Hydrogel microstructures with immiscible fluid isolation for small reaction volumes
Techniques for hydrogel microstructures with oil isolation for small reaction volumes include providing a hydrogel microstructure that has a plurality of pores and a hydrogel frame surrounding the pores. The microstructure has a volume in a range from about 1 picoliter to about 10,000 picoliters and is configured to repel an immiscible fluid. Each pore of the plurality of pores has a pore size configured to pass the target molecule in a first solution. The microstructure is contacted with the first solution, and with a second solution that includes a reactant molecule that reacts with the target molecule to produce an observable product molecule. The microstructure is encompassed with the immiscible fluid for an extended observation duration from about 1 to about 10,000 seconds, wherein the immiscible fluid does not pass into the pores but traps the product in the microstructure. The observable product molecule is measured at some time during the observation duration.
1. An apparatus comprising a structural substrate and one or more hydrogel microstructures, each comprising a plurality of pores and a hydrogel frame surrounding the pores,
wherein:
the structural substrate is a microchannel;
the hydrogel frame comprises a plurality of covalently embedded molecules of a probe species selected to bind to a target molecule;
each hydrogel microstructure has a volume confined to a range from about 1 picoliter to about 10,000 picoliters;
the one or more microstructures are fixed to the structural substrate; and
the plurality of pores have a pore size configured to pass the target molecule in solution through the microstructure by diffusion for binding to the probe species and prevent advection of a hydrophobic fluid through the pores when the device is flushed with the hydrophobic fluid at pressures in a range from about 0.2 pounds per square inch to about 25 pound per square inch,
the device further comprising:
a first fluid container configured to flush the microchannel with the first solution including the target molecule;
a second fluid container configured to flush the microchannel with a second solution that comprises a reactant molecule that reacts with the target molecule to produce an observable product molecule;
a third fluid container configured to flush the hydrogel microstructure with a hydrophobic fluid for an extended observation duration selected in a range from about 1 second to about 10,000 seconds; and
a port configured for observing the observable product molecule at some time during the observation duration.
2. A hydrogel microstructure as recited in claim 1 , wherein the pore size is greater than about 5 nanometers whereby large biomolecules may pass into the microstructure.
3. A hydrogel microstructure as recited in claim 1 , wherein the covalently embedded molecules of the probe species include an acrylate group.
4. An apparatus as recited in claim 1 , wherein the hydrogel frame comprises a plurality of covalently embedded molecules of a probe species selected to bind to the target molecule, whereby the target molecule in the first solution binds with a molecule of the plurality of covalently embedded molecules of the probe species.
5. An apparatus as recited in claim 1 , further comprising a fourth source configured to flush the microchannel with a fourth solution that comprises an enzyme that binds to the target molecule, wherein the reactant molecule is a substrate molecule that the enzyme converts to the observable product molecule.
6. An apparatus as recited in claim 1 , further comprising a rinse source configured to flush the microchannel with a rinse solution selected to remove at least one of the hydrophobic fluid or the target molecule or the reactant molecule or the observable product molecule.
7. An apparatus as recited in claim 1 , wherein the hydrogel microstructure is shaped to form an external reservoir of the second solution when the hydrogel microstructure is flushed with the hydrophobic fluid flowing past the microstructure.
8. A device comprising a microchannel and one or more hydrogel microstructures, each comprising a plurality of pores and a hydrogel frame surrounding the pores,
wherein:
the hydrogel frame comprises a plurality of covalently embedded molecules of a probe species selected to bind to a target molecule;
each hydrogel microstructure has a volume confined to a range from about 1 picoliter to about 10,000 picoliters;
the plurality of pores have a pore size configured to pass the target molecule in solution through the microstructure by diffusion for binding to the probe species and prevent advection of a hydrophobic fluid through the pores when the device is flushed with the hydrophobic fluid at pressures in a range from about 0.2 pounds per square inch to about 25 pound per square inch; and
each hydrogel microstructure is a microparticle not affixed to the device,
the device further comprising:
a first fluid container configured to flush the microchannel with the first solution including the target molecule;
a second fluid container configured to flush the microchannel with a second solution that comprises a reactant molecule that reacts with the target molecule to produce an observable product molecule;
a fluid source configured to flush the hydrogel microstructure with the hydrophobic fluid for an extended observation duration selected in a range from about 1 second to about 10,000 seconds, wherein the fluid source comprises a vortex that emulsifies the second solution around the microparticle in the hydrophobic fluid; and
a port configured for observing the observable product molecule at some time during the observation duration.
9. An apparatus as recited in claim 1 , wherein the one or more hydrogel microstructures are a plurality of hydrogel microstructures.
10. An apparatus as recited in claim 4 , further comprising a different hydrogel microstructure having at least one of a different probe species selected to bind to a different target molecule or a different volume or a different pore size.
11. The device as recited in claim 1 , wherein the microchannel has a total height h and the pore size is characterized by a size r and a ratio of h 2 divided by r 2 is much greater than 1.