Multiplexed Catalyzed Reporter Deposition

Abstract

A method for testing a sample for the presence of one or more targets comprises multiplexed catalyzed reporter deposition (CARD) is provided.

What is claimed is: 
     
         1 . A method for testing a sample for the presence of one or more targets comprising
 (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid strand and wherein target-specific binding partners of different specificity are linked to different nucleic acid strands;   (2) in one or two steps, contacting the sample with (a) an enzyme linked to a nucleic acid strand and (b) an intermediate moiety comprising a first domain capable of specifically binding to the nucleic acid strand linked to the target-specific binding partner; and a second domain capable of specifically binding to the nucleic acid strand linked to the enzyme;   (3) contacting the sample from step (2) with a substrate conjugate comprised of a detectably labeled substrate, where the substrate and detectable label are linked optionally with a releasable linker;   (4) optionally deactivating the bound enzyme;   (5) optionally detect signals from the bound detectable labels; and   (6) optionally repeating steps (1)-(5) or a subset thereof and optionally repeating steps (3)-(4) or any subset thereof prior to step (5) of imaging.   
     
     
         2 . The method of  claim 1 , wherein the second region of the intermediate moiety comprises repeated sequences, each of the sequences that specifically binds directly or indirectly to a corresponding sequence of the nucleic acid strand linked to the enzyme. 
     
     
         3 . The method of  claim 1 , wherein the method further comprises amplifying the intermediate moiety bound to the nucleic acid strand linked to the target-specific binding partner of step (1) by a nucleic acid amplification reaction to form an amplicon of repeated sequences, each of the sequences that specifically binds directly or indirectly to a corresponding sequence of the nucleic acid strand linked to the enzyme. 
     
     
         4 . The method of  claim 3 , wherein the nucleic acid amplification reaction comprises hairpin-based concatemerization reaction or hairpin-based dendrimerization reaction, optionally the amplification reaction being primer exchange reaction. The method of  claim 1 , wherein the enzyme is deactivated using an enzyme deactivator, wherein the enzyme deactivator comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), a reduced glutathione, a peroxide, a cyanide, a fluoride, or an azide. 
     
     
         6 . The method of  claim 1 , wherein the step of deactivating the bound enzyme is performed for less than 20 minutes, for less than 10 minutes, or for less than 2 minutes. 
     
     
         7 . The method of  claim 1 , wherein in step (3), the substrate conjugate comprised of the detectably labeled substrate reacts with the enzyme to form an activated substrate conjugate, and the activated substrate conjugate binds to a receptor for the activated substrate conjugate. 
     
     
         8 . The method of  claim 7 , wherein the receptor for the activated substrate conjugate is present in the sample, resulting in the deposition of the detectable labels. 
     
     
         9 . The method of  claim 1 , wherein the bound label is released by cleaving the releasable linker between the label and the substrate or by cleaving the releasable linker between the substrate and the nucleic acid. 
     
     
         10 . The method of  claim 1 , wherein the releasable linkers include at least one of disulfide bonds, esters, vicinal diols, sulfones, and photocleavable linkers. 
     
     
         11 . The method of  claim 1 , wherein the enzyme is chosen from horseradish peroxidase, glucose oxidase, alkaline phosphatase and beta-galactosidase. 
     
     
         12 . The method of  claim 11 , wherein the enzyme is horseradish peroxidase. 
     
     
         13 . The method of  claim 1 , wherein the detectable label is chosen from enzymes, radioactive isotopes, fluorogenic, chemiluminescent, and electrochemical materials. 
     
     
         14 . The method of  claim 1 , wherein the enzyme is horseradish peroxidase (HRP) and the substrate is a phenolic substrate. 
     
     
         15 . The method of  claim 1 , wherein the enzyme is a hydrolytic enzyme and the substrate is an ester, amide, or glycocide substrate. 
     
     
         16 . A method for testing a sample for the presence of one or more targets comprising
 (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid strand and wherein target-specific binding partners of different specificity are linked to different nucleic acid strands;   (2) optionally removing unbound target-specific binding partners;   (3) performing (3-a) or (3-b):
 (3-a) contacting the sample from step (1) or optionally step (2) with an enzyme linked to a nucleic acid strand complementary to the nucleic acid strand linked to the target-specific binding partner; 
 (3-b) contacting the sample from step (1) or optionally step (2) with a first member of a binding pair linked to a nucleic acid strand complementary to the nucleic acid strand linked to the target-specific binding partner; contacting the sample with a second member of the binding pair linked to an enzyme; and optionally removing unbound second member of the binding pair linked to the enzyme and/or optionally removing unbound second member of the binding pair linked to the enzyme; 
   (4) optionally removing unbound enzyme linked to complimentary nucleic acid strands;   (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprised of a detectably labeled substrate, where the substrate and detectable label are linked optionally with a releasable linker;   (6) optionally removing unbound substrate conjugate;   (7) optionally deactivating the bound enzyme;   (8) optionally imaging the sample to detect the bound detectable labels; and   (9) optionally repeating steps (1)-(8) or any subset thereof and optionally if more than one target-specific binding partner is used, repeating steps (1)-(7) or any subset thereof prior to step (8) of imaging.   
     
     
         17 . The method of  claim 16 , further comprising releasing the bound detectable labels after the step of imaging by cleaving the releasable linker between the label and the substrate. 
     
     
         18 . The method of  claim 16 , wherein the method further comprises amplifying the nucleic acid strand or a portion thereof linked to the target-specific binding partner of step (1), optionally wherein the amplifying step is performed prior to step (3). 
     
     
         19 . A method for testing a sample for the presence of one or more targets comprising
 (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid strand and wherein target-specific binding partners of different specificity are linked to different nucleic acid strands;   (2) optionally removing unbound target-specific binding partners;   (3) performing one of the following (3-a) or (3-b):
 (3-a) contacting the sample from step (1) or optionally step (2) with an enzyme linked to a nucleic acid strand complementary to the nucleic acid strand linked to the target-specific binding partner, wherein the following (i) or (ii) or both is met: 
 (i) the nucleic acid strand is linked to the target-specific binding partner with a first releasable linker; and 
 (ii) the enzyme is linked to the complementary nucleic acid strand with a second releasable linker; 
 (3-b) contacting the sample from step (1) with a first member of a binding pair linked to a nucleic acid strand complimentary to the nucleic acid strand linked to the target-specific binding partner, wherein:
 (i) the nucleic acid strand is linked to the target-specific binding partner with a first releasable linker; and 
 (ii) the first member of the binding pair is linked to the complimentary nucleic acid strand with a second releasable linker; 
 
 contacting the sample with a second member of the binding pair linked to an enzyme optionally with a third releasable linker; 
   (4) optionally removing unbound enzyme linked to complementary nucleic acid strands;   (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprised of a detectably labeled substrate, where the substrate and detectable label are linked optionally with a fourth releasable linker;   (6) optionally removing unbound substrate conjugate;   (7) optionally releasing the bound enzyme by cleaving the first, second, third, or fourth releasable linker;   (8) optionally imaging the sample to detect the bound detectable labels; and   (9) optionally repeating steps (1)-(8) or any subset thereof and optionally if more than one target-specific binding partner is used, repeating steps (1)-(7) or any subset thereof prior to step (8) of imaging.