Compositions and Methods for Analyte Detection

Abstract

The inventions provided herein relate to detection reagents, compositions, methods, and kits comprising the detection reagents for use in detection, identification, and/or quantification of analytes in a sample. Such detection reagents and methods described herein allow multiplexing of many more labeled species in the same procedure than conventional methods, in which multiplexing is limited by the number of available and practically usable colors.

What is claimed is: 
     
         1 . A method for detecting a plurality of analytes in a sample, comprising:
 a. contacting the sample with a composition comprising a plurality of detection reagents, wherein each subpopulation of the detection reagents targets at least one different analyte, and wherein each detection reagent comprises:   at least one probe reagent targeting an analyte and at least one nucleic acid label comprising one or a plurality of pre-determined subsequences, wherein said at least one probe reagent and said at least one nucleic acid label are conjugated together; and wherein at least a portion of said one or the plurality of pre-determined subsequences form an identifier of said at least one probe reagent; and   b. detecting in a temporally-sequential manner said one or the plurality of the pre-determined subsequences of said detection reagent, wherein said detection of the subsequences each generates a signal signature corresponding to said subsequence, and wherein a temporal order of the signal signatures corresponding to said one or the plurality of the subsequences of said detection reagent identifies a subpopulation of the detection reagents.   
     
     
         2 . The method of  claim 1 , wherein said each subpopulation of the detection reagents targets a set of analytes. 
     
     
         3 . The method of  claim 1  or  2 , wherein the temporal order of the signal signatures corresponding to said one or the plurality of the subsequences of said detection reagent is unique for each subpopulation of the detection reagents. 
     
     
         4 . The method of any of  claims 1 - 3 , wherein said detection reagents are present in a soluble phase. 
     
     
         5 . The method of any of  claims 1 - 4 , further comprising processing said sample before said contacting with said plurality of detection reagents. 
     
     
         6 . The method of any of  claims 1 - 5 , further comprising removing any unbound detection reagents before the detecting step (b). 
     
     
         7 . The method of any of  claims 1 - 6 , further comprising comparing said temporal order of the signal signatures with different identifiers of said at least one probe reagent, wherein an agreement between the temporal order of the signal signatures and a particular identifier of said at least one probe reagent identifies the analyte in the sample. 
     
     
         8 . The method of any of  claims 1 - 7 , further comprising measuring the intensity of the signal signatures generated from each subpopulation of the detection reagents. 
     
     
         9 . The method of  claim 8 , wherein the intensity of the signal signatures generated from each subpopulation of the detection reagents indicates an amount of the analyte. 
     
     
         10 . The method of  claim 8  or  9 , wherein the intensity of the signal signatures generated from each subpopulation of the detection reagents is used in identification of the subpopulation of the detection reagents. 
     
     
         11 . The method of any of  claims 1 - 10 , wherein said detecting of step (b) comprises sequencing. 
     
     
         12 . The method of  claim 11 , wherein said sequencing is performed via ligation, hybridization, synthesis, amplification, or single-base extension. 
     
     
         13 . The method of any of  claims 1 - 12  wherein said detecting of step (b) comprises hybridizing a decoder probe with said subsequence, wherein said decoder probe comprises a detectable label. 
     
     
         14 . The method of any of  claims 1 - 13 , wherein said detecting of step (b) comprises:
 a. hybridizing a set of decoder probes with a subsequence of the detection reagents, wherein each subpopulation of the decoder probes comprises a detectable label, each detectable label producing a signal signature;   b. detecting said signal signature produced by the hybridization of said set of decoder probes;   c. optionally removing said different signal signature produced by the hybridization of said set of decoder probes; and   d. repeating steps (a) through (c) for other subsequences of said detection reagents, thereby producing a temporal order of the signal signatures corresponding to said each detection reagent.   
     
     
         15 . The method of  claim 14 , wherein said each subpopulation of the decoder probes comprises a different detectable label, each different detectable label producing a different signal signature. 
     
     
         16 . The method of  claim 14  or  15 , wherein said each subpopulation of the decoder probes is at least partially or completely complementary to said subsequence of the detection reagents. 
     
     
         17 . The method of any of  claims 14 - 16 , wherein at least two or more subpopulations of the decoder probes are at least partially or completely complementary to the same subsequence of the detection reagents. 
     
     
         18 . The method of any of  claims 14 - 17 , wherein said removing step is performed by washing, heating, photobleaching, displacement, cleavage, enzymatic digestion, quenching, chemical degradation, bleaching, oxidation or any combinations thereof. 
     
     
         19 . The method of any of  claims 14 - 18 , wherein said detectable label comprises or is an optical label selected from the group consisting of a small-molecule dye, a fluorescent molecule, a fluorescent protein, a quantum dot, Raman label, a chromophore, and any combinations thereof. 
     
     
         20 . The method of any of  claims 14 - 19 , wherein said detectable label comprises or is a colorimetric reagent. 
     
     
         21 . The method of any of  claims 14 - 20 , wherein said detectable label comprises or is a Raman label. 
     
     
         22 . The method of any of  claims 1 - 21 , wherein said signal signatures comprise or are optical signatures. 
     
     
         23 . The method of  claim 22 , wherein said optical signatures comprise signatures of fluorescent color, visible light, no-color, Raman label, or any combinations thereof. 
     
     
         24 . The method of  claim 22 , wherein said optical signatures comprise signatures of one or more fluorescent colors, one or more visible lights, one or more no-colors, one or more Raman labels, or any combinations thereof. 
     
     
         25 . The method of any of  claims 22 - 24 , wherein said optical signatures are detected by optical imaging or spectroscopy. 
     
     
         26 . The method of any of  claims 1 - 25 , wherein said analytes are selected from the group consisting of antigens, receptors, proteins, peptides, sugars, glycoproteins, peptidoglycans, lipids, nucleic acids, oligonucleotides, cells, viruses, and any combinations thereof. 
     
     
         27 . The method of any of  claims 1 - 26 , wherein said nucleic acids are selected from the group consisting of cellular DNA or RNA, messenger RNA, microRNA, ribosomal RNA, and any combinations thereof. 
     
     
         28 . The method of any of  claims 1 - 27 , wherein said sample is a protein sample immobilized on a solid support. 
     
     
         29 . The method of  claim 28 , wherein the solid support is a blotting membrane. 
     
     
         30 . The method of any of  claims 1 - 27 , wherein said sample is a biological sample. 
     
     
         31 . The method of  claim 30 , wherein said biological sample comprises one or more cells, one or more tissues, one or more fluids or any combinations thereof. 
     
     
         32 . The method of  claim 30  or  31 , wherein said biological sample comprises blood, sputum, cerebrospinal fluid, urine, saliva, sperm, sweat, mucus, nasal discharge, vaginal fluids or any combinations thereof. 
     
     
         33 . The method of any of  claims 30 - 31 , wherein the said biological sample comprises a biopsy, a surgically removed tissue, a swap or any combinations thereof. 
     
     
         34 . The method of  claims 1 - 27 , wherein said sample comprises an environmental sample, food, food byproduct, soil, an archaeological sample, an extraterrestrial sample, or any combinations thereof. 
     
     
         35 . The method of any of  claims 1 - 34 , wherein said at least one probe reagent and said at least one nucleic acid label are conjugated together by at least one linker. 
     
     
         36 . The method of  claim 35 , wherein said linker is a bond. 
     
     
         37 . The method of  claim 35  or  36 , wherein said linker is a linker molecule. 
     
     
         38 . The method of  claim 37 , wherein said linker molecule is a polymer, sugar, nucleic acid, peptide, protein, hydrocarbon, lipid, polyethylene glycol, crosslinker, or any combinations thereof. 
     
     
         39 . The method of any of  claims 35 - 38 , wherein said linker is a particle. 
     
     
         40 . The method of  claim 39 , wherein said particle is selected from a group consisting of a gold nanoparticle, a magnetic bead or nanoparticle, a polystyrene bead, a nanotube, a nanowire, a microparticle, and any combinations thereof. 
     
     
         41 . The method of  claim 40 , wherein said particle is a nanoparticle. 
     
     
         42 . The method of any of  claims 39 - 41 , wherein said particle is modified. 
     
     
         43 . The method of any of  claims 39 - 42 , wherein said particle is coated with streptavidin or a derivative thereof. 
     
     
         44 . The method of any of  claims 39 - 43 , wherein said particle is modified with at least one functional group. 
     
     
         45 . The method of  claim 44 , wherein said at least one functional group is selected from the group consisting of amine, carboxyl, hydroxyl, aldehyde, ketone, tosyl, silanol, chlorine, hydrazine, hydrazide, photoreactive groups, and any combinations thereof. 
     
     
         46 . The method of any of  claims 35 - 45 , wherein said linker is multivalent. 
     
     
         47 . The method of  claim 46 , wherein when the multivalent linker is an avidin-like molecule, both the probe reagent and the nucleic acid label are biotinylated. 
     
     
         48 . The method of any of  claims 1 - 47 , wherein said at least one probe reagent is selected from the group consisting of a nucleic acid, an antibody or a portion thereof, an antibody-like molecule, an enzyme, a cell, an antigen, a small molecule, a protein, a peptide, a peptidomimetic, a sugar, a carbohydrate, a lipid, a glycan, a glycoprotein, an aptamer, and any combinations thereof. 
     
     
         49 . The method of any of  claims 1 - 48 , wherein said at least one probe reagent is modified. 
     
     
         50 . The method of any of  claims 1 - 49 , wherein said at least one probe reagent is biotinylated. 
     
     
         51 . The method of any of  claims 1 - 50 , wherein said at least one nucleic acid label is single-stranded, double-stranded, partially double-stranded, a hairpin, linear, circular, branched, a concatemer, or any combinations thereof. 
     
     
         52 . The method of any of  claims 1 - 51 , wherein said at least one nucleic acid label is modified. 
     
     
         53 . The method of any of  claims 1 - 52 , wherein said at least one nucleic acid label is designed for minimal cross-hybridization of bases with each other. 
     
     
         54 . The method of any of  claims 1 - 53 , wherein said at least one nucleic acid label is conjugated to at least one detectable molecule. 
     
     
         55 . The method of  claim 54 , wherein said at least one detectable molecule is an optical molecule selected from the group consisting of a small-molecule dye, a fluorescent protein, a quantum dot, a Raman label, a chromophore, and any combinations thereof. 
     
     
         56 . The method of any of  claims 1 - 55 , wherein each of said plurality of the pre-determined subsequences comprises at least one base. 
     
     
         57 . The method of any of  claims 1 - 56 , wherein each of said plurality of the pre-determined subsequences comprises from 1 to 100 nucleobases. 
     
     
         58 . The method of any of  claims 1 - 57 , wherein said plurality of the pre-determined subsequences are conjugated together by at least one sequence linker. 
     
     
         59 . The method of  claim 58 , wherein said sequence linker is a bond. 
     
     
         60 . The method of any of  claims 58 - 59 , wherein said sequence linker is a nucleotidic linker. 
     
     
         61 . The method of  claim 60 , wherein said nucleotidic linker is single-stranded, double-stranded, partially double-stranded, a hairpin or any combinations thereof. 
     
     
         62 . The method of  claim 60  or  61 , wherein said nucleotidic linker is at least one nucleotide long. 
     
     
         63 . The method of any of  claims 1 - 62 , wherein said detection reagent comprises one probe reagent and a plurality of nucleic acid labels. 
     
     
         64 . The method of any of  claims 1 - 62 , wherein said detection reagent comprises a plurality of probe reagents and a nucleic acid label. 
     
     
         65 . The method of any of  claims 1 - 62 , wherein said detection reagent comprises a plurality of probe reagents and a plurality of nucleic acid labels. 
     
     
         66 . The method of any of  claims 1 - 65 , wherein the method is adapted for use in immunofluorescence. 
     
     
         67 . The method of any of  claims 1 - 66 , wherein the method is adapted for use in immunohistochemistry. 
     
     
         68 . The method of any of  claims 1 - 67 , wherein the method is adapted for use in fluorescence in situ hybridization. 
     
     
         69 . The method of any of  claims 1 - 68 , wherein the method is adapted for use in western blot. 
     
     
         70 . A detection reagent comprising at least one probe reagent and at least one nucleic acid label,
 wherein said at least one nucleic acid label comprises at least one pre-determined subsequence to be detected in a temporally-sequential manner;   wherein said at least one pre-determined subsequence forms an identifier of said at least one probe reagent; and   wherein said at least one probe reagent and said at least one nucleic acid label are conjugated together.   
     
     
         71 . The detection reagent of  claim 70 , wherein the detection reagent is present in a soluble phase. 
     
     
         72 . The detection reagent of  claim 70  or  71 , wherein said at least one probe reagent and said at least one nucleic acid label are conjugated together by at least one linker. 
     
     
         73 . The detection reagent of  claim 72 , wherein said linker is a bond. 
     
     
         74 . The detection reagent of  claim 72  or  73 , wherein said linker is a linker molecule. 
     
     
         75 . The detection reagent of  claim 74 , wherein said linker molecule is a polymer, sugar, nucleic acid, peptide, protein, hydrocarbon, lipid, polyethelyne glycol, crosslinker or combination thereof. 
     
     
         76 . The detection reagent of any of  claims 72 - 75 , wherein said linker is a particle. 
     
     
         77 . The detection reagent of  claim 76 , wherein said particle is selected from a group consisting of a gold nanoparticle, a magnetic bead or nanoparticle, a polystyrene bead, a nanotube, a nanowire, a microparticle, and any combinations thereof. 
     
     
         78 . The detection reagent of  claim 76  or  77 , wherein said particle is a nanoparticle. 
     
     
         79 . The detection reagent of any of  claims 76 - 78 , wherein said particle is modified. 
     
     
         80 . The detection reagent of any of  claims 76 - 79 , wherein said particle is coated with streptavidin or a derivative thereof. 
     
     
         81 . The detection reagent of any of  claims 76 - 80 , wherein said particle is modified with at least one functional group. 
     
     
         82 . The detection reagent of  claim 81 , wherein the at least one functional group is selected from the group consisting of amine, carboxyl, hydroxyl, aldehyde, ketone, tosyl, silanol, chlorine, hydrazine, hydrazide, photoreactive groups, and any combinations thereof. 
     
     
         83 . The detection reagent of any of  claims 72 - 82 , wherein said linker is multivalent. 
     
     
         84 . The detection reagent of  claim 83 , wherein when the multivalent linker is an avidin-like molecule, both the probe reagent and the nucleic acid label are biotinylated. 
     
     
         85 . The detection reagent of any of  claims 70 - 84 , wherein said at least one probe reagent is selected from the group consisting of a nucleic acid, an antibody or a portion thereof, an antibody-like molecule, an enzyme, a cell, an antigen, a small molecule, a protein, a peptide, a peptidomimetic, a sugar, a carbohydrate, a lipid, a glycan, a glycoprotein, an aptamer, and any combinations thereof. 
     
     
         86 . The detection reagent of any of  claims 70 - 85 , wherein said at least one probe reagent is modified. 
     
     
         87 . The detection reagent of any of  claims 70 - 86 , wherein said at least one probe reagent is biotinylated. 
     
     
         88 . The detection reagent of any of  claims 70 - 87 , wherein said at least one nucleic acid label is single-stranded, double-stranded, partially double-stranded, a hairpin, linear, circular, branched, a concatemer, or any combinations thereof. 
     
     
         89 . The detection reagent of any of  claims 70 - 88 , wherein said at least one nucleic acid label is modified. 
     
     
         90 . The detection reagent of any of  claims 70 - 89 , wherein said at least one nucleic acid label is designed for minimal cross-hybridization of bases with each other. 
     
     
         91 . The detection reagent of any of  claims 70 - 90 , wherein said at least one nucleic acid label is conjugated to at least one detectable molecule. 
     
     
         92 . The detection reagent of  claim 91 , wherein said at least one detectable molecule is an optical molecule selected from the group consisting of small-molecule dye, a fluorescent protein, a quantum dot, a Raman label, a chromophore, and any combinations thereof. 
     
     
         93 . The detection reagent of any of  claims 70 - 92 , wherein said at least one nucleic acid label comprises a plurality of pre-determined subsequences. 
     
     
         94 . The detection reagent of any of  claims 70 - 93 , wherein each of said plurality of predetermined subsequences comprises at least one base. 
     
     
         95 . The detection reagent of any of  claims 70 - 94 , wherein each of said plurality of predetermined subsequences comprises from 1 to 100 nucleobases. 
     
     
         96 . The detection reagent of any of  claims 70 - 95 , wherein said plurality of the pre-determined subsequences are conjugated together by at least one sequence linker. 
     
     
         97 . The detection reagent of  claim 96 , wherein said sequence linker is a bond. 
     
     
         98 . The detection reagent of any of  claims 96 - 97 , wherein said sequence linker is a nucleotidic linker. 
     
     
         99 . The detection reagent of  claim 98 , wherein said nucleotidic linker is single-stranded, double-stranded, partially double-stranded, a hairpin, or any combinations thereof. 
     
     
         100 . The detection reagent of  claim 98  or  99 , wherein said nucleotidic linker is at least one nucleotide long. 
     
     
         101 . The detection reagent of any of  claims 70 - 100 , wherein said detection reagent comprises one probe reagent and a plurality of nucleic acid labels. 
     
     
         102 . The detecton reagent of any of  claims 70 - 101 , wherein said detection reagent comprises a plurality of probe reagents and a nucleic acid label. 
     
     
         103 . The detection reagent of any of  claims 70 - 102 , wherein said detection reagent comprises a plurality of probe reagents and a plurality of nucleic acid labels. 
     
     
         104 . The detection reagent of any of  claims 70 - 103 , wherein the detection reagent is adapted for use in immunofluorescence. 
     
     
         105 . The detection reagent of any of  claims 70 - 104 , wherein the detection reagent is adapted for use in immunohistochemistry. 
     
     
         106 . The detection reagent of any of  claims 70 - 105 , wherein the detection reagent is adapted for use in fluorescence in situ hybridization. 
     
     
         107 . The detection reagent of any of  claims 70 - 106 , wherein the detection reagent is adapted for use in western blot. 
     
     
         108 . A kit comprising:
 a. a plurality of the detection reagents of any of  claims 70 - 107 ; and   b. at least one reagent.   
     
     
         109 . The kit of  claim 108 , wherein the kit further comprises at least one set of decoder probes complementary to at least a portion of subsequences of the detection reagents, wherein each subpopulation of the decoder probes comprises a different detectable label, each different detectable label producing a different signal signature. 
     
     
         110 . The kit of  claim 108  or  109 , wherein said detection reagents are present in a soluble phase. 
     
     
         111 . The kit of any of  claims 108 - 110 , wherein said detection reagents are immobilized in a multi-well plate. 
     
     
         112 . The kit of any of  claims 108 - 111 , wherein said at least one reagent is selected from the group consisting of a readout reagent, a wash buffer, a signal removal buffer, and any combinations thereof. 
     
     
         113 . A kit comprising:
 a. a plurality of the nucleic acid labels of the detection reagents of any of  claims 70 - 107 ;   b. at least one coupling agent that allows a user to conjugate the nucleic acid labels to the user's probe reagents of interest, thereby forming the detection reagents comprising the user's probe reagents of interest; and   c. at least one reagent.   
     
     
         114 . The kit of  claim 113 , wherein the kit further comprises at least one set of decoder probes complementary to at least a portion of subsequences of the detection reagents, wherein each subpopulation of the decoder probes comprises a different detectable label, each different detectable label producing a different signal signature. 
     
     
         115 . The kit of  claim 113  or  114 , wherein the detection reagents comprising the user's probe reagents of interest are present in a soluble phase. 
     
     
         116 . The kit of any of  claims 113 - 115 , wherein the detection reagents comprising the user's probe reagents of interest are immobilized in a multi-well plate. 
     
     
         117 . The kit of any of  claims 113 - 116 , wherein said at least one reagent is selected from the group consisting of a readout reagent, a wash buffer, a signal removal buffer, and any combinations thereof.