Fluorescent polymer superquenching-based bioassays

Abstract

A chemical composition including a fluorescent polymer and a receptor that is specific for both a target biological agent and a chemical moiety including (a) a recognition element, (b) a tethering element, and (c) a property-altering element is disclosed. Both the fluorescent polymer and the receptor are co-located on a support. When the chemical moiety is bound to the receptor, the property-altering element is sufficiently close to the fluorescent polymer to alter the fluorescence emitted by the polymer. When an analyte sample is introduced, the target biological agent, if present, binds to the receptor, thereby displacing the chemical moiety from the receptor, resulting in an increase of detected fluorescence. Assays for detecting the presence of a target biological agent are also disclosed.

1 . A composition of matter comprising:
 a fluorescent polymer affixed to a support; and   a receptor specific for a target biological agent affixed to said support, said receptor being adapted for complexation with a chemical moiety comprising (a) a recognition element capable of binding to said receptor, (b) a tethering element, and (c) a property-altering element such that when said chemical moiety is bound to said receptor, said property-altering element is located sufficiently close to said fluorescent polymer such that the fluorescence emitted by said fluorescent polymer is altered from that emitted when said binding between said receptor and chemical moiety does not occur.   
     
     
         2 . The composition of matter of  claim 1 , wherein said polymer is anchored to said support via adsorption or a covalent linkage. 
     
     
         3 . The composition of matter of  claim 1 , wherein said polymer is selected from the group consisting of a neutral polymer and a charged polymer. 
     
     
         4 . The composition of matter of  claim 1 , wherein said polymer comprises chromophores which are covalently or non-covalently linked and aggregate to form a polymer ensemble. 
     
     
         5 . The composition of matter of  claim 4 , wherein said chromophores are similar. 
     
     
         6 . The composition of matter of  claim 4 , wherein said chromophores are dissimilar. 
     
     
         7 . The composition of matter of  claim 4 , wherein said polymer ensemble is covalently linked to a second polymer. 
     
     
         8 . The composition of matter of  claim 1 , wherein said polymer and receptor are covalently bound to said support. 
     
     
         9 . The composition of matter of  claim 1 , wherein said receptor is covalently bound to said polymer. 
     
     
         10 . The composition of matter of  claim 1 , wherein said fluorescent polymer is a conjugated or J-aggregated polymer assembly comprising assembled monomers or oligomers. 
     
     
         11 . The composition of matter of  claim 1 , wherein said polymer is selected from the group consisting of conjugated polyelectrolytes, functionalized conjugated oligomers, uncharged conjugated polymers, charged conjugated polymers and conjugated polymer blends. 
     
     
         12 . The composition of matter of  claim 1 , wherein said recognition element is selected from the group consisting of chemical ligands, hormones, antibodies, antibody fragments, oligonucleotides, antigens, polypeptides, glycolipids, proteins, protein fragments, enzymes, peptide nucleic acids and polysaccharides. 
     
     
         13 . The composition of matter of  claim 1 , wherein said tethering element is selected from the group consisting of a single bond, a single divalent atom, a divalent chemical moiety, a multivalent chemical moiety, polyethylene, polyethylene oxides, polyamides and non-polymeric organic structures. 
     
     
         14 . The composition of matter of  claim 1 , wherein said property-altering element is selected from the group consisting of methyl viologen, quinones, metal complexes, fluorescent dyes, non-fluorescent dyes, electron accepting moieties, electron donating moieties and energy accepting moieties. 
     
     
         15 . The composition of matter of  claim 1 , wherein said support is selected from the group consisting of streptavidin coated spheres, polymer microspheres, silica microspheres, organic nanoparticles, inorganic nanoparticles, magnetic beads, magnetic particles, semiconductor nanoparticles quantum dots, membranes, slides, plates and test tubes. 
     
     
         16 . The composition of matter of  claim 1 , wherein said target biological agent is selected from the group consisting of proteins, viruses, bacteria, spores, cells, microorganisms, antibodies, antibody fragments, nucleic acids and toxins. 
     
     
         17 . An assay for detecting the presence of a target biological agent in a sample comprising:
 determining the fluorescence emitted by said chemical composition of  claim 1  in the absence of a sample,   contacting said sample and said chemical moiety with said receptor; and   determining the fluorescence emitted by said fluorescent polymer after said contacting step;   wherein a difference in fluorescence emitted after said contacting step compared to that emitted in the absence of said sample is indicative of the presence of said biological agent.   
     
     
         18 . The assay of  claim 17 , wherein the amount of target biological agent present in said sample is correlated with the amount of said difference in fluorescence. 
     
     
         19 . The assay of  claim 17 , wherein said chemical moiety is added to said chemical composition prior to said sample. 
     
     
         20 . The assay of  claim 17 , wherein said chemical moiety and said sample are simultaneously added to said chemical composition. 
     
     
         21 . The assay of  claim 17 , wherein said sample is added to said chemical composition prior to said chemical moiety. 
     
     
         22 . The assay of  claim 17 , wherein said sample and chemical composition are incubated prior to the addition of said chemical moiety. 
     
     
         23 . The assay of  claim 17 , wherein said target biological agent is selected from the group consisting of proteins, viruses, bacteria, spores, cells, microorganisms, antibodies, antibody fragments, nucleic acids and toxins. 
     
     
         24 . A composition of matter comprising:
 a fluorescent polymer affixed to a support; and   a chemical moiety affixed to said support, said chemical moiety comprising a receptor specific for a target biological agent and a QTL bioconjugate including (a) a recognition element which binds to said receptor, (b) a tether, and (c) a property-altering element located sufficiently close to said fluorescent polymer such that the fluorescence emitted by said fluorescent polymer is altered when said property-altering element and said fluorescent polymer are complexed together to a distinguishable degree, said QTL bioconjugate being susceptible of subsequent separation from said polymer upon exposure to an agent having an affinity for binding to said receptor, wherein the separation of said QTL bioconjugate from said receptor results in a detectable change in fluorescence of said polymer.   
     
     
         25 . An assay for detecting the presence of a target biological agent in a sample comprising:
 (a) determining the fluorescence emitted by said chemical composition of  claim 1  in the absence of a sample;   (b) contacting said chemical composition of step (a) with said chemical moiety;   (c) determining the fluorescence emitted by said fluorescent polymer after said contacting step (b);   (d) contacting said chemical composition of step (a) with said chemical moiety and said sample; and   (e) determining the fluorescence emitted by the fluorescent polymer after said contacting step (d);   wherein a difference in fluorescence after said contacting step (d) compared to that emitted after said contacting step (b) is indicative of the presence of said target biological agent.   
     
     
         26 . The assay of  claim 25 , wherein the amount of target biological agent present in said sample is correlated with the amount of said difference in fluorescence. 
     
     
         27 . The assay of  claim 25 , wherein in step (d), said chemical moiety is added to said chemical composition prior to said sample. 
     
     
         28 . The assay of  claim 25 , wherein in step (d), said chemical moiety and said sample are simultaneously added to said chemical composition. 
     
     
         29 . The assay of  claim 25 , wherein in step (d), said sample is added to said chemical composition prior to said chemical moiety. 
     
     
         30 . The assay of  claim 25 , wherein in step (d), said sample and chemical composition are incubated prior to the addition of said chemical moiety. 
     
     
         31 . The assay of  claim 25 , wherein said target biological agent is selected from the group consisting of proteins, viruses, bacteria, spores, cells, microorganisms, antibodies, antibody fragments, nucleic acids and toxins.