SYSTEM, METHOD, AND APPARATUS FOR MEASURING AFFINITY CONSTANTS
First Claim
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1. A non-linear spectroscopic method of measuring an affinity constant without labeling, the method comprising:
- providing a plurality of free, charged biomolecules in an aqueous solution;
providing a plurality of bound biomolecules, the bound biomolecules being bound to respective surfaces of a plurality of polymer beads;
complexing the free charged biomolecules with the bound biomolecules;
probing the complexed biomolecules using second harmonic generation (“
SHG”
);
detecting a change in a net electric charge for solid/aqueous interfaces associated with the complexed biomolecules based on an SHG signal received from the solution; and
determining an affinity constant of the complexed biomolecules based on said detecting.
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Abstract
Nonlinear spectroscopic systems, methods, and apparatuses for measuring affinity constants of molecule-molecule reactions and/or for determining a maximum number of molecule-molecule complexes. Second harmonic generation (“SHG”) or sum-frequency generation is used to probe a sample. A magnitude of a net electric charge at a solid/water interface can be detected and used to determine characteristics of the molecule-molecule complex.
13 Citations
45 Claims
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1. A non-linear spectroscopic method of measuring an affinity constant without labeling, the method comprising:
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providing a plurality of free, charged biomolecules in an aqueous solution; providing a plurality of bound biomolecules, the bound biomolecules being bound to respective surfaces of a plurality of polymer beads; complexing the free charged biomolecules with the bound biomolecules; probing the complexed biomolecules using second harmonic generation (“
SHG”
);detecting a change in a net electric charge for solid/aqueous interfaces associated with the complexed biomolecules based on an SHG signal received from the solution; and determining an affinity constant of the complexed biomolecules based on said detecting.
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2. The method of claim 1, wherein said determining includes comparing a detected net electric charge with a previously detected net electric charge.
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3. The method of claim 1, wherein the plurality of bound biomolecules are charged.
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4. The method of claim 1, wherein the plurality of bound biomolecules are neutral.
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5. The method of claim 1, wherein the free charged biomolecules have a first negative charge, and the bound biomolecules have a second negative charge.
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6. The method of claim 5, wherein the first and second negative charges are the same.
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7. The method of claim 1, further comprising quantifying a maximum number of complexing biomolecules based on said determining the affinity constant.
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8. The method of claim 1, wherein the charged biomolecules are one of a protein, DNA, and RNA, and the bound molecules are one of a protein, DNA, and RNA.
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9. The method of claim 1, further comprising:
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providing a second plurality of free biomolecules in the aqueous solution; providing a second plurality of bound biomolecules; complexing the second free biomolecules with the second bound biomolecules; and selectively probing the first complexed biomolecules using second harmonic generation (“
SHG”
) in the presence of the second complexed biomolecules.
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10. The method of claim 1, wherein the determined affinity constant is used for one of drug discovery, for drug design, to investigate diseases and develop diagnostic methods at the biomolecular level, and for DNA sequencing.
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11. The method of claim 1, wherein 9 ng or less of the free, charged biomolecules is provided.
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12. The method of claim 1, further comprising measuring time-dependent processes associated with said complexing.
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13-26. -26. (canceled)
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27. A system comprising:
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means for providing light at a first frequency; means for outputting light at a second frequency different from the first frequency, said means for outputting including a sample of complexed molecules in an aqueous solution; means for detecting the light at the second frequency; and means for determining an affinity constant of complexed molecules at a solid/aqueous interface based on an output signal received from said means for detecting, wherein said means for providing light illuminates said means for outputting light with light at the first frequency.
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28. The system of claim 27, wherein said means for outputting includes means for filtering out frequencies other than the second frequency.
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29. The system of claim 27, wherein said means for providing light includes a laser.
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30-39. -39. (canceled)
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40. A method for detecting a change in net electric charge at a solid/water interface when free molecules from a medium complex with bound molecules that are bound to the solid side of the interface, the method comprising illuminating a complex formed by the bound and free molecules with light of a first frequency and capturing second harmonic light generated by an electric field near the complex;
- determining from the intensity of the second harmonic light, a characteristic of the complex.
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41. The method of claim 40, further comprising generating an output to a display device responsively to a result of the determining.
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42. The method of claim 40, wherein the medium is an aqueous medium.
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43. The method of claim 40, wherein the Free molecule is a biomolecule.
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44. The method of claim 40, wherein the bound molecule is a biomolecule.
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45-55. -55. (canceled)
Specification