Compositions and methods for performing assays

Abstract

The disclosure relates to compositions for use in assays, the compositions comprising at least one latent fluorophore including at least one enzyme-reactive quenching group and a conjugative group; and a support connectable to the latent fluorophore by the conjugative group. The disclosure further relates to methods of measuring the presence and/or concentration of an analyte, as well as methods of measuring the relative activity of at least two enzymes.

What is claimed is: 
     
         1 . A composition comprising:
 a. at least one latent fluorophore comprising:
 i. at least one enzyme-reactive quenching group, and 
 ii. at least one conjugative group; and 
   b. at least one support connectable to the latent fluorophore.   
     
     
         2 . The composition of  claim 1 , wherein the at least one support is connected to the latent fluorophore by the at least one conjugative group. 
     
     
         3 . The composition of  claim 1 , wherein the at least one support is chosen from microspheres. 
     
     
         4 . The composition of  claim 3 , wherein the at least one support is magnetic. 
     
     
         5 . The composition of  claim 1 , wherein the at least one conjugative group is chosen from bioconjugative groups, PEG linkers, biotin groups, and combinations thereof. 
     
     
         6 . The composition of  claim 1 , where the at least one latent fluorophore is chosen from trimethyl lock fluorophores. 
     
     
         7 . The composition of  claim 6 , wherein the trimethyl lock fluorophore is chosen from compounds of Chemical Formula 1, Chemical Formula 2, Chemical Formula 3, and combinations thereof: 
       
         
           
           
               
               
           
         
       
     
     
         8 . The composition of  claim 1 , wherein the at least one latent fluorophore comprises at least one diaphorase-reactive quenching group that is releasable from the latent fluorophore by a diaphorase. 
     
     
         9 . A method of detecting and/or measuring the concentration of an analyte in a sample, the method comprising:
 a. providing a composition comprising:
 i. at least one latent fluorophore comprising at least one enzyme-reactive quenching group and at least one conjugative group, and 
 ii. at least one support connectable to the at least one latent fluorophore by the at least one conjugative group; 
   b. providing a test sample and a reference sample;   c. contacting the test sample with the composition, at least one first unquenching enzyme capable of releasing the enzyme-reactive quenching group from the latent fluorophore, and at least one second enzyme capable of reacting with the analyte; and   d. contacting the reference sample with the latent fluorophore composition and the at least one first unquenching enzyme.   
     
     
         10 . The method of  claim 9 , further comprising:
 e. measuring the fluorescence signal of the test sample and the fluorescence signal of the reference sample.   
     
     
         11 . The method of  claim 10 , further comprising:
 f. comparing the fluorescence signal of the test sample with the fluorescence signal of the reference sample.   
     
     
         12 . The method of  claim 11 , further comprising:
 g. connecting the latent fluorophore to the support, and   h. isolating the latent fluorophore composition from the sample.   
     
     
         13 . The method of  claim 9 , wherein the at least one support is chosen from microspheres. 
     
     
         14 . The method of  claim 13 , wherein the at least one support is magnetic. 
     
     
         15 . The method of  claim 9 , wherein the at least one first unquenching enzyme and the at least one second enzyme are the same. 
     
     
         16 . The method of  claim 9 , wherein the at least one second enzyme is chosen from diaphorase, esterase, phosphatase, pyruvate oxidase, and combinations thereof. 
     
     
         17 . The method of  claim 9 , wherein the at least one first unquenching enzyme is capable of generating NADH or NADPH when reacted with the analyte. 
     
     
         18 . The method of  claim 9 , wherein the at least one first enzyme is chosen from a dehydrogenase, a protease, a phosphatase, and combinations thereof. 
     
     
         19 . The method of  claim 9 , wherein the at least one conjugative group is chosen from bioconjugative groups, PEG linkers, biotin groups, and combinations thereof. 
     
     
         20 . The method of  claim 9 , wherein the at least one latent fluorophore is chosen from trimethyl lock fluorophores. 
     
     
         21 . The method of  claim 20 , wherein the trimethyl lock fluorophore is chosen from compounds of Chemical Formula 1, Chemical Formula 2, Chemical Formula 3, and combinations thereof: 
       
         
           
           
               
               
           
         
       
     
     
         22 . A method of measuring the activities of at least two enzymes in a sample, the method comprising:
 a. providing a first fluorophore composition comprising:
 i. at least one first enzyme-reactive latent fluorophore comprising at least one first enzyme-reactive quenching group and at least one conjugative group, and 
 ii. at least one support connectable to the at least one first latent fluorophore by at least one conjugative group; 
   b. providing a second fluorophore composition comprising:
 i. at least one second enzyme-reactive latent fluorophore comprising at least one second enzyme-reactive quenching group and at least one conjugative group, wherein the at least one second enzyme-reactive latent fluorophore is different from said first enzyme-reactive latent fluorophore in said first fluorophore composition, and 
 ii. at least one support connectable to the at least one first latent fluorophore by at least one conjugative group; 
   c. providing a test sample to be analyzed and a reference sample to be analyzed;   d. contacting the test sample with the first and second latent fluorophore compositions, at least one first unquenching enzyme capable of releasing the enzyme-reactive quenching group from the first latent fluorophore, and at least one second unquenching enzyme capable of releasing the enzyme-reactive quenching group from the second latent fluorophore;   e. contacting the reference sample with the first and second latent fluorophore compositions.   
     
     
         23 . The method of  claim 22 , further comprising:
 f. measuring the fluorescence signal of the test sample and the fluorescence signal of the reference sample.   
     
     
         24 . The method of  claim 23 , further comprising:
 g. comparing the fluorescence signal of the test sample with the fluorescence signal of the reference sample.   
     
     
         25 . The method of  claim 24 , further comprising:
 h. distinguishing the fluorescence signal of the at least one first enzyme from the fluorescence signal of the at least one second enzyme.