Method for detecting an analyte by fluorescence

Abstract

Two-dimensional and three-dimensional arrays of a polydiacetylene backbone having a substrate incorporated are used in chemical sensing methods to detect the interaction of an analyte with the substrate by monitoring the change in the fluorescence of the array.

What is claimed is:  
     
         1 . A method for the detection of an analyte in a sample, which comprises contacting the sample to be tested with a three-dimensional array of a polydiacetylene backbone having a substrate incorporated in the array, wherein the substrate has direct affinity for the analyte or can function as a binder to the analyte or can react with the analyte; 
 and detecting the change in fluorescence to indicate the presence of the analyte.    
     
     
         2 . The method of  claim 1  wherein the array is in the form of a solution of liposomes or tubules.  
     
     
         3 . The method of  claim 1  wherein the analyte is an enzyme and the substrate is a reactive substrate of that enzyme.  
     
     
         4 . The method of  claim 1  or  2  wherein the analyte is an antigen and the substrate is the antibody of that antigen.  
     
     
         5 . The method of  claim 1  or  2  wherein the analyte is an antigen and the substrate is a fragment of the antibody of that antigen.  
     
     
         6 . The method of  claim 1  or  2  wherein the analyte is an antibody or antibody fragment and the substrate is the antigen of that antibody.  
     
     
         7 . The method of  claim 1  or  2  wherein the analyte is an antibody or antibody fragment and the substrate is the epitope of that antibody.  
     
     
         8 . The method of  claim 2  wherein the analyte is an enzyme and the substrate is a reactive substrate of that enzyme.  
     
     
         9 . The method of  claim 1  wherein the polydiacetylene of the array is in the non-fluorescent form, exhibiting a fluorescent signal that is about 1-3 times that of the background and less than that of the corresponding fluorescent form.  
     
     
         10 . The method of claim I wherein the substrate includes a ligand.  
     
     
         11 . The method of  claim 1  wherein the array is in the form of a solution of a liposome or tubule.  
     
     
         12 . The method of  claim 1  wherein the substrate includes a reactive substrate.  
     
     
         13 . The method of  claim 12  wherein the array is in the form of a solution of a liposome or tubule.  
     
     
         14 . The method of any one of  claims 1  to  13  wherein the three-dimensional array further comprises a fluorophore and wherein the change in fluorescence of the polydiacetylene array is monitored.  
     
     
         15 . The method of any one of  claims 1  to  13  wherein the three-dimensional array further comprises a fluorophore and wherein the change in fluorescence of the fluorophore is monitored.  
     
     
         16 . The method of  claim 1  wherein the array does not contain a further fluorophore.  
     
     
         17 . The method of  claim 1  wherein the change in fluorescence is detected by exposure to light having wavelengths below 550 nm and measurement of the emission.  
     
     
         18 . The method of  claim 1  wherein the change in fluorescence is detected by exposure to light having wavelengths between 450 and 500 nm and measurement of the emission.  
     
     
         19 . The method of  claim 1  wherein the polydiacetylene of the array exhibits fluorescence and the fluorescence increases as an indication of the presence of the analyte.  
     
     
         20 . A method for the detection of an analyte in a sample which comprises contacting the sample to be tested with a two-dimensional array of a polydiacetylene backbone having a substrate incorporated in the array, wherein the substrate has direct affinity for the analyte or can function as a binder to the analyte or can react with the analyte and wherein the two-dimensional array comprises a polymerized diacetylene array wherein no more than 90% of the diacetylenes are terminated with groups that specifically bind to the analyte; 
 and detecting the change in fluorescence to indicate the presence of the analyte.    
     
     
         21 . The method of  claim 20  wherein the two-dimensional array comprises a polymerized diacetylene array wherein no more than 60% of the diacetylenes are terminated with groups that specifically bind to the analyte.  
     
     
         22 . The method of  claim 20  wherein the array is coated onto a solid support.  
     
     
         23 . The method of  claim 22  wherein the solid support is a porous membrane.  
     
     
         24 . The method of  claim 20  wherein the array is unsupported.  
     
     
         25 . The method of  claim 20  which further comprises providing a filter or flow cell containing the array supported on or in a porous membrane; and passing a solution of the analyte through the filter or flow cell before or during the detect.  
     
     
         26 . The method of  claim 20  wherein the substrate includes a ligand.  
     
     
         27 . The method of  claim 26  wherein the array is on a nano-porous membrane.  
     
     
         28 . The method of  claim 20  wherein the substrate includes a reactive substrate.  
     
     
         29 . The method of  claim 28  wherein the array is on a nano-porous membrane.  
     
     
         30 . The method of  claim 20  wherein the two-dimensional array further comprises a fluorophore and wherein the change in fluorescence of the polydiacetylene array is monitored.  
     
     
         31 . The method of  claim 20  wherein the two-dimensional array further comprises a fluorophore and wherein the change in fluorescence of the fluorophore is monitored.  
     
     
         32 . The method of  claim 26  or  28  wherein the three-dimensional array further comprises a fluorophore and wherein the change in fluorescence of the polydiacetylene array is monitored.  
     
     
         33 . The method of  claim 26  or  28  wherein the three-dimensional array further comprises a fluorophore and wherein the change in fluorescence of the fluorophore is monitored.  
     
     
         34 . The method of  claim 20  wherein the array does not contain a further fluorophore.  
     
     
         35 . The method of  claim 20  wherein the change in fluorescence is detected by exposure to light having wavelengths below 550 nm and measurement of the emission.  
     
     
         36 . The method of  claim 20  wherein the change in fluorescence is detected by exposure to light having wavelengths between 450 and 500 nm and measurement of the emission.  
     
     
         37 . The method of  claim 20  wherein the polydiacetylene of the array exhibits fluorescence and the fluorescence increases as an indication of the presence of the analyte.  
     
     
         38 . A method for the detection of an analyte in a sample which comprises contacting the sample to be tested with three-dimensional arrays of a polydiacetylene backbone having a substrate incorporated in the arrays, wherein the substrate has direct affinity for the analyte or can function as a binder to the analyte or can react with the analyte, and the arrays are suspended in solution; and detecting the change in polarization of the fluorescence of the polydiacetylene arrays upon being excited with polarized light, to indicate the presence of the analyte.  
     
     
         39 . The method of  claim 38  wherein the three-dimensional arrays are liposomes.  
     
     
         40 . The method of  claim 38  or  claim 39 , wherein the substrate is an antibody and the analyte an antigen to that antibody.  
     
     
         41 . The method of  claim 38  or  claim 39 , wherein the substrate is an antibody fragment and the analyte an antigen to that antibody fragment.  
     
     
         42 . The method of  claim 38  or  claim 39 , wherein the analyte is immobilized onto a surface.  
     
     
         43 . The method of  claim 38  or  claim 39  wherein the analyte is attached to a microbead.  
     
     
         44 . The method of  claim 38  or  claim 39  wherein the analyte is attached to a macrobead.  
     
     
         45 . The method of  claim 38  wherein the three-dimensional arrays also contain fluorophores and the change in the polarization of the fluorophore emission upon the polydiacetylene array being excited with polarized light is detected to indicate the presence of the analyte.  
     
     
         46 . The method of  claim 45  wherein the three-dimensional arrays are liposomes.