Compositions and methods for analyte detection
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.
1. A method for analyte identification, comprising:
(a) contacting a sample with a plurality of detection reagents, wherein said plurality of detection reagents comprises a detection reagent that targets an analyte of a plurality of analytes immobilized in the sample, wherein said detection reagent comprises: (i) probe targeting said analyte and (ii) a nucleic acid label comprising a plurality of pre-determined subsequences, wherein said probe and said nucleic acid label are conjugated together, and wherein said plurality of pre-determined subsequences forms an identifier of said probe;
(b) with said analyte immobilized in the sample and said probe coupled to said analyte, (i) hybridizing a first decoder probe with a first subsequence of said plurality of pre-determined subsequences, wherein said first decoder probe comprises a first detectable label, (ii) detecting a first signal signature from said first detectable label, (iii) hybridizing a second decoder probe with a second subsequence of said plurality of pre-determined subsequences, wherein said second decoder probe comprises a second detectable label, and (iv) detecting a second signal signature from said second detectable label, to provide a set of signal signatures comprising said first signal signature and said second signal signature; and
(c) comparing said set of signal signatures against set of signal signatures assigned to different analytes including said analyte, to identify said probe, thereby identifying said analyte immobilized in the sample.
2. The method of claim 1 , further comprising
processing said sample before said contacting with said plurality of detection reagents.
3. The method of claim 1 , wherein
said first decoder probe and said second decoder probe comprise different detectable labels, each producing a different signal signature; and/or
(ii) said first decoder probe and said second decoder probe are at least partially or completely complementary to said first subsequence and said second subsequence.
4. The method of claim 1 , further comprising, prior to hybridizing said second decoder probe with said second subsequence, removing said first signal signature.
5. The method of claim 1 , wherein
said first signal signature and said second signal signature comprise optical signatures.
6. The method of claim 1 , wherein
said plurality of analytes are selected from the group consisting of antigens, receptors, proteins, peptides, sugars, glycoproteins, peptidoglycans, lipids, nucleic acids, oligonucleotides, cells, viruses, and any combination thereof.
7. The method of claim 1 , wherein
said sample is a protein sample immobilized on a solid support.
8. The method of claim 1 , wherein said probe and said nucleic acid label are conjugated together by at least one linker, wherein:
(i) said linker is a bond; and/or
(ii) said linker is a linker molecule, wherein said linker molecule is a polymer, sugar, nucleic acid, peptide, protein, hydrocarbon, lipid, polyethylene glycol, crosslinker, or any combination thereof; and/or
(iii) said linker is multivalent, wherein when the multivalent linker is an avidin-like molecule, both the probe and the nucleic acid label are biotinylated; and/or
(iv) said linker is a particle.
9. The method of claim 8 , wherein said linker is said particle, and wherein:
(i) said particle is selected from the group consisting of a gold nanoparticle, a magnetic bead or nanoparticle, a polystyrene bead, a nanotube, a nanowire, a microparticle, and any combination thereof; and/or
(ii) said particle is modified; and/or
(iii) said particle is coated with streptavidin or a derivative thereof; and/or
(iv) said particle is modified with at least one functional group, 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 combination thereof.
10. The method of claim 1 , wherein:
(i) said probe is selected from the group consisting of a nucleic acid, an antibody or a portion thereof, an antibody-like molecule, an enzyme, 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 combination thereof; and/or
(ii) said probe is modified; and/or
(iii) said probe is biotinylated.
11. The method of claim 1 , wherein:
(i) said nucleic acid label is single-stranded, double-stranded, partially double-stranded, a hairpin, linear, circular, branched, a concatemer, or any combination thereof; and/or
(ii) said nucleic acid label is modified; and/or
(iii) said nucleic acid label is configured for minimal cross-hybridization of bases with each other; and/or
(iv) said nucleic acid label is conjugated to at least one detectable molecule, 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 combination thereof.
12. The method of claim 1 , wherein
each of said plurality of the pre-determined subsequences comprises at least one base.
13. The method of claim 1 , wherein
each detection reagent of said plurality of detection reagents comprises one probe and a plurality of nucleic acid labels.
14. The method of claim 1 , further comprising measuring an intensity of said first signal signature and/or said second signature generated from said detection reagent, and using said intensity to (i) determine an amount of said analyte, and/or (ii) identify said detection reagent.
15. The method of claim 1 , further comprising hybridizing a third decoder probe with a third subsequence of said plurality of pre-determined subsequences, wherein said third decoder probe comprises a third detectable label, and detecting a third signal signature from said third detectable label.
16. The method of claim 1 , wherein said plurality of analytes comprises another analyte different than said analyte.
17. The method of claim 1 , wherein said plurality of detection reagents comprises another detection reagent that targets another analyte different than said analyte, and wherein said set of signal signatures corresponding to said detection reagent is different than another set of signal signatures corresponding to said another detection reagent targeting said another analyte.
18. The method of claim 17 , wherein said another detection reagent comprises: (i) another probe targeting said another analyte, and (ii) another nucleic acid label comprising another plurality of pre-determined subsequences, wherein said another plurality of pre-determined subsequences is different than said plurality of pre-determined subsequences.
19. The method of claim 1 , wherein said plurality of detection reagents is present in a soluble phase.
20. The method of claim 1 , wherein said plurality of analytes comprises ribonucleic acid (RNA) molecules.
21. The method of claim 1 , wherein said plurality of analytes comprises deoxyribonucleic acid (DNA) molecules.
22. The method of claim 1 , further comprising removing any unbound detection reagents prior to (b).
23. The method of claim 1 , wherein said first signal signature and said second signature are detected by optical imaging or spectroscopy.
24. The method of claim 1 , wherein said sample is a biological sample comprising one or more cells, one or more tissues, one or more fluids, or any combination thereof.
25. The method of claim 1 , wherein said sample comprises an environmental sample, food sample, food byproduct, soil sample, an archaeological sample, an extraterrestrial sample, or any combination thereof.
26. The method of claim 1 , wherein each of said plurality of pre-determined subsequences comprises from 1 to 100 nucleobases.
27. The method of claim 1 , wherein each of said plurality of pre-determined subsequences comprises greater than one nucleobase.
28. The method of claim 1 , wherein said first subsequence and said second subsequence are conjugated together by a sequence linker.
29. The method of claim 28 , wherein said sequence linker is a covalent bond.
30. The method of claim 28 , wherein said sequence linker is a nucleotidc linker.
31. The method of claim 30 , wherein said nucleotidic linker is single-stranded, double-stranded, partially double-stranded, a hairpin or any combination thereof.
32. The method of claim 30 , wherein said nucleotidic linker comprises at least one nucleotide.
33. The method of claim 1 , wherein said detection reagent comprises a plurality of probes, which plurality of probes comprises said probe.
34. The method of claim 1 , wherein said detection reagent comprises a plurality of nucleic acid labels, which plurality of nucleic acid labels comprises said nucleic acid label.
35. The method of claim 1 , wherein said detection reagent comprises a plurality of probes and a plurality of nucleic acid labels, wherein said plurality of probes comprises said probe and said plurality of nucleic acid labels comprises said nucleic acid label.
36. The method of claim 1 , wherein said analyte is an antigen, receptor, protein, peptide, sugar, glycoprotein, peptidoglycan, lipid, nucleic acid, oligonucleotide, cell, or virus.