;    (b) combining said sample with a semiconductor nanocrystal conjugate, wherein said combining is performed under conditions that allow formation of a complex comprising said conjugate and said analyte, when present;    (c) removing any unbound conjugate;    (d) detecting the presence of the complex, if present, by monitoring a spectral emission mediated by the semiconductor nanocrystal in the complex, wherein the emission indicates the presence of one or more target analytes in the sample.    
     
     
         2 . The method of  claim 1 , wherein there is a plurality of target analytes and the method further comprises: 
 (a) providing a conjugate specific for each target analyte, wherein each semiconductor nanocrystal conjugate has an emission spectrum distinct from the other semiconductor nanocrystal conjugates; and    (b) detecting the presence of the target analytes by monitoring the spectral emissions of the sample, wherein the emissions indicate the presence of the target analytes in the sample.    
     
     
         3 . The method of  claim 1 , wherein the one or more analytes is a nucleic acid molecule.  
     
     
         4 . The method of  claim 3 , wherein the nucleic acid molecule is contained within a chromosome or chromosomal fragment.  
     
     
         5 . The method of  claim 2 , wherein the one or more analytes is a nucleic acid molecule.  
     
     
         6 . The method of  claim 5 , wherein the nucleic acid molecule is contained within a chromosome or chromosomal fragment.  
     
     
         7 . The method of  claim 6 , wherein the target analytes are present on more than one chromosome or chromosomal fragments.  
     
     
         8 . The method of  claim 7 , wherein each of the target analytes is different from the others.  
     
     
         9 . The method of  claim 6 , wherein the target analytes are present on a single chromosome.  
     
     
         10 . The method of  claim 9 , wherein each of the target analytes is different from the others.  
     
     
         11 . The method of  claim 3 , wherein the nucleic acid molecule is a DNA molecule.  
     
     
         12 . The method of  claim 3 , wherein the nucleic acid molecule is a RNA molecule.  
     
     
         13 . The method of  claim 3 , wherein the conjugate comprises at least one polymerase chain reaction primer.  
     
     
         14 . The method of  claim 1 , wherein the one or more analytes is a polypeptide.  
     
     
         15 . The method of  claim 14 , wherein the semiconductor nanocrystal conjugate comprises an antibody.  
     
     
         16 . The method of  claim 1 , wherein the semiconductor nanocrystal conjugate comprises an aptamer.  
     
     
         17 . A method of detecting one or more target analytes in a sample containing or suspected of containing the one or more analytes, comprising the steps of: 
 (a) providing an unlabeled specific-binding molecule on a solid support;    (b) combining said sample with said specific-binding molecule, wherein said combining is performed under conditions that allow formation of a first complex comprising said specific-binding molecule and said analyte, when present;    (c) removing any unbound sample;    (d) combining said first complex with a semiconductor nanocrystal conjugate, wherein said combining is performed under conditions that allow formation of a second complex comprising said conjugate and said analyte, when present;    (e) removing any unbound conjugate;    (f) detecting the presence of the second complex, if present, by monitoring a spectral emission mediated by the semiconductor nanocrystal in the second complex, wherein the emission indicates the presence of one or more target analytes in the sample.    
     
     
         18 . The method of  claim 17 , wherein there is a plurality of target analytes and the method further comprises: 
 (a) providing an unlabeled specific-binding molecule on a solid support specific for each target analyte and providing a semiconductor nanocrystal conjugate specific for each target analyte, wherein each semiconductor nanocrystal conjugate has an emission spectrum distinct from the other semiconductor nanocrystal conjugates; and    (b) detecting the presence of the target analytes by monitoring the spectral emissions of the sample, wherein the emissions indicate the presence of the target analytes in the sample.    
     
     
         19 . The method of  claim 17 , wherein the one or more analytes is a nucleic acid molecule.  
     
     
         20 . The method of  claim 19 , wherein the nucleic acid molecule is a DNA molecule.  
     
     
         21 . The method of  claim 19 , wherein the nucleic acid molecule is a RNA molecule.  
     
     
         22 . The method of  claim 17 , wherein the one or more analytes is a polypeptide.  
     
     
         23 . The method of  claim 17 , wherein the semiconductor nanocrystal conjugate comprises an antibody.  
     
     
         24 . The method of  claim 17 , wherein the semiconductor nanocrystal conjugate comprises an aptamer.  
     
     
         25 . A method of detecting one or more target analytes in a sample containing or suspected of containing the one or more analytes, comprising the steps of: 
 (a) providing the sample on a solid support;    (b) combining with said sample a specific-binding molecule, wherein (i) said specific-binding molecule comprises a first member of a binding pair, and (ii) said combining is performed under conditions that allow formation of a first complex comprising said specific-binding molecule and said analyte, when present;    (c) removing any unbound specific-binding molecule;    (d) combining said first complex with a second member of the binding pair, wherein (i) said second member of the binding pair is linked to a first semiconductor nanocrystal; and (ii) said combining is performed under conditions that allow formation of a second complex comprising the binding pair and said one or more analytes; and    (e) detecting the presence of the second complex, if present, by monitoring a spectral emission mediated by the first semiconductor nanocrystal in the second complex, wherein the emission indicates the presence of one or more target analytes in the sample.    
     
     
         26 . The method of  claim 25 , wherein the one or more analytes is a nucleic acid molecule.  
     
     
         27 . The method of  claim 26 , wherein the nucleic acid molecule is contained within a chromosome or chromosomal fragment.  
     
     
         28 . The method of  claim 26 , wherein the nucleic acid molecule is a DNA molecule.  
     
     
         29 . The method of  claim 26 , wherein the nucleic acid molecule is a RNA molecule.  
     
     
         30 . The method of  claim 25 , wherein the one or more analytes is a polynucleotide.  
     
     
         31 . The method of  claim 30 , wherein the specific-binding molecule is a polymerase chain reaction amplification product and said first member of the binding pair is incorporated in the amplification product.  
     
     
         32 . The method of  claim 26 , wherein the specific-binding molecule is an aptamer.  
     
     
         33 . The method of  claim 25 , wherein the one or more analytes is a polypeptide.  
     
     
         34 . The method of  claim 33 , wherein the first member of the binding pair is a first antibody and the second member of the binding pair is a second antibody reactive with the first antibody.  
     
     
         35 . The method of  claim 25 , wherein the first member of the binding pair is biotin and the second member of the binding pair is streptavidin.  
     
     
         36 . The method of  claim 33 , wherein the first member of the binding pair is biotin and the second member of the binding pair is streptavidin.  
     
     
         37 . The method of  claim 25 , wherein the first member of the binding pair is digoxygenin and the second member of the binding pair is an antibody directed against digoxygenin.  
     
     
         38 . The method of  claim 25 , wherein the first member of the binding pair is flourescein and the second member of the binding pair is an antibody directed against flourescein.  
     
     
         39 . The method of  claim 25 , wherein there is more than one analyte, wherein the method further comprises: 
 combining with said sample a second specific-binding molecule, wherein (i) said second specific-binding molecule comprises a first member of a second binding pair, and (ii) said combining is performed under conditions that allow formation of a third complex comprising said second specific-binding molecule and said analyte, when present;    removing any unbound second specific-binding molecule;    combining said third complex with a second member of the second binding pair, wherein (i) said second member of the second binding pair is linked to a second semiconductor nanocrystal that has an emission spectrum distinct from the first semiconductor nanocrystal; and (ii) said combining is performed under conditions that allow formation of a fourth complex comprising the second binding pair and an analyte; and    detecting the presence of the fourth complex, if present, by monitoring a second spectral emission mediated by the second semiconductor nanocrystal in the fourth complex, wherein the second emission indicates the presence of more than one target analyte in the sample.    
     
     
         40 . A method of detecting one or more target analytes in a sample containing or suspected of containing the one or more analytes, comprising the steps of: 
 (a) providing a first complex comprising at least one specific-binding molecule to which is bound a semiconductor nanocrystal conjugate, wherein said semiconductor nanocrystal has a characteristic spectral emission and wherein said conjugate specifically binds to said specific-binding molecule;    (b) combining said sample with said first complex, wherein said combining is performed under conditions that allow formation of a second complex comprising said specific-binding molecule and said analyte, when present;    (c) detecting the presence of the second complex, if present, by monitoring the characteristic spectral emission of the semiconductor nanocrystal, wherein a change in the characteristic spectral emission indicates the presence of one or more target analytes in the sample.    
     
     
         41 . The method of  claim 40 , wherein a plurality of first complexes are provided each comprising a different specific-binding molecule each bound to a conjugate which specifically binds each specific-binding molecule and where each specific-binding molecule binds a different analyte, and wherein each conjugate bound to a different specific-binding molecule comprises a semiconductor nanocrystal that has a characteristic spectral emission distinct from the other semiconductor nanocrystals; and wherein changes in the spectral emission of any selected semiconductor nanocrystal associated with a particular specific-binding molecule in a first complex, indicates the presence of an analyte that binds to the particular specific-binding molecule.  
     
     
         42 . The method of  claim 40 , wherein said specific-binding molecule is radiolabeled, wherein when said conjugate is bound to said specific-binding molecule the semiconductor nanocrystal emits light.  
     
     
         43 . The method of  claim 42 , wherein a plurality of first complexes are provided each comprising a different specific-binding molecule each bound to a conjugate which specifically binds each specific-binding molecule and where each specific-binding molecule binds a different analyte, and wherein each conjugate bound to a different specific-binding molecule comprises a semiconductor nanocrystal that has a characteristic spectral emission distinct from the other semiconductor nanocrystals; and wherein changes in the spectral emission of any selected semiconductor nanocrystal associated with a particular specific-binding molecule in a first complex, indicates the presence of an analyte that binds to the particular specific-binding molecule.  
     
     
         44 . The method of  claim 40 , wherein said specific-binding molecules are selected from the group consisting of a protein, an oligonucleotide, a polysaccharide or a small molecule.  
     
     
         45 . The method of  claim 44 , wherein said specific-binding molecules are receptor molecules and a conjugate is provided that binds to each receptor.