Real-time, single-step bioassay using nanoplasmonic resonator with ultra-high sensitivity

Abstract

A nanoplasmonic resonator (NPR) comprising a metallic nanodisk with alternating shielding layer(s), having a tagged biomolecule conjugated or tethered to the surface of the nanoplasmonic resonator for highly sensitive measurement of enzymatic activity. NPRs enhance Raman signals in a highly reproducible manner, enabling fast detection of protease and enzyme activity, such as Prostate Specific Antigen (paPSA), in real-time, at picomolar sensitivity levels. Experiments on extracellular fluid (ECF) from paPSA-positive cells demonstrate specific detection in a complex bio-fluid background in real-time single-step detection in very small sample volumes.

1 . A nanoplasmonic resonator (NPR) comprising a plurality of metallic nanodisks with alternating shielding layer(s), having a tagged biomolecule conjugated or tethered to the surface of the nanoplasmonic resonator. 
     
     
         2 . The resonator of  claim 1  wherein the tag is a Raman active tag. 
     
     
         3 . The resonator of  claim 1  wherein the tag is a fluorescent tag. 
     
     
         4 - 5 . (canceled) 
     
     
         6 . The resonator of  claim 1  wherein the shielding layer comprises a material having a constant Raman spectra. 
     
     
         7 . The resonator of  claim 6  wherein the shielding layer is selected from the group consisting of silicon dioxide, quartz, polystyrene, silica, and dextran. 
     
     
         8 . The resonator of  claim 1  wherein the biomolecule is a peptide linked to a Raman active tag, wherein the peptide comprising an amino acid sequence that can be specifically modified or cleaved by an enzyme. 
     
     
         9 . (canceled) 
     
     
         10 . A nanoplasmonic resonance SERS detection platform comprising a patterned substrate featuring a surface enhanced Raman scattering (SERS) nanoplasmonic resonator (NPR) singly or in an array, wherein the nanoplasmonic resonator has a biomolecule conjugated thereto. 
     
     
         11 . The platform of  claim 10  wherein the substrate on which the nanoplasmonic resonator is patterned can be comprised of quartz, polystyrene, silica, dextran, or any other materials with constant Raman spectra. 
     
     
         12 . The platform of  claim 10  wherein the nanoplasmonic resonator comprising metallic nanodisks with alternating shielding layer(s), having a tagged biomolecule conjugated or tethered to the surface of the nanoplasmonic resonator, wherein the tag is a Raman active tag for SERS detection. 
     
     
         13 . The platform of  claim 12  wherein the nanodisk comprises a thin layer of gold or silver. 
     
     
         14 . The platform of  claim 12  wherein the shielding layer comprises silicon dioxide, quartz, polystyrene, silica, or dextran. 
     
     
         15 . The platform of  claim 10  wherein the biomolecule is a peptide linked to a Raman active tag, wherein the peptide comprising a specific sequence that can be specifically modified or cleaved by an enzyme. 
     
     
         16 . The platform of  claim 10  wherein the nanoplasmonic resonators in an array are the same or different. 
     
     
         17 . The platform of  claim 15 , wherein a plurality of members of a biomolecule library are conjugated to nanoplasmonic resonators comprising said array and spatially separated in either a random array or ordered microarray format. 
     
     
         18 .- 19 . (canceled) 
     
     
         20 . A method for in vitro detection and measurement of enzymatic activity using a nanosensor comprising a nanoplasmonic resonator (NPR) with at least picomolar sensitivity, wherein the NPR enhances Raman spectra intensity in Surface-Enhanced Raman Spectroscopy (SERS) and enables single-step detection of enzymatic activity. 
     
     
         21 . A method for real-time reaction monitoring of enzyme activity comprising:
 (a) providing an array of peptide-conjugated nanoplasmonic resonators patterned on a substrate, wherein each nanoplasmonic resonator (NPR) comprising metallic nanodisks with alternating shielding layer(s) and having a tagged biomolecule conjugated or tethered to the surface of the nanoplasmonic resonator, wherein the tag is a Raman tag molecules, each being the same or different, and wherein each peptide comprises a sequence recognized and modified by a specific enzyme using a specific reaction;   (b) providing a biological sample suspected of containing an enzyme,   (c) contacting said sample with said array of nanoplasmonic resonators;   (d) allowing said reaction to be carried out; and   (e) measuring detection of the enzyme by SERS detection.   
     
     
         22 . The method of  claim 21 , wherein a biomolecule library is conjugated to the nanoplasmonic resonators and spatially separated in either a random array or ordered microarray format. 
     
     
         23 . The method of  claim 22 , wherein the array of the biomolecule-nanoplasmonic resonators detect the activity of multiple enzymes simultaneously. 
     
     
         24 - 26 . (canceled)