Assays for measuring binding kinetics and binding capacity of acceptors for lipophilic or amphiphilic molecules
Aspects of the invention relate to methods for measuring the binding constant of a lipophilic or amphiphilic molecule acceptor for a lipophilic or amphiphilic molecule. Methods involve rapid, cell-free competition assays including a labeled lipophilic or amphiphilic molecule and nanoparticle.
1. A method for measuring the equilibrium constant of an acceptor for a lipophilic or amphiphilic molecule, comprising:
(a) providing the lipophilic or amphiphilic molecule having detectable signal;
(b) providing a structure, the structure comprising a nanostructure core and a lipid layer surrounding and attached to the nanostructure core, wherein the structure quenches the signal of the molecule when the structure and the molecule are proximate;
(c) providing an acceptor;
(d) allowing the acceptor to compete with the structure for binding with the molecule; and
(e) measuring the signal, wherein the level of the signal correlates with the equilibrium constant of the acceptor for the molecule,
wherein the method is a cell-free method.
2. The method of claim 1 , the method further comprising increasing the amount of acceptor provided.
3. The method of claim 1 , the method further comprising increasing the amount of structure provided.
4. The method of claim 2 , wherein increasing the amount of acceptor provided leads to an increase in signal.
5. The method of claim 3 , wherein increasing the amount of structure provided leads to a decrease in signal.
6. The method of claim 1 , wherein increasing or decreasing the amount of molecule improves the signal to noise ratio of the system.
7. The method of claim 1 , wherein the method is a component of an assay.
8. The method of claim 1 , wherein the method is a component of a diagnostic assay.
9. The method of claim 1 , wherein the method is a method for assessing cardiovascular risk in a subject.
10. The method of claim 1 , wherein the lipid layer is a bilayer.
11. The method of claim 1 , wherein the molecule is a steroid or a derivative or analog thereof.
12. The method of claim 1 , wherein the molecule is a lipopolysaccharide or a derivative or analog thereof.
13. The method of claim 1 , wherein the molecule is a Cholestane, Cholane, Pregnane, Androstane or Estrane or a derivative or analog thereof.
14. The method of claim 1 , wherein the molecule is BODIPY-cholesterol.
15. The method of claim 1 , wherein the signal is fluorescence.
16. The method of claim 1 , wherein the signal is fluorescence polarization.
17. The method of claim 1 , wherein the nanostructure core is an inorganic material.
18. The method of claim 1 , wherein the nanostructure core is a metal.
19. The method of claim 1 , wherein the nanostructure core is gold.
20. The method of claim 1 , wherein the structure further comprises apolipoprotein bound to at least the outer surface of the lipid layer.
21. The method of claim 20 , wherein the apolipoprotein is apolipoprotein A-I, apolipoprotein A-IL or apolipoprotein E.
22. The method of claim 1 , wherein the acceptor is a lipoprotein.
23. The method of claim 1 , wherein the acceptor is a high-density lipoprotein (HDL).
24. The method of claim 1 , wherein the acceptor is a component of serum.
25. The method of claim 24 , wherein the serum is human serum.
26. The method of claim 24 , wherein the serum is enriched for HDL.
27. The method of claim 24 , wherein the serum is depleted for ApoB.