Micro-fluidic device for measuring osmotic second virial coefficients
A micro-fluidic device having a cavity containing a solid support (e.g., a plurality of particles) and a biomolecule immobilized on a surface of the solid support is provided. The micro-fluidic device can be used to monitor interactions between a biomolecule in a sample and the biomolecule immobilized on the solid support. For example, the micro-fluidic device can be used to monitor protein self-interactions wherein the protein in the sample is the same protein as that immobilized on the surface of the solid support. A method of making the micro-fluidic device is also described. The micro-fluidic device can be used for chromatographic applications where sample consumption is crucial.
1. A method of measuring non-binding interactions between a first biomolecule and a second biomolecule to determine an osmotic second virial coefficient, the method comprising:
flowing a solution through a micro-fluidic device comprising a solid support contained in a cavity, wherein the first biomolecule is immobilized on a surface of the solid support;
injecting a sample comprising the second biomolecule into the flowing solution upstream of the cavity; and
measuring the time (retention time, t R ) required for the second biomolecule to emerge from the cavity;
wherein the cavity has an internal volume of 100 μl or less, and wherein the retention time (t R ) is a measure of a degree of non-binding interactions between the first biomolecule and the second biomolecule which correlates with the osmotic second virial coefficient for the first biomolecule and second biomolecule pair.
2. The method of claim 1 , further comprising:
determining the time required (t m ) for a non-interacting marker molecule to pass through the cavity, said marker molecule having a substantial absence of interactions with the first biomolecule; and
calculating a chromatographic capacity factor (k′) from t R and t m , wherein
k
′
=
t
R
-
t
m
t
m
.
3. The method of claim 1 , wherein the first and second biomolecules are the same biomolecule.
4. The method of claim 1 , wherein the first and second biomolecules are different biomolecules.
5. The method of claim 2 , further comprising:
correlating k′ with the osmotic second virial coefficient describing the non-binding interactions between the first and the second biomolecule.
6. The method of claim 1 , wherein the first and second biomolecules are both lysozyme.
7. The method of claim 1 , wherein the first and second biomolecules are proteins.
8. The method of claim 1 , wherein the first biomolecule is a ligand and the second biomolecule is a protein.
9. The method of claim 1 , wherein the first biomolecule is a drug and the second biomolecule is a protein.
10. The method of claim 1 , wherein the sample further comprises a compound, and wherein the time required for the second biomolecule to emerge from the cavity (t R ) is a measure of the degree of non-binding interactions described by the osmotic second viral coefficient between the first biomolecule and the second biomolecule in the presence of the compound.
11. The method of claim 1 , wherein the cavity has an internal volume of 10 μl or less.