Methods for single molecule analyte detection
Methods are provided for detecting a single compound analyte immobilized to a solid substrate by serially contacting and removing different probes to the same analyte.
1. A method of detecting a single compound analyte bound to a solid substrate, said method comprising:
contacting said single compound analyte with a first probe thereby binding said first probe to said single compound analyte, wherein said first probe comprises a first analyte binding component and a first detectable label;
detecting said first detectable label;
removing said first probe from said single compound analyte and said solid substrate;
contacting said single compound analyte with a second probe thereby binding said second probe to said single compound analyte, wherein said second probe comprises a second analyte binding component and a second detectable label;
detecting said second detectable label; removing said second probe from said single compound analyte and said solid substrate;
contacting said single compound analyte with a third probe thereby binding said third probe to said single compound analyte, wherein said third probe comprises a third analyte binding component and a third detectable label; and
detecting said third detectable label thereby detecting the single compound analyte using said first, second, and third detectable labels;
wherein said first detectable label, said second detectable label and said third detectable label are different and wherein said single compound analyte is individually detected.
2. The method of claim 1 , wherein said single compound analyte is spatially separated from other single compound analytes on said solid substrate.
3. The method of claim 1 , wherein said first analyte binding component, said second analyte binding component, and said third analyte binding component are the same.
4. The method of claim 3 , wherein the first analyte binding component, said second analyte binding component, or said third analyte binding component further comprises an oligonucleotide tail region.
5. The method of claim 4 , wherein the first analyte binding component, said second analyte binding component, or said third analyte binding component further comprises a linker region.
6. The method of claim 4 , wherein said oligonucleotide tail comprises a region for polynucleotide synthesis.
7. The method of claim 6 , wherein said oligonucleotide tail comprises a homopolymeric base region of at least 25 nucleotides.
8. The method of claim 7 , wherein said homopolymeric base region comprises one or more stop bases.
9. The method of claim 1 , wherein said first detectable label, said second detectable label and said third detectable label are fluorescent or chemiluminescent.
10. The method of claim 1 , wherein said first detectable label, said second detectable label and said third detectable label are detected optically.
11. The method of claim 1 , wherein said first detectable label, said second detectable label and said third detectable label are detected electrically.
12. The method of claim 1 , wherein said single compound analyte is a protein or oligonucleotide derived from a biological sample.
13. The method of claim 1 , wherein said first analyte binding component, said second analyte binding component, and said third analyte binding component is a protein or oligonucleotide.
14. The method of claim 1 , wherein said first analyte binding component, said second analyte binding component, and said third analyte binding component is an antibody, aptamer or oligonucleotide.
15. The method of claim 1 , wherein said single compound analyte is a protein, peptide, RNA, or DNA.
16. The method of claim 1 , wherein said single compound analyte is a protein, peptide or RNA.
17. The method of claim 1 , wherein said single compound analyte is a cDNA.
18. The method of claim 1 , wherein said single compound analyte is a DNA.