Raman-active reagents and the use thereof

Abstract

The present invention provides a new class of Raman-active reagents for use in biological and other applications, as well as methods and kits for their use and manufacture. Each reagent includes a Raman-active reporter molecule, a binding molecule, and a surface enhancing particle capable of causing surface enhanced Raman scattering (SERS). The Raman-active reporter molecule and the binding molecule are affixed to the particle to give both a strong SERS signal and to provide biological functionality, i.e. antigen or drug recognition. The Raman-active reagents can function as an alternative to fluorescence-labeled reagents, with advantages in detection including signal stability, sensitivity, and the ability to simultaneously detect several biological materials. The Raman-active reagents also have a wide range of applications, especially in clinical fields (e.g., immunoassays, imaging, and drug screening).

1 . A method for determining the presence or amount of a target analyte in a test sample, the method comprising the steps of:
 (a) contacting a test sample with a Raman-active reagent, said Raman-active reagent comprising a Raman-active reporter molecule, a binding molecule, and a surface enhancing particle capable of causing surface enhanced Raman scattering, wherein said Raman-active reporter molecule is operably linked to the surface enhancing particle and provides a detectable or measurable Raman scattering signal when illuminated by an excitation source capable of inducing a Raman scattering, and wherein said binding molecule is operably linked to the surface enhancing particle and is capable of specifically binding to a target analyte in the test sample;   (b) allowing the Raman-active reagent to bind to the target analyte to form a reagent/analyte complex;   (c) inducing a Raman scattering signal by illuminating the reagent/analyte complex with the excitation source to induce Raman scattering;   (d) measuring the intensity of the Raman scattering signal; and   (e) determining the presence or amount of the target analyte in the test sample.   
     
     
         2 . The method of  claim 1  wherein the binding molecule is selected from the group of lectins, lectin fragments, lectin derivatives, antigens, monoclonal antibodies, polyclonal antibodies, immunoreactive fragments, immunoreactive derivatives, peptides, haptens, aptamers, nucleic acid molecules, crown ethers, cyclodextrins, cryptands, and calixarenes. 
     
     
         3 . The method of  claim 1  wherein the Raman-active reporter molecule is selected from the group of dithiobisbenzonic acid, 4-mercaptobenzoic acid, 2-naphthalenethiol, thiophenol, 4,4′-dithiobis(succinimidylbenzoate), direct red 81, Chicago sky blue, p-dimethylaminoazobenzene, 4-(4-Aminophenylazo)phenylarsonic acid monosodium salt, 1,5-difluoro-2,4-dinitrobenzene, arsenazo I, basic fuchsin, disperse orange 3, HABA (2-(4-hydroxyphenylazo)-benzoic acid, erythrosine B, trypan blue, ponceau S, ponceau SS, 5,5′-dithiobis(2-nitrobenzoic acid), metal complexes and polymeric particles. 
     
     
         4 . The method of  claim 1  wherein the surface enhancing particle comprises a metallic material. 
     
     
         5 . The method of  claim 4  wherein the metallic material is either gold, silver, copper, platinum, aluminum, gallium, indium, zinc, cadmium, lithium, or sodium. 
     
     
         6 . The method of  claim 5  wherein the metallic material is gold. 
     
     
         7 . The method of  claim 1  wherein the surface enhancing particle is either a silica, plastic, glass, carbon, ceramic or magnetic material, coated with a metallic material. 
     
     
         8 . The method of  claim 7  wherein the metallic coating is either gold, silver, copper, platinum, aluminum, gallium, indium, zinc, cadmium, lithium or sodium. 
     
     
         9 . The method of  claim 8  wherein the metallic coating is gold. 
     
     
         10 . The method of  claim 1  wherein the binding molecule is operably linked to the Raman-active reporter molecule, the Raman-active reporter molecule being operably linked to the surface enhancing particle. 
     
     
         11 . The method of  claim 10  wherein the binding molecule is operably linked to the Raman-active reporter molecule by a linker molecule. 
     
     
         12 . The method of  claim 10  wherein the Raman-active reporter molecule is operably linked to the surface enhancing particle by a linker molecule. 
     
     
         13 . The method of  claim 1  wherein the binding molecule is operably linked to the surface enhancing particle by a linker molecule. 
     
     
         14 . The method of  claim 1  wherein the Raman-active reporter molecule is operably linked to the surface enhancing particle by a linker molecule. 
     
     
         15 . The method of  claim 1  further comprising the step of contacting the reagent/analyte complex to a substrate that binds the reagent/analyte complex prior to inducing the Raman scattering with the excitation source. 
     
     
         16 . The method of  claim 1  wherein the test sample is first placed in contact with a substrate prior to being placed in contact with the Raman-active reagent. 
     
     
         17 . The method of  claim 1  further comprising the step of exposing the reagent/analyte complex to a magnetic force that causes the reagent/analyte complex to be separated from the test sample. 
     
     
         18 . A method for determining the presence or amount of a target analyte, the method comprising the steps of:
 (a) contacting a target analyte with a Raman-active reagent, said Raman-active reagent comprising a Raman-active reporter molecule, a binding molecule, and a surface enhancing particle capable of causing surface enhanced Raman scattering, wherein said Raman-active reporter molecule is operably linked to the surface enhancing particle and provides a detectable or measurable Raman scattering signal when illuminated by an excitation source capable of inducing a Raman scattering, and wherein said binding molecule is operably linked to the surface enhancing particle and is capable of specifically binding to the target analyte;   (b) allowing the Raman-active reagent to bind to the target analyte to form a reagent/analyte complex;   (c) inducing a Raman scattering signal by illuminating the reagant/analyte complex with the excitation source to induce Raman scattering;   (d) measuring the intensity of the Raman scattering signal to determine the presence or amount of the target analyte.   
     
     
         19 . The method of  claim 18  wherein the binding molecule is selected from the group of lectins, lectin fragments, lectin derivatives, antigens, monoclonal antibodies, polyclonal antibodies, immunoreactive fragments, immunoreactive derivatives, peptides, haptens, aptamers, nucleic acid molecules, crown ethers, cyclodextrins, cryptands, and calixarenes. 
     
     
         20 . The method of  claim 18  wherein the Raman-active reporter molecule is selected from the group of dithiobisbenzonic acid, 4-mercaptobenzoic acid, 2-naphthalenethiol, thiophenol, 4,4′-dithiobis(succinimidylbenzoate), direct red 81, Chicago sky blue, p-dimethylaminoazobenzene, 4-(4-Aminophenylazo)phenylarsonic acid monosodium salt, 1,5-difluoro-2,4-dinitrobenzene, arsenazo I, basic fuchsin, disperse orange 3, HABA (2-(4-hydroxyphenylazo)-benzoic acid, erythrosine B, trypan blue, ponceau S, ponceau SS, 5,5′-dithiobis(2-nitrobenzoic acid), metal complexes and polymeric particles. 
     
     
         21 . The method of  claim 18  wherein the surface enhancing particle comprises a metallic material. 
     
     
         22 . The method of  claim 21  wherein the metallic material is either gold, silver, copper, platinum, aluminum, gallium, indium, zinc, cadmium, lithium, or sodium. 
     
     
         23 . The method of  claim 22  wherein the metallic material is gold. 
     
     
         24 . The method of  claim 18  wherein the surface enhancing particle is either a silica, plastic, glass, carbon, ceramic or magnetic material, coated with a metallic material. 
     
     
         25 . The method of  claim 24  wherein the metallic coating is either gold, silver, copper, platinum, aluminum, gallium, indium, zinc, cadmium, lithium or sodium. 
     
     
         26 . The method of  claim 25  wherein the metallic coating is gold. 
     
     
         27 . The method of  claim 18  wherein the binding molecule is operably linked to the Raman-active reporter molecule, the Raman-active reporter molecule being operably linked to the surface enhancing particle. 
     
     
         28 . The method of  claim 27  wherein the binding molecule is operably linked to the Raman-active reporter molecule by a linker molecule. 
     
     
         29 . The method of  claim 27  wherein the Raman-active reporter molecule is operably linked to the surface enhancing particle by a linker molecule. 
     
     
         30 . The method of  claim 18  wherein the binding molecule is operably linked to the surface enhancing particle by a linker molecule. 
     
     
         31 . The method of  claim 18  wherein the Raman-active reporter molecule is operably linked to the surface enhancing particle by a linker molecule. 
     
     
         32 . The method of  claim 18  further comprising the step of contacting the reagent/analyte complex to a substrate that binds the reagent/analyte complex prior to inducing the Raman scattering with the excitation source. 
     
     
         33 . The method of  claim 18  wherein the target analyte is first placed in contact with a substrate prior to being placed in contact with the Raman-active reagent. 
     
     
         34 . The method of  claim 18  further comprising the step of exposing the reagent/analyte complex to a magnetic force. 
     
     
         35 . A method for determining the presence or amount of a target analyte in a test sample, the method comprising the steps of:
 (a) contacting a test sample with a Raman-active reagent, said Raman-active reagent comprising a Raman-active reporter molecule, a binding molecule, and a surface enhancing particle capable of causing surface enhanced Raman scattering, wherein said Raman-active reporter molecule is operably linked to the surface enhancing particle and provides a detectable or measurable Raman scattering signal when illuminated by an excitation source capable of inducing a Raman scattering, and wherein said binding molecule is operably linked to the surface enhancing particle and is capable of specifically binding to a target analyte if the target analyte is present in the test sample;   (b) illuminating the test sample with the excitation source to induce Raman scattering;   (c) measuring the intensity of the Raman scattering signal to determine the presence or amount of the target analyte in the test sample.   
     
     
         36 . The method of  claim 35  wherein the binding molecule is selected from the group of lectins, lectin fragments, lectin derivatives, antigens, monoclonal antibodies, polyclonal antibodies, immunoreactive fragments, immunoreactive derivatives, peptides, haptens, aptamers, nucleic acid molecules, crown ethers, cyclodextrins, cryptands, and calixarenes. 
     
     
         37 . The method of  claim 35  wherein the Raman-active reporter molecule is selected from the group of dithiobisbenzonic acid, 4-mercaptobenzoic acid, 2-naphthalenethiol, thiophenol, 4,4′-dithiobis(succinimidylbenzoate), direct red 81, Chicago sky blue, p-dimethylaminoazobenzene, 4-(4-Aminophenylazo)phenylarsonic acid monosodium salt, 1,5-difluoro-2,4-dinitrobenzene, arsenazo I, basic fuchsin, disperse orange 3, HABA (2-(4-hydroxyphenylazo)-benzoic acid, erythrosine B, trypan blue, ponceau S, ponceau SS, 5,5′-dithiobis(2-nitrobenzoic acid), metal complexes and polymeric particles. 
     
     
         38 . The method of  claim 35  wherein the surface enhancing particle comprises a metallic material. 
     
     
         39 . The method of  claim 38  wherein the metallic material is either gold, silver, copper, platinum, aluminum, gallium, indium, zinc, cadmium, lithium, or sodium. 
     
     
         40 . The method of  claim 39  wherein the metallic material is gold. 
     
     
         41 . The method of  claim 35  wherein the surface enhancing particle is either a silica, plastic, glass, carbon, ceramic or magnetic material, coated with a metallic material. 
     
     
         42 . The method of  claim 41  wherein the metallic coating is either gold, silver, copper, platinum, aluminum, gallium, indium, zinc, cadmium, lithium or sodium. 
     
     
         43 . The method of  claim 42  wherein the metallic coating is gold. 
     
     
         44 . The method of  claim 35  wherein the binding molecule is operably linked to the Raman-active reporter molecule, the Raman-active reporter molecule being operably linked to the surface enhancing particle. 
     
     
         45 . The method of  claim 44  wherein the binding molecule is operably linked to the Raman-active reporter molecule by a linker molecule. 
     
     
         46 . The method of  claim 44  wherein the Raman-active reporter molecule is operably linked to the surface enhancing particle by a linker molecule. 
     
     
         47 . The method of  claim 35  wherein the binding molecule is operably linked to the surface enhancing particle by a linker molecule. 
     
     
         48 . The method of  claim 35  wherein the Raman-active reporter molecule is operably linked to the surface enhancing particle by a linker molecule. 
     
     
         49 . The method of  claim 35  further comprising a step of contacting the test sample to a substrate capable of binding to the target analyte or a reagent/analyte complex formed by the specific binding of a target analyte to the Raman-active reagent. 
     
     
         50 . A method for determining the presence or amount of a plurality of different target analytes in a test sample, the method comprising the steps of:
 (a) contacting a test sample with a plurality of different Raman-active reagents, each Raman-active reagent comprising a Raman-active reporter molecule, a binding molecule, and a surface enhancing particle capable of causing surface enhanced Raman scattering, the Raman-active reporter molecule being operably linked to the surface enhancing particle and capable of providing a detectable or measurable Raman scattering signal when illuminated by an excitation source capable of inducing Raman scattering, and the binding molecule being operably linked to the surface enhancing particle and capable of specifically binding to a target analyte in the test sample, wherein the binding molecule of a first Raman-active reagent has a specific binding affinity to a first target analyte and wherein the binding molecule of a second Raman-active reagent has a specific binding affinity to a second target analyte;   (b) allowing the first Raman-active reagent to bind to the first target analyte to form a first reagent/analyte complex and the second Raman-active reagent to bind to the second target analyte to form a second reagent/analyte complex;   (c) inducing a Raman scattering signal by illuminating the first reagant/analyte complex and the second reagant/analyte complex with the excitation source;   (d) measuring the intensity of the Raman scattering signal to determine the presence or amount of the first target analyte and second target analyte.   
     
     
         51 . The method of  claim 50  further comprising the step of contacting the first reagent/analyte complex and the second reagent/analyte complex to a substrate capable of binding either or both the first reagent/analyte complex or the second reagent/analyte complex, prior to inducing the Raman scattering with the excitation source. 
     
     
         52 . The method of  claim 50  wherein the test sample is first placed in contact with a substrate prior to being placed in contact with the plurality of different Raman-active reagents. 
     
     
         53 . The method of  claim 50  further comprising the step of exposing the first reagent/analyte complex and the second reagent/analyte to a magnetic force to separate the first reagent/analyte complex from the second reagent/analyte.