RAPID AND CONTINUOUS ANALYTE PROCESSING IN DROPLET MICROFLUIDIC DEVICES
The compositions and methods described herein are designed to introduce functionalized microparticles into droplets that can be manipulated in microfluidic devices by fields, including electric (dielectrophoretic) or magnetic fields, and extracted by splitting a droplet to separate the portion of the droplet that contains the majority of the microparticles from the part that is largely devoid of the microparticles. Within the device, channels are variously configured at Y- or T junctions that facilitate continuous, serial isolation and dilution of analytes in solution. The devices can be limited in the sense that they can be designed to output purified analytes that are then further analyzed in separate machines or they can include additional channels through which purified analytes can be further processed and analyzed.
1 .- 17 . (canceled)
18 . A method of analyzing an analyte in a sample, the method comprising introducing the sample into a microfluidic device comprising:
(a) a substrate comprising a series of contiguous channels and;
(b) a field generator,
wherein the series of contiguous channels comprises a first channel having an initial segment configured to receive a tagged droplet comprising an analyte bound to a functionalized particle, a middle segment through which the tagged droplet travels, and a terminal segment that bifurcates into a second channel and a third channel,
wherein the field generator is positioned adjacent to the first channel and marginalizes the analyte toward a side of the tagged droplet such that, upon reaching the bifurcation, a portion of the tagged droplet that includes the majority of the analyte enters the second channel and the remainder of the tagged droplet enters the third channel, thereby producing, in the second channel, a smaller droplet that contains the majority of the analyte while excluding at least some of the complex mixture.
19 . A method of manipulating an analyte within a sample, the method comprising:
providing the sample;
encapsulating the sample within a droplet comprising a functionalized particle, wherein the functionalized particle comprises a field-responsive element and a tag that specifically binds the analyte, thereby generating an analyte-tagged droplet;
marginalizing the analyte by exposing the analyte-tagged droplet to a field generator;
cleaving the analyte-tagged droplet so the portion of the tagged droplet that includes the majority of the marginalized analyte becomes contained within a smaller droplet; and
diluting the analyte by fusing the smaller droplet with a droplet comprising a buffer or reagent, thereby generating an analyte-diluted droplet.
20 . The method of claim 19 , wherein the analyte-tagged droplet is one of a plurality and the method is carried out by continuously and rapidly moving the plurality of droplets through the channels of a microfluidic device.
21 . The method of claim 19 , wherein the analyte is a biomolecule selected from the group consisting of a nucleic acid, a protein, a sugar, and a fat.
22 . The method of claim 19 , wherein the sample is a single, lysed biological cell.
23 . The method of claim 22 , wherein the single, lysed biological cell is prepared by:
encapsulating a single biological cell in a droplet, thereby generating a cell-containing droplet; and
fusing the cell-containing droplet with a droplet comprising a lysis buffer, thereby generating a droplet comprising a single, lysed biological cell.
24 . The method of claim 19 , further comprising the steps of
exposing the analyte-diluted droplet to a field generator, wherein the field generator marginalizes the analyte toward a side of the analyte-diluted droplet; and
cleaving the analyte-diluted droplet so the portion of the dilution droplet that includes the majority of the analyte becomes contained within a smaller analyte-diluted droplet.
25 . A kit comprising instructions for use, a microfluidic device comprising a plurality of channels configured to form junctions for facilitating continuous, serial separation and dilution of an analyte, and one or more of the following items:
(a) a fluid for encapsulating a sample comprising the analyte in a droplet;
(b) a fluid for sheathing the droplet;
(c) a lysis buffer;
(d) a functionalized particle;
(e) a dilution buffer; and
(f) a solution comprising a reagent.
26 . The kit of claim 25 , wherein the junctions comprise one or more of a T-shaped, W-shaped, X-shaped, or Y-shaped junction.
27 . The kit of claim 25 , wherein the microfluidic device further comprises a microchannel for encapsulating a single cell comprising an analyte in a droplet.
28 . The kit of claim 25 , wherein the microfluidic device further comprises a field generator.
29 . The kit of claim 25 , wherein the fluid for encapsulating the sample is an aqueous fluid and the fluid for sheathing the droplet is a non-aqueous fluid.
30 . The kit of claim 25 , wherein the reagent is an enzyme or a reagent required for carrying out PCR.