Processing polynucleotide-containing samples
View Patent ↗Methods and systems for processing polynucleotides (e.g., DNA) are disclosed. A processing region includes one or more surfaces (e.g., particle surfaces) modified with ligands that regain polynucleotides under a first set of conditions (e.g., temperature and pH) and release the polynucleotides under a second set of conditions (e.g., higher temperature and/or more basic pH). The processing region can be used to, for example, concentrate polynucleotides of a sample and/or separate inhibitors of amplification reactions from the polynucleotides. Microfluidic devices with a processing region are disclosed.
1. A microfluidic device, comprising:
a processing region having a vertical central axis therethrough, the processing region comprised of an inlet, a retention member, a filter, and an outlet;
said retention member being configured to preferentially retain one or more polynucleotides in a sample as compared to polymerase chain reaction inhibitors in the sample, wherein said retention member comprises a plurality of polynucleotide binding particles, and said plurality of binding particles having surfaces that comprise a poly-cationic polyamide bound thereto, further wherein the retention member is configured to retain polynucleotides from a sample preferentially to inhibitors in the sample as the sample passes through the retention member from the processing region inlet to the processing region outlet;
wherein the vertical central axis of the processing region is perpendicular to a horizontal plane of the microfluidic device;
wherein the processing region outlet is disposed along the vertical central axis above the processing region inlet;
wherein the filter is configured to prevent the plurality of binding particles from passing downstream of the processing region;
a device inlet in communication with, and upstream of, the processing region;
a device outlet in communication with, and downstream of, the processing region; and
a detection region in fluid communication with and downstream of the retention member.
2. The microfluidic device of claim 1 , wherein the plurality of particles have a volume less than 5 microliters.
3. The microfluidic device of claim 1 , wherein the sample has a volume from 0.5 microliters to 3 milliliters.
4. The microfluidic device of claim 1 , wherein the poly-cationic polyamide comprises at least one of poly-DL-ornithine, PEI, poly-L-lysine and poly-D-lysine.
5. The microfluidic device of claim 1 , wherein the filter is disposed between the retention member and the processing region outlet.
6. The microfluidic device of claim 1 , further comprising at least one channel having at least one sub-millimeter cross-sectional dimension.
7. The microfluidic device of claim 1 , further comprising a valve, wherein the valve includes a mass of thermally responsive substance that is relatively immobile at a first temperature and more mobile at a second temperature.
8. The microfluidic device of claim 1 , further comprising a gate, wherein the gate includes a mass of thermally responsive substance which is relatively immobile at a first temperature and which, upon heating to a second temperature, changes state.
9. The microfluidic device of claim 1 , further comprising a fluid reservoir configured to hold a liquid.
10. The microfluidic device of claim 9 , wherein the reservoir comprises a liquid having a pH of at least about 10.
11. The microfluidic device of claim 9 , wherein the device is configured to contact the retention member with the liquid.
12. The microfluidic device of claim 1 , further comprising an actuator, wherein the actuator comprises a chamber having a mass of thermally expansive material therein, and a gas.
13. The microfluidic device of claim 1 , further comprising a hydrophobic vent, wherein the vent comprises a layer of porous hydrophobic material.
14. The microfluidic device of claim 1 , having three layers of polymeric material.
15. The microfluidic device of claim 1 , thermally associated with an array of heat sources.
16. The microfluidic device of claim 15 , wherein at least one of the heat sources is configured to heat an aqueous liquid in contact with the retention member to at least about 65° C.
17. The microfluidic device of claim 16 , wherein the liquid is held in a pouch, isolated from the reservoir by a generally impermeable membrane.
18. The microfluidic device of claim 17 , wherein the pouch additionally comprises a piercing member configured to puncture the impermeable membrane.
19. The microfluidic device of claim 1 , further comprising one or more PCR reagents stored as one or more lyophilized particles.
20. The microfluidic device of claim 19 , wherein the lyophilized particles have an average volume of about 5 microliters or less.
21. The microfluidic device of claim 1 , further comprising one or more lysing reagents stored as one or more lyophilized particles.
22. The microfluidic device of claim 21 , wherein the lyophilized particles have an average volume of about 35 microliters or less.
23. The microfluidic device of claim 1 , additionally comprising a lysing chamber and a waste chamber.
24. The microfluidic device of claim 1 , wherein the particles are made of a polymeric material selected from the group consisting of: polystyrene, latex polymers, polyacrylamide, and polyethylene oxide.
25. The microfluidic device of claim 24 , wherein the polymeric material is modified to include one or more carboxylic groups and/or one or more amino groups covalently bound to the polymeric material, wherein the groups provide an attachment point for one or more ligands of the poly-cationic polyamide.
26. The microfluidic device of claim 1 , wherein the particles have an average diameter of between about 4 microns and about 20 microns.
27. The microfluidic device of claim 1 , wherein the particles are present in a density of about 10 8 particles per milliliter.
28. The microfluidic device of claim 1 , wherein at least some of the particles are magnetic.