Digital Analysis of Molecular Analytes Using Electrical Methods

Abstract

Electrical detection methods are used to identify and further characterize single-molecule target analytes such as proteins and nucleic acids. A composition including a probe region and a tail region is contacted with a target analyte. The probe region specifically binds to the target analyte. The tail region is coupled to the probe region, and includes a nucleic acid template for polynucleotide synthesis. When conditions are such that polynucleotide synthesis occurs along the tail region, one hydrogen ion is released for every nucleotide that is incorporated into the tail region. A transistor such as an ISFET detects and measures changes in ion concentration, and these measurements can be used to identify the tail region and thus characterize the corresponding target analyte.

What is claimed is: 
     
         1 . A composition, comprising:
 a probe region, wherein the probe region is configured to specifically bind to a target analyte;   a tail region, the tail region comprising a homopolymeric base region comprising at least 25 consecutive nucleotides; and   optionally a linker region located between the probe region and the tail region, wherein the linker region comprises a nucleotide sequence configured to specifically bind to a portion of the tail region, and wherein the probe region and the tail region each comprises a separate nucleic acid molecule when the optional linker region is present.   
     
     
         2 . A composition, comprising:
 a probe region, wherein the probe region is configured to specifically bind to a target analyte; and   at least one linker region attached to the probe region, wherein the linker region comprises a nucleotide sequence configured to specifically bind to a portion of at least one tail region, the tail region comprising a homopolymeric base region comprising at least 25 consecutive nucleotides, wherein the probe region and the tail region each comprises a separate nucleic acid molecule.   
     
     
         3 . The composition of  claim 2 , further comprising at least one tail region, wherein a portion of each tail region is configured to specifically bind to a distinct linker region. 
     
     
         4 . The composition of  claim 1 , wherein the tail region and the probe region are covalently linked through a nucleic acid backbone. 
     
     
         5 . The composition of  claim 1  or  3 , wherein the tail region further comprises one or more nucleotides comprising one or more bases that are distinct from the bases within the homopolymeric base region. 
     
     
         6 . The composition of  claim 1  or  2 , wherein the linker region is configured to specifically bind to portions of multiple tail regions. 
     
     
         7 . The composition of  claim 1  or  2 , wherein the homopolymeric base region comprises a poly-A tail, a poly-T tail, a poly-C tail, or a poly-G tail. 
     
     
         8 . The composition of  claim 1  or  2 , wherein the homopolymeric base region comprises at least 100 consecutive nucleotides. 
     
     
         9 . The composition of  claim 1  or  2 , wherein the homopolymeric base region comprises at least 200 consecutive nucleotides. 
     
     
         10 . The composition of  claim 1  or  2 , wherein the target analyte comprises a protein, a peptide, or a nucleic acid. 
     
     
         11 . The composition of  claim 1  or  2 , wherein the probe region comprises a protein, a peptide, or a nucleic acid. 
     
     
         12 . The composition of  claim 1  or  2 , wherein the probe region comprises an antibody. 
     
     
         13 . The composition of  claim 1  or  2 , wherein the linker region sequence comprises at least 10 nucleotides. 
     
     
         14 . The composition of  claim 1  or  2 , wherein the linker region sequence comprises 20-25 nucleotides. 
     
     
         15 . The composition of  claim 1  or  3 , wherein the tail region further comprises:
 a nucleotide adjacent to the homopolymeric base region, wherein the nucleotide comprises a base that is distinct from the bases within the homopolymeric base region; 
 a second homopolymeric base region adjacent to the nucleotide, wherein the second homopolymeric base region comprises bases that are different from the nucleotide base; and 
 optionally a plurality of additional homopolymeric base regions each separated from an adjacent homopolymeric base region, by an intervening nucleotide, wherein the intervening nucleotide base is different from the bases of each adjacent homopolymeric base region. 
 
     
     
         16 . The composition of  claim 15 , wherein each homopolymeric base region comprises the same base. 
     
     
         17 . The composition of  claim 15 , wherein the nucleotide and each optional intervening nucleotide comprise the same base. 
     
     
         18 . A library comprising a plurality of compositions according to  claim 15 , wherein (1) each probe region is associated with a plurality of linker regions, and (2) each linker region specifically binds to a portion of a distinct tail region. 
     
     
         19 . The library of  claim 18 , wherein the lengths of all of the tail regions in the library are constant. 
     
     
         20 . A method of characterizing at least one target analyte, comprising:
 obtaining a plurality of ordered tail region sets, each of the ordered tail region sets comprising one or more tail regions of any of  claims 1  and  3 - 18 , and directed to a defined subset of N distinct target analytes, wherein the N distinct target analytes are immobilized on spatially separate regions of a substrate;   contacting the N distinct target analytes with the probe regions of any of  claims 1 - 18  under conditions designed to promote specific binding of the probe regions of the probe regions to one or more of the immobilized N distinct target analytes;   performing at least M cycles, wherein the performing comprises:
 (1) if the tail regions are not covalently attached to probe regions, a hybridization step comprising contacting the bound probe regions with the tail regions, wherein each tail region specifically binds to a linker region of a probe region; 
 (2) a synthesis step, the synthesis step comprising contacting the bound tail regions with a reaction mixture comprising reagents and under conditions that result in synthesis of a polynucleotide strand using the tail region as a template; and 
 (3) a stripping step, the stripping step comprising stripping the tail regions or the probe regions from the N distinct target analytes; 
   detecting during each of the at least M cycles a plurality of output signals from the spatially separate regions of the substrate; and   determining from the detected plurality of output signals at least K bits of information per cycle for one or more of the N distinct target analytes, wherein the at least K bits of information are used to determine L total bits of information, wherein K×M=L bits of information and L≧log 2  (N), and wherein the L bits of information are used to identify one or more of the N distinct target analytes.   
     
     
         21 . The method of  claim 20 , wherein L>log 2  (N), and wherein L comprises bits of information used for correcting errors in the plurality of signals. 
     
     
         22 . The method of  claim 20 , wherein L>log 2  (N), and wherein L comprises bits of information that are ordered in a predetermined order. 
     
     
         23 . The method of  claim 22 , wherein the predetermined order is a random order. 
     
     
         24 . The method of  claim 20 , wherein L>log 2  (N), and wherein L comprises bits of information used for determining an identification code for each of the N distinct target analytes. 
     
     
         25 . The method of  claim 20 , wherein L>log 2  (N), and wherein L comprises bits of information comprising a key for decoding an order of the ordered tail region sets for each cycle in the at least M cycles. 
     
     
         26 . The method of  claim 20 , further comprising using a key to decode the identity of one or more of the N distinct target analytes. 
     
     
         27 . The method of  claim 20 , further comprising digitizing the plurality of signals to expand a dynamic range of detection of the plurality of signals. 
     
     
         28 . The method of  claim 20 , further comprising comparing the L bits of information determined for an N target analyte with an expected bits of information for provided by a key, wherein the comparison is used to determine an identity of the N target analyte. 
     
     
         29 . The method of  claim 20 , wherein the method is computer implemented. 
     
     
         30 . The method of  claim 20 , further comprising determining from the L bits of information an error correction for the plurality of output signals. 
     
     
         31 . The method of  claim 30 , wherein the error correction comprises using a Reed-Solomon code. 
     
     
         32 . The method of  claim 20 , further comprising determining a number of ordered tail region sets based on the number of N distinct target analytes. 
     
     
         33 . The method of  claim 20 , wherein the substrate contains at least one transistor, the transistor detecting the plurality of output signals. 
     
     
         34 . The method of  claim 33 , wherein the transistor is an ion-sensitive field-effect transistor (ISFET) structure. 
     
     
         35 . A kit for characterizing at least one target analyte, comprising:
 a plurality of probe region containers, each probe region container holding a distinct molecule comprising the probe region and the linker region of  claim 1 ;   a plurality of ordered tail region containers, each tail region container holding a distinct nucleic acid molecule comprising the tail region of  claim 1 ;   a reaction mixture container holding a reaction mixture comprising enzymes and polynucleotides used for synthesizing a polynucleotide strand template from one of the tail regions; and   instructions for use comprising instructions for contacting the target analyte with the contents of at least one probe region container, or a portion thereof, the contents of at least one tail region container, or a portion thereof, and the contents of the reaction mixture container, or a portion thereof, under conditions that result in the synthesis of a polynucleotide strand reaction product.   
     
     
         36 . A kit for characterizing at least one target analyte, comprising:
 a plurality of composition containers, each composition container holding a distinct composition of any of  claims 1 - 18 ;   a reaction mixture container holding a reaction mixture comprising enzymes and polynucleotides used for synthesizing a polynucleotide strand template from one of the tail regions if a tail region is present; and   instructions for use comprising instructions for contacting the target analyte with the contents of at least one probe region container, or a portion thereof, and the contents of the reaction mixture container, or a portion thereof, under conditions that result in the synthesis of a polynucleotide strand reaction product.   
     
     
         37 . The kit of  claim 35  or  36 , the instructions for use further comprising:
 instructions for performing at least M cycles, wherein the performing comprises:
 (1) if the tail regions are not covalently attached to probe regions, a hybridization step comprising contacting the bound probe regions with the tail regions, wherein each tail region specifically binds to a linker region of a probe region; 
 (2) a synthesis step, the synthesis step comprising contacting the bound tail regions with a reaction mixture comprising reagents and under conditions that result in synthesis of a polynucleotide strand using the tail region as a template; and 
 (3) a stripping step, the stripping step comprising stripping the tail regions or the probe regions from the N distinct target analytes; 
 
 instructions for detecting during each of the at least M cycles a plurality of output signals from the spatially separate regions of the substrate; and 
 instructions for determining from the plurality of signals at least K bits of information per cycle for one or more of the N distinct target analytes, wherein the at least K bits of information are used to determine L total bits of information, wherein K×M=L bits of information and L≧log 2  (N), and wherein the L bits of information are used to determine a presence or an absence of one or more of the N distinct target analytes. 
 
     
     
         38 . The kit of  claim 37 , wherein L>log 2  (N). 
     
     
         39 . The kit of  claim 37 , further comprising instructions for determining an identification of each of the N distinct target analytes using the L bits of information, wherein L comprises bits of information for target identification. 
     
     
         40 . The kit of  claim 37 , further comprising instructions for determining an order of the plurality of ordered probe reagent sets using the L bits of information, wherein L comprises bits of information that are ordered in a predetermined order. 
     
     
         41 . The kit of  claim 37 , wherein the predetermined order is a random order. 
     
     
         42 . The kit of  claim 37 , further comprising instructions for using a key for decoding an order of the plurality of ordered probe reagent sets.