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, the Raman-active reagent including a reactive group, a binding molecule, and a surface enhancing particle capable of causing surface enhanced Raman scattering, the Raman-active reporter molecule being chemically linked to the surface enhancing particle and providing a detectable or measurable Raman scattering signal when illuminated by an excitation source capable of inducing a Raman scattering, the reactive group operably linked to the binding molecule, and wherein binding molecule is capable of specifically binding to a 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; 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 binding molecule is anti-prostate-specific antigen (anti-PSA). 
     
     
         4 . 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-hydrozyphenylazo)-benzoic acid, erythrosine B, trypan blue, ponceau S, ponceau SS, 5,5′-dithiobis(2-nitrobenzoic acid), metal complexes and polymeric particles. 
     
     
         5 . The method of  claim 1  wherein the reactive group is a terminal functional group of a reactive compound that is selected from the group consisting of succinimides, maleimides, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes carbodiimides, carbonates, arylating agents, acryloyl derivatives, diazoalkanes, diazoacetyl compounds, anhydrides, aziridines, imidoesters, or carbonyldiimidazole. 
     
     
         6 . The method of  claim 1  wherein the reactive group is a terminal succinimide group of the reactive compound N-hydroxysuccinimide (NHS). 
     
     
         7 . The method of  claim 1  wherein the surface enhancing particle comprises a metallic material. 
     
     
         8 . The method of  claim 1  wherein the metallic material is either gold, silver, copper, platinum, aluminum, gallium, indium, zinc, cadmium, lithium, or sodium. 
     
     
         9 . The method of  claim 8  wherein the metallic material is gold. 
     
     
         10 . 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. 
     
     
         11 . The method of  claim 10  wherein the metallic coating is either gold, silver, copper, platinum, aluminum, gallium, indium, zinc, cadmium, lithium or sodium. 
     
     
         12 . The method of  claim 11  wherein the metallic coating is gold. 
     
     
         13 . 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. 
     
     
         14 . 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. 
     
     
         15 . 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. 
     
     
         16 . The method of  claim 1  wherein the Raman-active reagent permits the separation between the reporter molecule and particle surface to be minimized and maximizes the number of reporter molecules on each particle.