Systems and methods for multiplexed analyte detection using antibody-oligonucleotide conjugates

Abstract

Provided herein, in some embodiments, are systems, methods, compositions, and kits for detecting and quantifying analytes using a primary analyte binding molecule conjugated to a nucleic acid template.

What is claimed is: 
     
         1 . A method of measuring the amount of analyte in a sample comprising the steps of
 a) Contacting a sample comprising at least one analyte with at least one primary analyte binding molecule; wherein the primary analyte binding molecule is immobilized on a solid support; wherein the analyte binds to the at least one primary analyte binding molecule;   b) Contacting at least one secondary analyte binding molecule with the at least one analyte,
 wherein the 5′ end of at least one nucleic acid template is conjugated to the at least one secondary analyte binding molecule; and 
 wherein the at least one nucleic acid template comprises an oligonucleotide sequence at the 3′ end that hybridizes to a primer; wherein the primer is immobilized at the 5′ end on the solid support adjacent to each of the at least one primary analyte binding molecules; 
   c) Hybridizing the oligonucleotide sequence to the primer;   d) Performing one or more nucleic acid amplification reactions on the at least one nucleic acid template, so that nucleic acid colonies are generated;   e) Measuring the presence of the nucleic acid colonies to determine the amount of the specified analyte in a sample.   
     
     
         2 .- 19 . (canceled) 
     
     
         20 . A kit for measuring the amount of analyte in a sample comprising:
 a) at least one primary analyte binding molecule configured to be immobilized on a solid support;   b) at least one primer configured to be immobilized on the solid support; and   c) at least one secondary analyte binding molecule;
 wherein the 5′ end of at least one nucleic acid template is conjugated to the at least one secondary analyte binding molecule; 
 wherein the at least one nucleic acid template comprises an oligonucleotide sequence at the 3′ end that hybridizes to the at least one primer; 
   wherein the solid support is a flow cell with at least one peak region and one valley region, wherein the primary analyte binding molecule is bound to the valley region of the flow cell, and wherein the primer is bound at a higher position than said primary analyte binding molecule on the flow cell.   
     
     
         21 . The kit of  claim 20 , wherein the primary analyte binding molecule is selected from the group consisting of: antibodies, antibody fragments, Fab fragments, Fab′ fragments, F(ab′)2 fragments, scFv proteins, analyte specific trapping agents, and nanoparticles. 
     
     
         22 . The kit of  claim 20 , wherein the secondary analyte binding molecule is selected from the group consisting of: antibodies, antibody fragments, Fab fragments, Fab′ fragments, F(ab′)2 fragments, scFv proteins, analyte specific trapping agents, and nanoparticles. 
     
     
         23  - 27 . (canceled) 
     
     
         28 . The kit of  claim 20 , wherein the primer is modified to reduce enzymatic digestion. 
     
     
         29 . The kit of  claim 29 , wherein the modification is selected from the group consisting of: modifications of the phosphodiester backbone, modifications to the sugar ring, 3′ capping with inverted thymidine, and modifications on the nucleotide bases. 
     
     
         30 . The kit of  claim 20 , wherein the nucleic acid template sequence is modified to reduce enzymatic digestion. 
     
     
         31 . The kit of  claim 30 , wherein the modification is selected from the group consisting of: modifications of phosphodiester backbone, modifications to the sugar ring, 3′ capping with inverted thymidine, and modifications on the nucleotide bases. 
     
     
         32 . The kit of  claim 20 , wherein the nucleic acid sequence template at its 5′ end further comprises an oligonucleotide sequence that is identical to the primer sequence. 
     
     
         33 . The kit of  claim 20 , wherein the nucleic acid template sequence is the reverse mirror image of its complementary sequence. 
     
     
         34 . The kit of  claim 20 , wherein the wherein said wherein the 5′ end of a filler is attached to the secondary analyte binding molecule and the 3′ end of said filler is attached to the 5′ end of the nucleic acid template sequence. 
     
     
         35 . The kit of  claim 34 , wherein said filler sequence is double-stranded. 
     
     
         36 . The kit of  claim 20 , wherein the amount of analyte in a sample is determined by the presence of a unique recognition sequence associated with a particular analyte binding molecule. 
     
     
         37 . The kit of claim  24 , wherein the amount of analyte in a sample is determined by the presence of the nucleic acid colonies in a specific region of the flow cell. 
     
     
         38 . The kit of  claim 20 , where the presence of the nucleic acid colonies is measured using DNA sequencing methods. 
     
     
         39 . A system for measuring the amount of analyte in a sample, the system comprising:
 a) a source unit configured to house
 a sample comprising a least one analyte, the sample configured to contact at least one primary analyte binding molecule, and 
 at least one secondary analyte binding molecule, wherein the 5′ end of at least one nucleic acid template is conjugated to the at least one secondary analyte binding molecule, and wherein the at least one nucleic acid template comprises an oligonucleotide sequence at the 3′ end; 
   b) a reaction unit configured to receive
 a solid support configured to include (1) at least one primer immobilized on the solid support at the primer 5′ end and (2) the at least one primary analyte binding molecule immobilized on the solid support; 
 the sample comprising the at least one analyte from the source unit, the at least one analyte configured to bind to the at least one primary analyte binding molecule, and 
 the at least one secondary analyte binding molecule from the source unit, the at least one secondary analyte binding molecule configured to bind to the at least one analyte, wherein the oligonucleotide sequence at the 3′ end of the nucleic acid template hybridizes to the primer; 
   wherein the solid support is a flow cell with at least one peak region and one valley region, wherein the primary analyte binding molecule is bound to the valley region of the flow cell, and wherein the primer is bound at a higher position than said primary analyte binding molecule on the flow cell;   c) an amplification unit configured to perform one or more nucleic acid amplification reactions on the at least one nucleic acid template, so that a nucleic acid colony is generated; and   d) an identification unit configured to identify the specific nucleic acid colony associated with the specific analyte to determine the amount of analyte in a sample.   
     
     
         40 .- 43 . (canceled) 
     
     
         44 . The system of claim  40 , wherein the at least one primer is bound to the peak region. 
     
     
         45 . The system of claim  40 , wherein the amount of analyte in a sample is determined by the presence of the nucleic acid colonies in a specific region of the flow cell. 
     
     
         46 . (canceled) 
     
     
         47 . The system of  claim 39 , comprising a measuring unit that comprises a sequencing unit configured to determine the presence of the nucleic acid colonies using DNA sequencing. 
     
     
         48 . The system of  claim 39 , wherein the identification unit is configured to identify each specific nucleic acid colony from a plurality of nucleic acid colonies, each nucleic acid colony associated with a specific analyte, to determine the amount of each analyte present.