Detection of enhanced multiplex signals by surface enhanced raman spectroscopy (sers)

Abstract

Various methods of using Raman-active or SERS-active probe constructs to detect analytes in biological samples, such as the nucleic acid and/or protein-containing analytes in a body fluid are provided.

1 - 26 . (canceled) 
   
   
       27 . A biological target complex comprising:
 a target analyte bound to a first specific binding member;   a second specific binding member that binds to the first specific binding member forming a target complex, wherein the second specific binding member comprises a seed particle suitable for catalyzing the formation of a surface enhanced Raman scattering (SERS) substrate, wherein the SERS substrate is suitable to be activated to provide a SERS effect;   a capture reagent bound to a solid substrate, wherein the capture reagent comprises a Raman label, wherein the target analyte binds to the capture reagent forming a biological target complex; and   a layer of roughened metal over the substrate or the biological target complex.   
   
   
       28 . The biological target complex of  claim 27 , wherein the layer of roughened metal comprises roughness features on the order of tens of nanometers. 
   
   
       29 . The biological target complex of  claim 28 , wherein plasmon excitation due to electromagnetic irradiation of the biological target complex is confined to the roughness features. 
   
   
       30 . The biological target complex of  claim 29 , wherein the layer of roughened metal comprises a thickness of approximately one-half the wavelength of the electromagnetic irradiation. 
   
   
       31 . The biological target complex of  claim 27 , wherein the layer is transparent. 
   
   
       32 . The biological target complex of  claim 27 , wherein the layer of roughened metal comprises gold, silver, copper, or aluminum. 
   
   
       33 . The biological target complex of  claim 27 , wherein the target analyte is a DNA, RNA, polypeptide, antibody, antigen, carbohydrate or small molecule. 
   
   
       34 . The biological target complex of  claim 27 , wherein the capture reagent is a DNA, RNA, polypeptide, antibody, antigen, carbohydrate or small molecule. 
   
   
       35 . The biological target complex of  claim 27 , wherein the first or second specific binding member is a DNA, RNA, antibody, antigen, polypeptide or carbohydrate. 
   
   
       36 . The biological target complex of  claim 27 , wherein the target analyte further comprises an ancillary specific binding member. 
   
   
       37 . A method comprising:
 a) providing a target analyte bound to a first specific binding member;   b) providing a capture reagent bound to a solid substrate, wherein the capture reagent comprises a Raman label;   c) contacting the target analyte of a) with the capture reagent of (b) under conditions suitable for forming a target analyte-capture reagent complex;   d) contacting, prior to, concurrently with, or subsequent to c) the first specific binding partner with a second specific binding member functionally associated with a seed particle suitable for associating with a SERS substrate, wherein the first specific binding member binds to the second specific binding member; and   e) coating either the substrate or the target analyte-capture reagent complex with a layer of roughened metal;   f) contacting the target analyte-capture reagent complex with electromagnetic radiation suitable for detecting a specific property associated with the analyte-capture reagent complex by Raman spectroscopy.   
   
   
       38 . The method of  claim 37 , wherein the layer of roughened metal comprises roughness features on the order of tens of nanometers. 
   
   
       39 . The method of  claim 37 , wherein plasmon excitation due to the electromagnetic irradiation is confined to the roughness features. 
   
   
       40 . The method of  claim 37 , wherein the layer comprises a thickness of approximately one-half the wavelength of the electromagnetic irradiation. 
   
   
       41 . The method of  claim 37 , wherein the layer of roughened meta is transparent. 
   
   
       42 . The method of  claim 37 , wherein the layer of roughened metal comprises gold, silver, copper, or aluminum. 
   
   
       43 . The method of  claim 37 , comprising forming the layer of roughened metal by vapor deposition of metal particles or application of metal colloids. 
   
   
       44 . The method of  claim 43 , wherein application of metal colloids comprises subjecting a colloidal solution of metal cations to reducing conditions to form metal nanoparticles in situ. 
   
   
       45 . The method of  claim 43 , wherein application of metal colloids comprises using seed particle to precipitate nanoparticles from a metal colloid solution. 
   
   
       46 . The method of  claim 45 , wherein the seed particle is selected from the group consisting of gold, Ag, Cu, Pt, Ag/Au, Pt/Au, Cu/Au coreshell and alloy particles. 
   
   
       47 . The method of  claim 37 , further comprising detecting a single analyte molecule. 
   
   
       48 . A system comprising:
 biological target complex comprising a target analyte bound to a first specific binding member, a second specific binding member that binds to the first specific binding member forming a target complex, the second specific binding member comprising a seed particle suitable for catalyzing the formation of a surface enhanced Raman scattering (SERS) substrate, a capture reagent bound to a solid substrate, the capture reagent comprising a Raman label, wherein the target analyte binds to the capture reagent forming the biological target complex and a layer of roughened metal over the substrate or the biological target complex; and   an electromagnetic radiation source.   
   
   
       49 . The system of  claim 48 , further comprising a Raman detection unit. 
   
   
       50 . The system of  claim 48 , wherein the electromagnetic radiation source comprises a frequency doubled Nd:YAG laser, a frequency doubled Ti:sapphire laser, a nitrogen laser, a helium-cadmium laser a light emitting diode, an Nd:YLF laser, ion lasers, or dye lasers. 
   
   
       51 . The system of  claim 48 , wherein the radiation source is either pulsed or continuous. 
   
   
       52 . The system of  claim 48 , further comprising confocal optics and a microscope objective. 
   
   
       53 . The system of  claim 48 , further comprising a flow through cell. 
   
   
       54 . The system of  claim 48 , further comprising a monochromator. 
   
   
       55 . The system of  claim 52 , wherein the confocal optics comprises one or more of dichroic filters, barrier filters, holographic notch filters, confocal pinholes, lenses, and mirrors. 
   
   
       56 . The system of  claim 49 , wherein the Raman detection unit comprises an avalanche photodiode interfaced with a computer. 
   
   
       57 . The system of  claim 56 , wherein the Raman detection unit is configured to count and digitize a signal. 
   
   
       58 . The system of  claim 49 , wherein the Raman detection unit comprises a double-grating spectrophotometer with a gallium-arsenide photomultiplier tube, Fourier-transform spectrographs, charged injection devices, photodiode arrays, InGaAs detectors, electronmultiplied CCD, intensified CCD or phototransistor arrays. 
   
   
       59 . The system of  claim 49 , wherein the Raman detection unit comprises a red-enhanced intensified charge-coupled device (RE-ICCD) detection system.