Thermally resolved molecule assays
Provided herein are compositions and methods including the step of thermally scanning a sample that can be used and implemented to detect the presence of and/or concentration of a molecule in a sample.
1. A method of target molecule detection comprising:
contacting a sample with a first probe, wherein the first probe is configured to bind a target molecule;
contacting the sample with a second probe, wherein the second probe is configured to bind the target molecule;
forming a signal complex, wherein the signal complex comprises a target molecule that is bound to the first probe and the second probe;
forming a background complex, wherein the background complex does not comprise a target molecule that is simultaneously bound to the first probe and the second probe;
thermally scanning the signal complex and the background complex to form a temperature melting curve, the temperature melting curve comprising:
a first melting temperature peak, wherein the first melting temperature peak corresponds to the melting temperature of the signal complex; and
a second melting temperature peak, wherein the second temperature melting peak corresponds to the melting temperature of the background complex; and
detecting the target molecule via measuring a characteristic of the first melting temperature peak.
2. The method of claim 1 , further comprising quantifying the amount of signal complex by the peak area of the first melting temperature peak.
3. The method of claim 1 , wherein the first probe is selected from the group consisting of: aptamers, antibodies or fragments thereof, proteins, and oligonucleotides.
4. The method of claim 1 , wherein the second probe is selected from the group consisting of: aptamers, antibodies or fragments thereof, proteins, and oligonucleotides.
5. The method of claim 4 , wherein the first probe and the second probe are each directly bound to the target molecule in the signal complex.
6. The method of claim 5 , wherein the first probe and the second probe are each selected from the group consisting of: aptamers, antibodies or fragments thereof, proteins, and oligonucleotides.
7. The method of claim 6 , further comprising contacting the signal complex with an intercalating agent.
8. The method of claim 7 , wherein the step of thermally scanning further comprises measuring a signal produced by the intercalating agent.
9. The method of claim 6 , wherein the first probe, the second probe, or each of the first and the second probes comprise an optically active molecule.
10. The method of claim 9 , wherein the step of thermally scanning further comprises measuring a signal produced by the optically active molecule.
11. The method of claim 5 , further comprising contacting the sample with a proximity oligonucleotide.
12. The method of claim 11 , wherein the first probe is configured to bind the target molecule and the proximity oligonucleotide.
13. The method of claim 12 , wherein the second probe is configured to bind the target protein and the proximity oligonucleotide, and wherein the first and the second probes form a paired bivalent probe.
14. The method of claim 13 , wherein the first probe and the second probe each comprise a protein binding moiety independently selected the group consisting of: aptamers, antibodies or fragments thereof, proteins, and oligonucleotides.
15. The method of claim 14 , wherein the binding moiety is an antibody or a fragment thereof and wherein the antibody or a fragment thereof further comprises a connector oligonucleotide, wherein the connector oligonucleotide is coupled to the antibody or fragment thereof and is configured to bind the proximity nucleotide.
16. The method of claim 13 , wherein the first probe comprises a quencher molecule.
17. The method of claim 16 , wherein the second probe comprises an optically active molecule.
18. The method of claim 17 , wherein the step of thermally scanning further comprises measuring a signal produced by the optically active molecule.
19. The method of claim 13 , wherein the proximity oligonucleotide is coupled to an electrically conductive substrate.
20. The method of claim 19 , wherein the first probe, the second probe, or each of the first probe and the second probe further comprises a redox-active molecule.
21. The method of claim 20 , wherein the step of thermally scanning further comprises measuring the current produced by the electrically conductive substrate.
22. The method of claim 12 , further comprising contacting the sample with a connector oligonucleotide, wherein the connector oligonucleotide is configured to bind the proximity oligonucleotide.
23. The method of claim 22 , wherein the second probe is configured to bind the target molecule and the connector oligonucleotide.
24. The method of claim 23 , wherein the proximity oligonucleotide is coupled to an electrically conductive substrate.
25. The method of claim 24 , wherein the first probe, the second probe, the connector oligonucleotide, or combinations thereof, further comprise a redox active molecule.
26. The method of claim 25 , wherein the step of thermally scanning further comprises measuring the current produced by the electrically conductive substrate.