Plasmonic substrates for metal-enhanced fluorescence based sensing, imaging and assays

Abstract

Techniques for metal enhanced fluorescence include determining a calibration curve that relates concentration of a particular analyte to at least one of intensity or lifetime of fluorescent emissions at a functionalized substrate in response to incident light, for a plurality of known concentrations of the particular analyte mixed with a reagent. The functionalized substrate comprises a plasmonic substrate and a bioactive target molecule that has an affinity for the particular analyte. The reagent comprises a detection molecule. A concentration of the particular analyte in a sample is determined directly from the calibration curve and measurements, in response to the incident light, of at least one of intensity or lifetime of fluorescent emissions at the functionalized substrate in contact with the sample and reagent.

1 . A method comprising:
 providing a functionalized substrate comprising a plasmonic substrate that is functionalized with a bioactive target molecule that has an affinity for a particular analyte;   providing a reagent comprising a detection molecule for the particular analyte;   determining a calibration curve that relates concentration of the particular analyte to at least one of intensity or lifetime of fluorescent emissions at the functionalized substrate in response to incident light for a plurality of known concentrations of the particular analyte mixed with the reagent,   contacting a sample and the reagent to the functionalized substrate;   obtaining measurements of at least one of intensity or lifetime of fluorescent emissions at the functionalized substrate in contact with the sample and reagent in response to the incident light; and   determining a concentration of the particular analyte in the sample directly from the calibration curve and the measurements.   
     
     
         2 . A method as recited in  claim 1 , wherein the sample and reagent are not rinsed from the functionalized substrate before obtaining the measurements. 
     
     
         3 . A method as recited in  claim 1 , wherein the sample comprises a living cell and the analyte is secreted by the living cell. 
     
     
         4 . A method as recited in  claim 1 , wherein the analyte is a cytokine. 
     
     
         5 . A method as recited in  claim 1 , further comprising;
 obtaining second measurements of at least one of intensity or lifetime of fluorescent emissions at the functionalized substrate in contact with the sample and reagent in response to the incident light at a later time; and   determining a concentration of the particular analyte in the sample at the later time directly from the calibration curve and the second measurements.   
     
     
         6 . A method as recited in one of  claim 1  wherein:
 the particular analyte comprises a plurality of different analytes; 
 the fluorescent emissions comprise emissions in a corresponding plurality of emission wavelength bands associated with a corresponding plurality of detection molecules comprising a corresponding plurality of different fluorophores; and 
 the bioactive target molecule comprise a corresponding plurality of different target molecules that have affinities for the plurality of different analytes. 
 
     
     
         7 . A method as recited in  claim 6 , wherein the plurality of different analytes consists of four different analytes. 
     
     
         8 . A method as recited in  claim 3 , further comprising determining viability of the living cell. 
     
     
         9 . A method as recited in  claim 3 , further comprising determining phenotype of the living cell. 
     
     
         10 . A method as recited in  claim 1 , wherein the plasmonic substrate comprises:
 a layer of metal configured as a mirror to reflect light,   a layer of dielectric material disposed on the mirror; and   a layer of metal nanoparticles disposed on the layer of dielectric material.   
     
     
         11 . A method as recited in  claim 10 , wherein the layer of metal nanoparticles has an optical density below about 1. 
     
     
         12 . A method as recited in  claim 10 , wherein a thickness of the dielectric layer is greater than about 20 nanometers. 
     
     
         13 . A method as recited in  claim 10 , wherein a thickness of the dielectric layer is in a range from about 20 nanometers to about 80 nanometers. 
     
     
         14 . A method as recited in  claim 10 , wherein a thickness of the dielectric layer is in a range from about 25 nanometers to about 80 nanometers. 
     
     
         15 . A method as recited in  claim 10 , wherein a thickness of the dielectric layer is selected to maximize fluorescent enhancement for a particular fluorophore in the detection molecule. 
     
     
         16 . A plasmonic substrate comprising:
 a layer of metal configured as a mirror to reflect light,   a layer of dielectric material having a thickness greater than about 20 nanometers disposed on the mirror; and   a layer of metal nanoparticles disposed on the layer of dielectric material.   
     
     
         17 . A plasmonic substrate as recited in  claim 16 , wherein the layer of metal nanoparticles has an optical density below about 1. 
     
     
         18 . A plasmonic substrate as recited in  claim 16 , wherein a thickness of the dielectric layer is in a range from about 20 nanometers to about 80 nanometers. 
     
     
         19 . A plasmonic substrate as recited in  claim 16 , wherein a thickness of the dielectric layer is in a range from about 25 nanometers to about 80 nanometers. 
     
     
         20 . A fluorescence affinity assay kit for determining the quantity of a particular analyte, comprising:
 a plasmonic substrate that comprises a layer of metal nanoparticles affixed to a substrate;   a solution comprising a bioactive target molecule that has affinity for a particular analyte, wherein the target molecule includes a ligand for affixing to the plasmonic substrate; and   a reagent comprising at least one plurality of substantively identical detection molecules, wherein
 the detection molecule comprises a fluorophore, and 
 the detection molecule has affinity for the particular analyte. 
   
     
     
         21 . A fluorescence affinity assay kit as recited in  claim 20 , wherein:
 the plasmonic substrate further comprises
 a layer of metal configured as a mirror to reflect light, and 
 a layer of dielectric material having a thickness greater than about 20 nanometers disposed on the mirror; and 
   the layer of metal nanoparticles is disposed on the layer of dielectric material.   
     
     
         22 . A computer-readable not-transitory medium carrying one or more sequences of instructions, wherein execution of the one or more sequences of instructions by one or more processors causes an apparatus to perform the steps of:
 determining a calibration curve that relates concentration of a particular analyte to at least one of intensity or lifetime of fluorescent emissions at a functionalized substrate in response to incident light for a plurality of known concentrations of the particular analyte mixed with a reagent, wherein the functionalized substrate comprises a plasmonic substrate and a bioactive target molecule that has an affinity for the particular analyte and the reagent comprises a detection molecule; and   determining a concentration of the particular analyte in a sample directly from the calibration curve and measurements of at least one of intensity or lifetime of fluorescent emissions at the functionalized substrate in contact with the sample and reagent in response to the incident light.   
     
     
         23 . An apparatus comprising:
 a source of incident light,   an optical coupler configured to direct incident light onto a functionalized substrate in contact with a mixture of a sample and a reagent, wherein the functionalized substrate comprises a plasmonic substrate and a bioactive target molecule that has an affinity for a particular analyte and the reagent comprises a detection molecule for the particular analyte;   a detector configured to measure fluorescent emissions from the functionalized substrate;   at least one processor; and   at least one memory including one or more sequences of instructions,   the at least one memory and the one or more sequences of instructions configured to, with the at least one processor, cause the apparatus to perform at least the following,
 determining a calibration curve that relates concentration of a particular analyte to at least one of intensity or lifetime of fluorescent emissions at the functionalized substrate in response to the incident light for a plurality of known concentrations of the particular analyte mixed with the reagent; and 
 determining a concentration of the particular analyte in a sample directly from the calibration curve and measurements of at least one of intensity or lifetime of fluorescent emissions at the functionalized substrate in contact with the sample and reagent in response to the incident light.