Digital Analysis of Molecular Analytes Using Single Molecule Detection

Abstract

Methods and systems are provided for small molecule analyte detection using digital signals, key encryption, and communications protocols. The methods provide detection of a large numbers of proteins, peptides, RNA molecules, and DNA molecules in a single optical or electrical detection assay within a large dynamic range.

What is claimed is: 
     
         1 . A method for detecting a plurality of analytes, comprising:
 obtaining a plurality of ordered probe reagent sets, each of said ordered probe reagent sets comprising one or more probes directed to a defined subset of N distinct target analytes, wherein said N distinct target analytes are immobilized on spatially separate regions of a substrate, and each of said probes is detectably labeled;   performing at least M cycles of probe binding and signal detection, each cycle comprising one or more passes, wherein a pass comprises use of at least one of said ordered probe reagent sets;   detecting from said at least M cycles a presence or an absence of a plurality of signals from said spatially separate regions of said substrate; and   determining from said plurality of signals at least K bits of information per cycle for one or more of said N distinct target analytes, wherein said 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 said L bits of information are used to determine a presence or an absence of one or more of said N distinct target analytes.   
     
     
         2 . The method of  claim 1 , wherein L>log 2  (N), and wherein L comprises bits of information for target identification. 
     
     
         3 . The method of  claim 1 , wherein L>log 2  (N), and wherein L comprises bits of information that are ordered in a predetermined order. 
     
     
         4 . The method of  claim 3 , wherein said predetermined order is a random order. 
     
     
         5 . The method of  claim 1 , wherein L>log 2  (N), and wherein L comprises bits of information comprising a key for decoding an order of said plurality of ordered probe reagent sets. 
     
     
         6 . The method of  claim 1 , further comprising digitizing said plurality of signals to expand a dynamic range of detection of said plurality of signals. 
     
     
         7 . The method of  claim 1 , wherein said at least K bits of information comprise information about the number of passes in a cycle. 
     
     
         8 . The method of  claim 1 , wherein said at least K bits of information comprise information about the absence of a signal for one of said N distinct target analytes. 
     
     
         9 . The method of  claim 1 , wherein said detectable label is a fluorescent label. 
     
     
         10 . The method of  claim 1 , wherein said probe comprises an antibody. 
     
     
         11 . The method of  claim 10 , wherein antibody is conjugated directly to a label. 
     
     
         12 . The method of  claim 10 , wherein said antibody is bound to a secondary antibody conjugated to a label. 
     
     
         13 . The method of  claim 1 , wherein said probe comprises an aptamer. 
     
     
         14 . The method of  claim 13 , wherein said aptamer comprises a homopolymeric base region. 
     
     
         15 . The method of  claim 1 , wherein said plurality of analytes comprises a protein, a peptide aptamer, or a nucleic acid molecule. 
     
     
         16 . The method of  claim 1 , wherein said detecting from said at least M cycles a presence or an absence of a plurality of signals comprises optically detecting said plurality of signals. 
     
     
         17 . The method of  claim 1 , wherein said detecting from said at least M cycles a presence or an absence of a plurality of signals comprises electrically detecting said plurality of signals. 
     
     
         18 . The method of  claim 1 , wherein said method is computer implemented. 
     
     
         19 . The method of  claim 1 , wherein K is one bit of information per cycle. 
     
     
         20 . The method of  claim 19 , wherein K is two bits of information per cycle. 
     
     
         21 . The method of  claim 20 , wherein K is three or more bits of information per cycle. 
     
     
         22 . The method of  claim 1 , further comprising determining from said L bits of information an error correction for said plurality of output signals. 
     
     
         23 . The method of  claim 22 , wherein said error correction comprises using a Reed-Solomon code. 
     
     
         24 . The method of  claim 1 , further comprising determining a number of ordered probe reagent sets based on the number of N distinct target analytes. 
     
     
         25 . The method of  claim 1 , further comprising determining a type of probe reagent sets based on the type of N distinct target analytes. 
     
     
         26 . The method of  claim 1 , wherein said N distinct target analytes are present in a sample, and wherein the sample is divided into a plurality of aliquots that are diluted to a plurality of distinct final dilutions, each of said plurality of aliquots being immobilized onto a distinct section of the substrate. 
     
     
         27 . The method of  claim 26 , wherein one of the distinct final dilutions is determined based on a probable naturally-occurring concentration of at least one of the N distinct target analytes. 
     
     
         28 . The method of  claim 26 , wherein a concentration of one of the N distinct target analytes is determined by counting the occurrences of the target analyte within one of the distinct sections and adjusting the count according to the dilution of the respective aliquot. 
     
     
         29 . A kit for detecting a plurality of analytes, comprising:
 a plurality of ordered probe reagent sets, each of said ordered probe reagent sets comprising one or more probes directed to a defined subset of N distinct target analytes, wherein said N distinct target analytes are immobilized on spatially separate regions of a substrate, and each of said probes is detectably labeled;   instructions for detecting said N distinct analytes based on a plurality of detectable signals, said instructions comprising:
 instructions for performing at least M cycles of probe binding and signal detection, each cycle comprising one or more passes, wherein a pass comprises use of at least one of said ordered probe reagent sets; 
 instructions for detecting from said at least M cycles a presence or an absence of a plurality of signals from said spatially separate regions of said substrate; and 
 instructions for determining from said plurality of signals at least K bits of information per cycle for one or more of said N distinct target analytes, wherein said 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 said L bits of information are used to determine a presence or an absence of one or more of said N distinct target analytes. 
   
     
     
         30 . The kit of  claim 29 , wherein said one or more probes comprises an antibody. 
     
     
         31 . The kit of  claim 29 , wherein said label is a fluorescent label. 
     
     
         32 . The kit of  claim 29 , wherein said probe comprises an antibody. 
     
     
         33 . The kit of  claim 32 , wherein said antibody is conjugated directly to a label. 
     
     
         34 . The kit of  claim 32 , wherein said antibody is bound to a secondary antibody conjugated to a label. 
     
     
         35 . The kit of  claim 29 , wherein said probe comprises an aptamer. 
     
     
         36 . The kit of  claim 35 , wherein said aptamer comprises a homopolymeric base region. 
     
     
         37 . The kit of  claim 29 , wherein said plurality of analytes comprises a protein, a peptide aptamer, or a nucleic acid molecule. 
     
     
         38 . The kit of  claim 29 , wherein L>log 2  (N). 
     
     
         39 . The kit of  claim 29 , wherein M≦N. 
     
     
         40 . The kit of  claim 29 , further comprising instructions for determining an identification of each of said N distinct target analytes using said L bits of information, wherein L comprises bits of information for target identification. 
     
     
         41 . The kit of  claim 29 , further comprising instructions for determining an order of said plurality of ordered probe reagent sets using said L bits of information, wherein L comprises bits of information that are ordered in a predetermined order. 
     
     
         42 . The kit of  claim 41 , wherein said predetermined order is a random order. 
     
     
         43 . The kit of  claim 29 , further comprising instructions for using a key for decoding an order of said plurality of ordered probe reagent sets.