Reagents, Kits And Methods, For Detecting Biological Molecules By Energy Transfer From An Activated Chemiluminescent Substrate To An Energy Acceptor Dye

Abstract

Reagents, kits and methods for detecting biological molecules by energy transfer from an activated chemiluminescent substrate to an energy acceptor dye such as a J-aggregated dye are described.

What is claimed is: 
     
         1 . An article of manufacture comprising;
 a support comprising a surface;   a chemiluminescent enhancing material on the surface of the support;   an energy acceptor dye on the surface of the support; and   one or more biomolecular probes on the surface of the support.   
     
     
         2 . The article of  claim 1 , wherein the energy acceptor dye is a J-aggregated dye. 
     
     
         3 . The article of  claim 1 , wherein the chemiluminescent enhancing material is a cationic homopolymer or copolymer comprising positively charged onium groups. 
     
     
         4 . The article of  claim 3 , wherein the chemiluminescent enhancing material comprises poly(vinylbenzyldimethylbenzylammonium chloride) (BDMQ), poly(vinylbenzyltrimethylammonium chloride) (TMQ), poly(vinylbenzyltributylammonium chloride) (TBQ), poly(vinylbenzyltri(n-pentyl)ammonium chloride) (TPQ), poly(vinylbenzyltributylphosphonium chloride) (TB), poly(vinylbenzyltrioctylphosphonium chloride) (TO), two or more of any of the foregoing or a copolymer comprising one or more of the foregoing. 
     
     
         5 . The article of  claim 1 , wherein the support is a particle. 
     
     
         6 . The article of  claim 1 , wherein the support comprises latex, polystyrene, nylon, polyacrylamide or poly(styrenedivinyl benzene) beads. 
     
     
         7 . The article of  claim 2 , wherein the J-aggregated dye is a cyanine dye. 
     
     
         8 . The article of  claim 1 , wherein the one or more biomolecular probes comprise antibodies, polynucleotide, oligonucleotides, polypeptides, proteins, receptors, lectins or aptamers. 
     
     
         9 . The article of  claim 2 , wherein the support has an anionic surface, the chemiluminescent enhancing material is a cationic homopolymer or copolymer on the support surface and the J-aggregated dye is an anionic dye on the cationic homopolymer or copolymer. 
     
     
         10 . The article of  claim 9 , wherein the anionic dye is an anionic cyanine dye having the structure as set forth below: 
       
         
           
           
               
               
           
         
       
     
     
         11 . A kit for detecting analyte in a sample comprising:
 the article of  claim 1 , wherein the one or more surface-bound biomolecular probes comprises a probe which is capable of binding to the analyte or, when the analyte is present, which binds to the analyte;   a chemiluminescent substrate; and   optionally, an enzyme-labeled biomolecule or an enzyme-labeled analyte.   
     
     
         12 . The kit of  claim 11 , wherein the chemiluminescent substrate is a dioxetane, an acridinium ester, an acridinium sulfonimide, an acridan, an acridan enolphosphate, luciferin or a luminol. 
     
     
         13 . The kit of  claim 12 , wherein the chemiluminescent substrate is a 1,2-dioxetane substrate. 
     
     
         14 . The kit of  claim 13 , wherein the chemiluminescent substrate has a structure as set forth below: 
       
         
           
           
               
               
           
         
       
     
     
         15 . The kit of  claim 11 , wherein the enzyme label is a hydrolytic enzyme. 
     
     
         16 . The kit of  claim 11 , wherein the enzyme label is selected from the group consisting of alkaline phosphatase, β-galactosidase, glucuronidase and neuraminidase. 
     
     
         17 . The kit of  claim 11 , wherein the surface-bound biomolecular probe is an antibody and the analyte is an antigen for the antibody and wherein the kit comprises an enzyme-labeled antibody which is capable of binding to the analyte when the analyte is bound to the surface-bound biomolecular probe. 
     
     
         18 . The kit of  claim 14 , wherein the kit comprises the enzyme-labeled biomolecule or the enzyme-labeled analyte and wherein the enzyme label is alkaline-phosphatase. 
     
     
         19 . A kit for detecting analyte in a sample comprising:
 the article of  claim 9 , wherein the one or more surface-bound biomolecular probes comprises a probe which is capable of binding to the analyte or, when the analyte is present, which binds to the analyte;   a chemiluminescent substrate, wherein the activated chemiluminescent substrate excites the energy acceptor dye resulting in emissions therefrom; and   optionally, an enzyme-labeled biomolecule or enzyme-labeled analyte.   
     
     
         20 . The kit of  claim 19 , wherein the anionic cyanine dye has a structure as set forth below: 
       
         
           
           
               
               
           
         
       
     
     
         21 . A method for detecting analyte in a sample comprising:
 contacting the sample with the article of  claim 1 , wherein the one or more biomolecular probes comprises a probe which binds to the analyte;   allowing analyte in the sample to bind to the surface-bound probe; wherein: (a) the analyte is an enzyme; (b) the analyte is labeled with an enzyme; (c) the support surface is contacted with an enzyme-labeled biomolecule which binds to the analyte; or (d) the analyte is unlabeled and enzyme-labeled analyte is added to the sample to allow the enzyme-labeled analyte in the sample to compete with the unlabeled analyte for binding to the surface-bound probe;   contacting the support surface with a chemiluminescent substrate which is activated by the enzyme, wherein the activated chemiluminescent substrate excites the energy acceptor dye resulting in emissions therefrom; and   detecting emissions from the energy acceptor dye.   
     
     
         22 . The method of  claim 21 , wherein the energy acceptor dye is a J-aggregated dye. 
     
     
         23 . The method of  claim 21 , wherein the enzyme or enzyme label is a hydrolytic enzyme. 
     
     
         24 . The method of  claim 23 , wherein the enzyme or enzyme label is selected from the group consisting of alkaline phosphatase, β-galactosidase, glucuronidase and neuraminidase. 
     
     
         25 . The method of  claim 21 , wherein the enzyme or enzyme label is an oxidative enzyme. 
     
     
         26 . The method of  claim 21 , wherein the surface-bound probe is an antibody and the analyte is an antigen for the antibody or the surface-bound probe is an antigen for an antibody and the analyte is the antibody to the antigen. 
     
     
         27 . The method of  claim 26 , wherein the support surface is contacted with an enzyme-labeled biomolecule subsequent to allowing analyte in the sample to bind to the support-bound probe and wherein the enzyme-labeled biomolecule is an antibody which binds the analyte when the analyte is bound to the support-bound probe. 
     
     
         28 . The method of  claim 21 , wherein the chemiluminescent substrate is a dioxetane, an acridinium ester, an acridinium sulfonimide, an acridan, an acridan enolphosphate, luciferin or a luminol. 
     
     
         29 . The method of  claim 28 , wherein the chemiluminescent substrate is a 1,2-dioxetane. 
     
     
         30 . The method of  claim 29 , wherein the chemiluminescent substrate has a structure as set forth below: 
       
         
           
           
               
               
           
         
       
     
     
         31 . The method of  claim 30 , wherein the enzyme or enzyme label is alkaline phosphatase. 
     
     
         32 . A method for detecting analyte in a sample comprising:
 contacting the sample with the article of  claim 9 , wherein the one or more biomolecular probes comprises a probe which binds to the analyte;   allowing analyte in the sample to bind to the surface-bound probe; wherein: (a) the analyte is an enzyme; (b) the analyte is labeled with an enzyme; (c) the support surface is contacted with an enzyme-labeled biomolecule which binds to the analyte; or (d) the analyte is unlabeled and enzyme-labeled analyte is added to the sample to allow the enzyme-labeled analyte in the sample to compete with the unlabeled analyte for binding to the surface-bound probe;   contacting the support surface with a chemiluminescent substrate which is activated by the enzyme, wherein the activated chemiluminescent substrate excites the energy acceptor dye resulting in emissions therefrom; and   detecting emissions from the energy acceptor dye.   
     
     
         33 . The method of  claim 32 , wherein the enzyme or enzyme label is a hydrolytic enzyme. 
     
     
         34 . The method of  claim 33 , wherein the enzyme or enzyme label is selected from the group consisting of alkaline phosphatase, β-galactosidase, glucuronidase and neuraminidase. 
     
     
         35 . The method of  claim 32 , wherein the surface-bound probe is an antibody and the analyte is an antigen for the antibody or the surface-bound probe is an antigen for an antibody and the analyte is the antibody to the antigen. 
     
     
         36 . The method of  claim 35 , wherein the support surface is contacted with an enzyme-labeled biomolecule subsequent to allowing analyte in the sample to bind to the surface-bound probe and wherein the enzyme-labeled biomolecule is an antibody which binds the analyte when the analyte is bound to the support-bound probe. 
     
     
         37 . The method of  claim 32 , wherein the chemiluminescent substrate is a dioxetane, an acridinium ester, an acridinium sulfonimide, an acridan, an acridan enolphosphate, luciferin or a luminol. 
     
     
         38 . The method of  claim 37 , wherein the chemiluminescent substrate is a 1,2-dioxetane. 
     
     
         39 . The method of  claim 38 , wherein the chemiluminescent substrate has a structure as set forth below: 
       
         
           
           
               
               
           
         
       
     
     
         40 . The method of  claim 35 , wherein the enzyme or enzyme label is alkaline phosphatase. 
     
     
         41 . A kit for detecting analyte in a sample comprising:
 an article of manufacture comprising a support having a surface, an energy acceptor dye on the surface of the support, and one or more biomolecular probes on the surface of the support, wherein the one or more biomolecular probes is capable of binding to the analyte or, when the analyte is present, which binds to the analyte;   a chemiluminescent substrate; and   optionally, an enzyme-labeled biomolecule or enzyme-labeled analyte.   
     
     
         42 . The kit of  claim 41 , wherein the energy acceptor dye is a J-aggregated dye. 
     
     
         43 . The kit of  claim 41 , further comprising a chemiluminescent enhancing material on the surface of the support. 
     
     
         44 . The kit of  claim 41 , wherein the chemiluminescent substrate is a dioxetane, an acridinium ester, an acridinium sulfonimide, an acridan, an acridan enolphosphate, luciferin or a luminol. 
     
     
         45 . The kit of  claim 44 , wherein the chemiluminescent substrate is a 1,2-dioxetane substrate. 
     
     
         46 . The kit of  claim 45 , wherein the chemiluminescent substrate has a structure as set forth below: 
       
         
           
           
               
               
           
         
       
     
     
         47 . The kit of  claim 41 , wherein the kit comprises an enzyme-labeled biomolecule or an enzyme-labeled analyte. 
     
     
         48 . The kit of  claim 47 , wherein the enzyme label is a hydrolytic enzyme. 
     
     
         49 . The kit of  claim 48 , wherein the enzyme label is selected from the group consisting of alkaline phosphatase, β-galactosidase, glucuronidase and neuraminidase. 
     
     
         50 . The kit of  claim 47 , wherein the surface-bound probe is an antibody and the analyte is an antigen for the antibody or the surface-bound probe is an antigen for an antibody and the analyte is the antibody to the antigen. 
     
     
         51 . The kit of  claim 46 , wherein the kit comprises an enzyme-labeled biomolecule which binds to the analyte when the analyte is bound to the probe on the support surface or an enzyme-labeled analyte which competes with unlabeled analyte in the sample for binding to the support-bound probe and wherein the enzyme label is an alkaline phosphatase label. 
     
     
         52 . A method for detecting multiple analytes in a sample comprising:
 contacting the sample with a first article of manufacture comprising: a support having a surface; a first chemiluminescent enhancing material on the surface of the support; a first energy acceptor dye on the surface of the support; and a first biomolecular probe on the surface of the support, wherein the first biomolecular probe is capable of binding to a first analyte;   contacting the sample with a second article of manufacture comprising: a support having a surface; a second chemiluminescent enhancing material on the surface of the support; a second energy acceptor dye on the surface of the support; and a second biomolecular probe on the surface of the support, wherein the second biomolecular probe is capable of binding to a second analyte;   allowing first analyte in the sample to bind to the first probe, wherein: (a) the first analyte is a first enzyme; (b) the first analyte is labeled with a first enzyme; (c) the support surface of the first article of manufacture is contacted with a biomolecule which is labeled with a first enzyme and which binds to the first analyte on the support surface; or (d) the first analyte is unlabeled and first analyte labeled with a first enzyme is added to the sample to allow the enzyme-labeled first analyte in the sample to compete with the unlabeled first analyte for binding to the first biomolecular probe of the first article of manufacture;   allowing second analyte in the sample to bind to the second probe wherein: (a) the second analyte is a second enzyme; (b) the second analyte is labeled with a second enzyme; (c) the second support surface of the second article of manufacture is contacted with a biomolecule which is labeled with a second enzyme and which binds to the second analyte on the support surface; or (d) the second analyte is unlabeled and second analyte labeled with a second enzyme is added to the sample to allow the enzyme-labeled second analyte in the sample to compete with the unlabeled second analyte for binding to the second biomolecular probe of the second article of manufacture;   contacting the first article of manufacture with a first chemiluminescent substrate which is activated by the first enzyme, wherein the activated first chemiluminescent substrate excites the first energy acceptor dye resulting in emissions therefrom and contacting the second article of manufacture with a second chemiluminescent substrate which is activated by the second enzyme, wherein the activated second chemiluminescent substrate excites the second energy acceptor dye resulting in emissions therefrom; and   detecting emissions from the first energy acceptor dye and detecting emissions from the second energy acceptor dye, wherein the emissions from the first energy acceptor dye are distinguishable from those of the second energy acceptor dye.   
     
     
         53 . The method of  claim 52 , wherein the first and second chemiluminescent substrates are the same or different. 
     
     
         54 . The method of  claim 52 , wherein the first and second chemiluminescent enhancing materials are the same or different. 
     
     
         55 . The method of  claim 52 , wherein the first and second articles of manufacture are contacted with the sample simultaneously or sequentially. 
     
     
         56 . The method of  claim 52 , wherein the first and second articles of manufacture are contacted with the first and second chemiluminescent substrates simultaneously or sequentially. 
     
     
         57 . The method of  claim 52 , wherein emissions from the first energy acceptor dye and emissions from the second energy acceptor dye are detected simultaneously or sequentially. 
     
     
         58 . A kit for detecting multiple analytes in a sample comprising:
 a first article of manufacture comprising: a first support having a surface; a first chemiluminescent enhancing material on the surface of the support; a first energy acceptor dye on the surface of the support; and a first biomolecular probe on the surface of the support, wherein the first biomolecular probe comprises a first probe capable of binding to a first analyte or, when the first analyte is present, which binds to the first analyte;   a second article of manufacture comprising: a support having a surface; a second chemiluminescent enhancing material on the surface of the support; a second energy acceptor dye on the surface of the support; and a second biomolecular probe on the surface of the support, wherein the second biomolecular probe comprises a first probe capable of binding to a second analyte or, when the second analyte is present, which binds to the second analyte;   a first chemiluminescent substrate, wherein the activated first chemiluminescent substrate excites the first energy acceptor dye resulting in emissions therefrom;   a second chemiluminescent substrate, wherein the activated second chemiluminescent substrate excites the second energy acceptor dye resulting in emissions therefrom, wherein the emissions from the first energy acceptor dye are distinguishable from those of the second energy acceptor dye;   optionally, a first enzyme-labeled biomolecule or first analyte labeled with the first enzyme;   optionally, a second enzyme-labeled biomolecule or second analyte labeled with the second enzyme.   
     
     
         59 . The kit of  claim 58 , wherein the first and second chemiluminescent substrates are the same or different. 
     
     
         60 . The kit of  claim 58 , wherein the first and second chemiluminescent enhancing materials are the same or different. 
     
     
         61 . The article of  claim 1 , wherein the energy acceptor dye is a fluorescent dye. 
     
     
         62 . The article of  claim 1 , wherein the energy acceptor dye is an aromatic compound selected from the group consisting of naphthalenes, anthracenes, pyrenes, biphenyls, acridine, coumarins, xanthenes, phthalocyanines, stilbenes, furans, oxazoles, oxadiazoles, and benzothiazoles. 
     
     
         63 . The article of  claim 1 , further comprising a molecular filter on the surface of the support, wherein the molecular filter masks or quenches light emissions that interfere with the detection of luminescent emissions from the energy acceptor dye. 
     
     
         64 . The article of  claim 63 , wherein the molecular filter is hemoglobin or a quenching dye. 
     
     
         65 . The kit of  claim 11 , further comprising:
 a molecular filter, wherein the molecular filter masks or quenches light emissions that interfere with the detection of luminescent emissions from the energy acceptor dye.   
     
     
         66 . The kit of  claim 65 , wherein the chemiluminescent substrate, when activated, excites the energy acceptor dye resulting in luminescent emissions therefrom and wherein the molecular filter masks or quenches luminescent emissions that interfere with the detection of the luminescent emissions from the energy acceptor dye. 
     
     
         67 . The method of  claim 21 , wherein a molecular filter is on the support surface during detecting emissions from the energy acceptor dye, wherein the molecular filter masks or quenches light emissions that interfere with the detection of luminescent emissions from the energy acceptor dye.