Nanoparticle probs with Raman spectrocopic fingerprints for analyte detection

Abstract

The invention encompasses reagents comprising particles with at least one Raman dye and a specific binding members bound thereto and methods of using such reagents. The invention also encompases reagents of a specific binding member and two or more different Raman dyes and methods for using such reagents. New types of particle probes having a specific binding member bound thereto are described. These reagents are used in a novel detection strategy that utilizes the catalytic properties of the Au nanoparticles to generate a silver coating that can behave as a surface-enhanced Raman scattering (SERS) promoter for the dye-labeled particles that have been captured by target and an underlying chip in microarray format. The strategy provides the high sensitivity and high selectivity attributes of grey-scale scanometric detection but provides a route to multiplexing and ratioing capabilities since a very large number of probes can be designed based upon the concept of using a Raman tag as a spectroscopic fingerprint in detection. These spectra are used as fingerprints to differentiate oligonucleotide or other targets in one solution. This method has been used to distinguish six dissimilar DNA targets with six Raman labeled nanoparticle probes, and also two RNA targets with single nucleotide polymorphisms (SNPs).

What is claimed:  
     
         1 . A reagent comprising a particle having bound there to at least one Raman label and a specific binding member wherein the Raman label can be activated to provide a SERS effect.  
     
     
         2 . The reagent of  claim 1  wherein the particle has two or more different Raman labels.  
     
     
         3 . The reagent of  claim 1  wherein the specific binding member is a DNA, RNA, polypeptide, antibody, antigen, carbohydrate or small molecule.  
     
     
         4 . The reagent of  claim 1  wherein the particle is a gold, Ag, Cu, Pt, Ag/Au, Pt/Au, Cu/Au coreshell and alloy particles.  
     
     
         5 . The reagent of  claim 1  where the Raman label is activated by a silver, gold or copper stain.  
     
     
         6 . A reagent comprising a specific binding member having two or more different known labels bound thereto.  
     
     
         7 . A reagent of  claim 6  wherein the specific binding member is a DNA, RNA, antibody, antigen, polypeptide or carbohydrate.  
     
     
         8 . A method for detecting an analyte comprising: 
 (a) forming a complex of the reagent of  claim 1  and the analyte;    (b) binding the complex to a substrate;    (c) staining the complex on the substrate to activate the SERS effect in the Raman label; and    (d) measuring the SERS effect.    
     
     
         9 . The method of  claim 8  wherein the complex is bound to the substrate through one or more specific binding substances.  
     
     
         10 . A method for detecting an analyte comprising: 
 (a) binding the analyte to a substrate;    (b) complexing the reagent of  claim 1  with the analyte on the substrate    (c) staining the complex on the substrate to activate the SERS effect in the Raman label;    (d) measuring the SRS effect.    
     
     
         11 . The method of  claim 10  wherein the reagent is indirectly bound to the analyte on the substrate through one or more specific binding substances.  
     
     
         12 . The method of  claim 8  or  10  wherein the target analyte comprises an antibody, an antigen, a hapten, a receptor, a ligand, a protein, a peptide, a polypeptide, a nucleic acid, a membrane or membrane fraction, a lipid, a membrane-protein complex, a carbohydrate, a virus, a cell or macromolecule or molecular complex.  
     
     
         13 . The method of  claim 8  or  10  wherein the specific binding member comprises an antibody, an antigen, a receptor, a ligand, a protein, a polypeptide, small molecule or a nucleic acid.  
     
     
         14 . The method of  claim 8  or  10  wherein the substrate has a plurality of different first specific binding member attached thereto in an array to allow for the detection of multiple types of target analytes.  
     
     
         15 . The method of  claim 8  or  10  wherein the substrate comprises a glass slide, microplate well, beads or optical fiber.  
     
     
         16 . A method for detecting for the presence or absence of one or more target analytes in a sample, the target analytes having at least two binding sites, said method comprising: 
 providing a substrate having bound thereto one or more types of a first specific binding member for immobilizing the target analyte onto said substrate;    providing one or more types of particles having bound thereto (a) one or more Raman labels; and (b) a second specific binding member for binding to a specific target analyte, wherein (i) the Raman active label bound to each type of particle is different and serves as an identifier for a specific target analyte; (ii) the second specific binding member bound to each type of particle is different and is targeted to a specific target analyte; and (iii) the Raman label comprises at least one Raman label.    contacting the particles, the sample and the substrate under conditions effective for specific binding interactions between the target analyte and first and second specific binding member so as to form a test substrate having particles complexed thereto in the presence of one or more target analytes in the sample;    contacting the test substrate with a staining material to produce a detection substrate having a surface capable of causing surface-enhanced Raman scattering (SERS); and    determining for the presence of said particle complexes on said detection substrate as an indication of the presence of one or more target analytes in the sample by obtaining and analyzing a SERS spectrum.    
     
     
         17 . A method for detecting for the presence or absence of one or more target nucleic acids in a sample, the sequence of the nucleic acid having at least two portions, said method comprising: 
 providing a substrate having oligonucleotides bound thereto, the oligonucleotides bound to the substrate having a sequence that is complementary to a first portion of the nucleic acid;    providing one or more types of particles comprising oligonucleotides bound thereto and a Raman active label bound to a portion of the oligonucleotides, wherein (i) at least some of the oligonucleotides attached to each type of particle have a sequence that is complementary to a second portion of the sequence of a specific target nucleic acid; and (ii) the Raman label bound to each type of particles is different and serves as an identifier for a specific target nucleic acid, said Raman label comprising at least one Raman label.    contacting the particles, the substrate, and the sample under conditions effective for hybridization of the oligonucleotides bound to the substrate with the first portion of the nucleic acid and for hybridization of the oligonucleotides attached to the particle with the second portion of the nucleic acid so as to form a test substrate having one or more particle complexes bound thereto when one or more target nucleic acids are present in said sample;    contacting the test substrate with a staining material to produce a detection substrate having a surface capable of causing surface-enhanced Raman scattering (SERS); and    determining for the presence of said particle complexes on said detection substrate as an indication of the presence of one or more target nucleic acids in the sample by obtaining and analyzing a SERS spectrum.    
     
     
         18 . A method for detecting for the presence or absence of a target nucleic acid in a sample, the sequence of the nucleic acid having at least two portions, said method comprising: 
 providing a substrate having oligonucleotides bound thereto, the oligonucleotides bound to the substrate having a sequence that is complementary to a first portion of the nucleic acid;    providing a particle comprising oligonucleotides bound thereto and a Raman label bound to a portion of the oligonucleotides, wherein (i) at least some of the oligonucleotides attached to the particle have a sequence that is complementary to a second portion of the nucleic acid; and (ii) the Raman label bound to particles serves as an identifier for the target nucleic acid, said Raman label comprising at least one Raman label providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;    contacting the particles, the substrate, and the sample under conditions effective for hybridization of the oligonucleotides bound to the substrate with the first portion of the nucleic acid and for hybridization of the oligonucleotides attached to the particle with the second portion of the nucleic acid so as to form a test substrate having a particle complex bound thereto when said target nucleic acid is present in said sample;    contacting the test substrate with a staining material to produce a detection substrate having a surface capable of causing surface-enhanced Raman scattering (SERS); and    determining for the presence of said particle complex on said detection substrate as an indication of the presence of the target nucleic acid in the sample by obtaining and analyzing a SERS spectrum.    
     
     
         19 . A method for screening one or more molecules to determine whether the molecule is a ligand to one or more specific receptors, the molecules are present in a sample, said method comprising: 
 providing a substrate having bound thereto one or more specific receptors;    providing conjugates comprising particles, oligonucleotides bound to the particles, a Raman active label bound to a portion of the oligonucleotides, and the molecule from said sample bound to a portion of the oligonucleotides, wherein said Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;    contacting the particles, sample and substrate under conditions effective for specific binding interactions between the molecule bound to the particles with the specific receptor bound to the substrate so as to form a test substrate having particles complexed thereto when the molecule is a ligand to a specific receptor;    contacting the test substrate with a staining material to produce a detection substrate having a surface capable of causing surface-enhanced Raman scattering (SERS); and    determining for the presence of said particle complexes on said detection substrate as a confirmation of a ligand to a specific receptor by obtaining and analyzing a SERS spectrum.    
     
     
         20 . A test kit comprising the reagent of  claim 1  in one container and a Raman enhancer stain in another container.  
     
     
         21 . A test kit comprising the reagent of  claim 1  in one container and a silver, gold or copper stain Raman enhancer in another container.  
     
     
         22 . A fiber optic detection device comprising a bundle of optical fibers terminating with ends of the optical fiber wherein a plurality of the optical fibers have a reagent of  claim 1  located at the ends of the optical fiber.  
     
     
         23 . The fiber optic detection device of  claim 20  wherein two or more of the reagents of claims  1  at the ends of the optical fiber have different specific binding member and different Raman labels.