INTEGRATION OF EX SITU FABRICATED POROUS POLYMER MONOLITHS INTO FLUIDIC CHIPS

Abstract

Bare porous polymer monoliths, fluidic chips, methods of incorporating bare porous polymer monoliths into fluidic chips, and methods for functionalizing bare porous polymer monoliths are described. Bare porous polymer monoliths may be fabricated ex situ in a mold. The bare porous polymer monoliths may also be functionalized ex situ. Incorporating the bare preformed porous polymer monoliths into the fluidic chips may include inserting the monoliths into channels of channel substrates of the fluidic chips. Incorporating the bare preformed porous polymer monoliths into the fluidic chips may include bonding a capping layer to the channel substrate. The bare porous polymer monoliths may be mechanically anchored to channel walls and to the capping layer. The bare porous polymer monoliths may be functionalized by ex situ immobilization of capture probes on the monoliths. The monoliths may be functionalized by direct attachment of chitosan.

Claims

1. A method of incorporating one or more porous polymer monoliths into a fluidic chip, the method comprising: inserting one or more bare preformed porous polymer monoliths into one or more channels of a channel substrate of the fluidic chip.

2. The method of claim 1, further comprising fabricating the one or more bare porous polymer monoliths.

3. The method of claim 2, wherein fabricating the one or more bare porous polymer monoliths comprises fabricating one or more porous polymer monoliths in a mold.

4. The method of claim 3, wherein fabricating the one or more monoliths in the mold comprises: adding a pre-monolith solution to one or more channels of a molding substrate; photopolymerizing the pre-monolith solution; and removing the polymerized solution from the one or more channels of the molding substrate.

5. The method of claim 1, further comprising chemically functionalizing one or more porous polymer monoliths, wherein the one or more inserted monoliths comprise the one or more functionalized porous polymer monoliths.

6. The method of claim 5, wherein chemically functionalizing one or more porous polymer monoliths comprises immobilizing a capture probe on the one or more porous polymer monoliths.

7. The method of claim 6, wherein the capture probe is an antibody, protein, amino acid, or peptide.

8. The method of claim 6, wherein the capture probe is labeled with a fluorescent marker.

9. The method of claim 6, wherein the capture probe is chitosan.

10. The method of claim 9, wherein the chitosan is immobilized on the one or more porous polymer monoliths using a bifunctional cross-linker.

11. The method of claim 9, wherein the chitosan is immobilized on the one or more porous polymer monoliths through a direct reaction of the chitosan with the one or more porous polymer monoliths.

12. The method of claim 1, further comprising bonding a capping layer to the channel substrate of the fluidic chip, wherein bonding the capping layer to the channel substrate seals the one or more bare porous polymer monoliths in the one or more channels of the channel substrate of the fluidic chip.

13. The method of claim 1, wherein inserting the one or more monoliths into one or more channels of the channel substrate comprises: depositing a bare porous polymer monolith within a droplet of water onto the channel substrate; and seating the deposited monolith into a channel of the channel substrate.

14. The method of claim 13, further comprising: suspending a bare porous polymer monolith in water; and drawing the suspended monolith into a pipette; wherein the monolith deposited onto the channel substrate is deposited from the pipette.

15. The method of claim 13, further comprising: removing the water droplet from the channel substrate; and drying the channel substrate.

16. The method of claim 13, wherein seating the deposited monolith into the channel comprises agitating the deposited monolith.

17. The method of claim 1, wherein the one or more monoliths have cross-sectional dimensions larger than the cross-sectional dimensions of the one or more channels.

18. The method of claim 1, wherein the one or more monoliths are oversized relative to the one or more channels.

19. The method of claim 1, wherein inserting the one or more bare porous polymer monoliths into the one or more channels of the channel substrate of the fluidic chip comprises: inserting a first bare porous polymer monolith in a channel of the one or more channels of the channel substrate; and inserting a second bare porous polymer monolith in the channel of the one or more channels of the channel substrate.

20. The method of claim 19, wherein the first monolith has a first functionalization, the second monolith has a second functionalization, and the first functionalization is different than the second functionalization.

21. The method of claim 20, wherein the first monolith comprises a first monolith chemistry, the second monolith comprises a second monolith chemistry, and the first monolith chemistry and the second monolith chemistry are different.

22. The method of claim 21, wherein the first monolith chemistry is hydrophilic, and the second monolith chemistry is hydrophobic.

23. The method of claim 1, further comprising anchoring the one or more inserted monoliths to walls of the one or more channels.

24. The method of claim 23, wherein anchoring the one or more inserted monoliths comprises softening the one or more channels of the channel substrate of the fluidic chip.

25. The method of claim 24, wherein softening the one or more channels of the channel substrate of the fluidic chip comprises exposing at least a portion of the one or more channels to a solvent.

26. The method of claim 25, wherein the solvent comprises decahydronaphthalene (decalin).

27. The method of claim 26, wherein the solvent comprises a solution of decalin in ethanol.

28. The method of claim 23, wherein anchoring the one or more inserted monoliths to the walls of the one or more channels results in mechanical interlocking of the one or more inserted monoliths and the walls of the one or more channels.

29. The method of claim 23, wherein anchoring the one or more inserted monoliths to the walls of the one or more channels does not result in covalent attachment of the one or more inserted monoliths and the walls of the one or more channels.

30. A chitosan-functionalized porous polymer monolith comprising: a porous polymer monolith; and a chitosan anchored to the porous polymer monolith.

31. The monolith of claim 30, wherein the chitosan is anchored to the porous polymer monolith using a bifunctional cross-linker.

32. The monolith of claim 31, wherein the bifunctional cross-linker is N-[-maleimidobutyryloxy]succinimide ester.

33. The monolith of claim 30, wherein the chitosan is anchored to the porous polymer monolith through a direct reaction of the chitosan with the porous polymer monolith.

34. The monolith of claim 30, wherein the porous polymer monolith comprises glycidyl methacrylate.

35. A method for manufacturing a chitosan-functionalized porous polymer monolith, the method comprising: anchoring chitosan to a porous polymer monolith.

36. The method of claim 35, wherein anchoring the chitosan comprises using a bifunctional cross-linker to couple amines from the chitosan with epoxy groups on the porous polymer monolith.

37. The method of claim 36, wherein the bifunctional cross-linker is N-[-maleimidobutyryloxy]succinimide ester.

38. The method of claim 35, wherein the anchoring the chitosan comprises directly attaching chitosan on the porous polymer monolith through a direct reaction of the chitosan with the porous polymer monolith.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings, which are incorporated herein and form part of the specification, illustrate various embodiments of the present invention. In the drawings, like reference numbers indicate identical or functionally similar elements. Additionally, the left-most digit(s) of the reference number identifies the drawing in which the reference number first appears.

[0040] FIG. 1A illustrates a perspective view of top, side, and end of a portion of a channel substrate of a fluidic chip embodying aspects of the present invention.

[0041] FIG. 1B illustrates a cross-sectional view of a fluidic chip embodying aspects of the present invention.

[0042] FIG. 1C illustrates a perspective view of a bottom, side, and end of a bare preformed porous polymer monolith embodying some aspects of the present invention.

[0043] FIG. 1D illustrates a perspective view of a bottom and end of a bare preformed porous polymer monolith embodying some aspects of the present invention.

[0044] FIG. 2A illustrates a perspective view of top, side, and end of a portion of a channel substrate of a fluidic chip embodying aspects of the present invention.

[0045] FIG. 2B illustrates a cross-sectional view of a fluidic chip embodying aspects of the present invention.

[0046] FIG. 2C illustrates a perspective view of a bottom, side, and end of a bare preformed porous polymer monolith embodying aspects of the present invention.

[0047] FIG. 2D illustrates a perspective view of a bottom and end of a bare preformed porous polymer monolith embodying aspects of the present invention.

[0048] FIG. 3 is a flowchart illustrating a process for incorporating one or more porous polymer monoliths into a fluidic chip embodying aspects of the present invention.

[0049] FIG. 4 is a flowchart illustrating a process for ex situ fabrication of one or more porous polymer monoliths embodying aspects of the present invention.

[0050] FIG. 5 is a schematic diagram illustrating the addition of pre-monolith solution to a mold embodying aspects of the present invention.

[0051] FIG. 6 is a schematic diagram illustrating monolith de-molding embodying aspects of the present invention.

[0052] FIG. 7 is an optical micrograph of a trapezoidal monolith element embodying aspects of the present invention.

[0053] FIGS. 8A and 8B are a schematic diagrams illustrating functionalization of one or more porous polymer monoliths embodying aspects of the present invention.

[0054] FIG. 9 is a flowchart illustrating a process for integration of one or more bare preformed monoliths embodying aspects of the present invention.

[0055] FIG. 10 is a schematic diagram illustrating insertion of one or more bare preformed porous polymer monoliths into one or more channels of a fluidic chip embodying aspects of the present invention.

[0056] FIG. 11 is a top view of bare preformed porous polymer monoliths inserted into a channel of a fluidic chip embodying aspects of the present invention.

[0057] FIG. 12 is a schematic diagram illustrating bonding of a capping layer to a channel substrate embodying aspects of the present invention.

[0058] FIG. 13A-13D are schematic diagrams illustrating a monolith self-assembly process for insertion of a bare preformed porous polymer monolith into a channel of a fluidic chip embodying aspects of the present invention.

[0059] FIG. 14 is a scanning electron microscope image of a trapezoidal porous polymer monolith integrated in a channel of a fluidic chip embodying aspects of the present invention.

[0060] FIG. 15 is a scanning electron microscope image of mechanical interlocking of a porous polymer monolith with a wall of a channel of a channel substrate of a fluidic chip embodying aspects of the present invention.

[0061] FIGS. 16A-16C are fluorescence images of a fluorescein solution being pumped through an ex situ fabricated bare porous polymer monolith integrated in a channel of a fluidic chip embodying aspects of the present invention at different times. FIG. 16D is a graph illustrating fluorescence intensity profiles at the different times for the ex situ fabricated bare porous polymer monolith integrated in the channel embodying aspects of the present invention.

[0062] FIGS. 17A-17C are fluorescence images of a fluorescein solution being pumped through a porous polymer monolith formed in situ in a channel of a fluidic chip. FIG. 17D is a graph illustrating fluorescence intensity profiles at the different times for the porous polymer monolith formed in situ in the channel.

[0063] FIG. 18 is a top view image of porous polymer monoliths integrated in a channel of a fluidic chip embodying aspects of the present invention.

[0064] FIGS. 19A and 19B are top view images of a multiple porous polymer monoliths integrated in a channel of a fluidic chip before and after analyte exposure, respectively, embodying aspects of the present invention. FIG. 19C is a graph illustrating the fluorescence intensity as a function of position in the channel having the multiple porous polymer monoliths integrated therein before and after analyte exposure embodying aspects of the present invention.

[0065] FIGS. 20A-20F are top view images of a wettability-based bubble separator separating air and water from a two phase flow embodying aspects of the present invention.

[0066] FIG. 21A is flat side view of the chitosan functionalized triangular porous polymer monolith in the triangular channel of the channel substrate of a microfluidic chip embodying aspects of the present invention.

[0067] FIG. 21B is a scanning electron microscope cross-section image of a chitosan functionalized triangular porous polymer monolith in a triangular channel of a channel substrate of a microfluidic chip embodying aspects of the present invention.

[0068] FIG. 22 is a schematic diagram illustrating a direct reaction between a GMA monolith and chitosan embodying aspects of the present invention.

[0069] FIG. 23 is a graph illustrating average DNA concentrations at a chip outlet during loading, washing, and elution embodying aspects of the present invention.

[0070] FIG. 24 is a graph showing a comparison between outlet DNA concentration for a control monolith and chitosan coated monolith during the capture, wash and elution of 40 ng of DNA.

[0071] FIG. 25 is a graph showing DNA capture capacity of the non-limiting sample of a chitosan functionalized monolith embodying aspects of the present invention.

[0072] FIG. 26 illustrates monolith-bound DNA visualized with intercalating dye in a fluidic chip embodying aspects of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0073] FIG. 1A illustrates a perspective view of top, side, and end of a portion of a channel substrate 101 of a fluidic chip 100 embodying aspects of the present invention. In some non-limiting embodiments, the fluidic chip 100 may be a microfluidic chip. In some embodiments, the fluidic chip 100 may include a channel substrate 101. In some non-limiting embodiments, the channel substrate 101 may comprise a thermoplastic, such as, for example and without limitation, cyclic olefin copolymer (COC). In some embodiments, the channel substrate 101 may include one or more channels 102. In some non-limiting embodiments, the one or more channels 102 may comprise one or more microchannels. In some non-limiting embodiments, as illustrated in FIG. 1A, the one or more channels 102 of the channel substrate 101 may include a first channel 102a and a second channel 102b.

[0074] In some non-limiting embodiments, the one or more channels 102 may have a width within a range greater than or equal to 10 micrometers and less than or equal to 1 centimeter, and this range should be understood as describing and disclosing all widths (including all decimal or fractional widths) within this range. In some non-limiting embodiments, the one or more channels 102 may have a width within a range greater than or equal to 1 millimeter and less than or equal to 1 centimeter, and this range should be understood as describing and disclosing all widths (including all decimal or fractional widths) within this range. In some non-limiting embodiments, the one or more channels 102 may have a width within a range greater than or equal to 100 micrometers and less than or equal to 1 millimeter, and this range should be understood as describing and disclosing all widths (including all decimal or fractional widths) within this range. In some non-limiting embodiments, the one or more channels 102 may have a width within a range greater than or equal to 10 micrometers and less than or equal to 100 micrometers, and this range should be understood as describing and disclosing all widths (including all decimal or fractional widths) within this range. However, this is not required, and, in some alternative embodiments, the one or more channels 102 may have widths outside these ranges.

[0075] In some non-limiting embodiments, the one or more channels 102 may have a height (i.e., depth) within a range greater than or equal to 10 micrometers and less than or equal to 1 centimeter, and this range should be understood as describing and disclosing all heights (including all decimal or fractional heights) within this range. In some non-limiting embodiments, the one or more channels 102 may have a height within a range greater than or equal to 1 millimeter and less than or equal to 1 centimeter, and this range should be understood as describing and disclosing all heights (including all decimal or fractional heights) within this range. In some non-limiting embodiments, the one or more channels 102 may have a height within a range greater than or equal to 100 micrometers and less than or equal to 1 millimeter, and this range should be understood as describing and disclosing all heights (including all decimal or fractional heights) within this range. In some non-limiting embodiments, the one or more channels 102 may have a height within a range greater than or equal to 10 micrometers and less than or equal to 100 micrometers, and this range should be understood as describing and disclosing all heights (including all decimal or fractional heights) within this range. However, this is not required, and, in some alternative embodiments, the one or more channels 102 may have heights outside these ranges.

[0076] In some embodiments, the fluidic chip 100 may include one or more porous polymer monoliths 103. In some embodiments, the one or more porous polymer monoliths 103 may be integrated in one or more channels 102 of the channel substrate 101. In some embodiments, a channel 102 may include one or more integrated porous polymer monoliths 103. For example, in the non-limiting embodiment illustrated in FIG. 1A, the first channel 102a may include porous polymer monoliths 103a and 103c, and the second channel 102b may include porous polymer monolith 103b.

[0077] In some non-limiting embodiments, the one or more porous polymer monoliths 103 may have a length within a range greater than or equal to 10 micrometers and less than or equal to 1 centimeter, and this range should be understood as describing and disclosing all lengths (including all decimal or fractional lengths) within this range. In some non-limiting embodiments, the one or more porous polymer monoliths 103 may have a length within a range greater than or equal to 1 millimeter and less than or equal to 1 centimeter, and this range should be understood as describing and disclosing all lengths (including all decimal or fractional lengths) within this range. In some non-limiting embodiments, the one or more porous polymer monoliths 103 may have a length within a range greater than or equal to 100 micrometers and less than or equal to 1 millimeter, and this range should be understood as describing and disclosing all lengths (including all decimal or fractional lengths) within this range. In some non-limiting embodiments, the one or more porous polymer monoliths 103 may have a length within a range greater than or equal to 10 micrometers and less than or equal to 100 micrometers, and this range should be understood as describing and disclosing all lengths (including all decimal or fractional lengths) within this range. However, this is not required, and, in some alternative embodiments, the one or more monoliths 103 may have lengths outside these ranges.

[0078] In some embodiments, the one or more porous polymer monoliths 103 may include one or more functionalized porous polymer monoliths. In some non-limiting embodiments, the functionalized porous polymer monoliths may be functionalized, for example and without limitation, as immunosensors, bubble separators, or nucleic acid purifiers. In some non-limiting embodiments, porous polymer monoliths 103 may be chemically functionalized. In some non-limiting embodiments, chemical functionalization of a porous polymer monolith 103 may comprise immobilization of a capture probe on the one or more porous polymer monoliths. In some embodiments, the capture probe may be an antibody, protein (e.g., protein G, which may be from Streptococcus), aptamer, amino acid, peptide, or synthetic capture probe. In some embodiments, the capture probe may labeled with a fluorescent marker (e.g., rhodamine, fluorescein isothiocyanate (FITC), or quantum dots). In some embodiments, the capture probe may be capable of electrostatically driven, pH modulated nucleic acid capture (e.g., chitosan).

[0079] In some embodiments, the fluidic chip 100 may include two or more porous polymer monoliths 103, and the two or more porous polymer monoliths 103 may have differing chemistries, porosity, and/or functionality. For example, in some non-limiting embodiments, one or more of the porous polymer monoliths 103 may have a chemistry comprising glycidyl methacrylate (GMA), and one or more of the porous polymer monoliths 103 may have a chemistry comprising butylmethacrylate (BMA). In another example, in some non-limiting embodiments, the porous polymer monoliths 103 may have the same chemistry but be functionalized with different fluorescent markers (e.g., rhodamine and FITC).

[0080] FIG. 1B illustrates a cross-sectional view of the fluidic chip 100 illustrated in FIGS. 1A and 2B, respectively. In some embodiments, as illustrated in FIG. 1B, the fluidic chip 100 may include a capping layer (i.e., cover plate or second substrate) 104. In some embodiments, the capping layer 104 may be bonded to the channel substrate 101. In some embodiments, the capping layer 104 may enclose the channels 102 of the channel substrate 101. In some embodiments, the capping layer 104 may seal the one or porous polymer monoliths 103 within the one or more channels 102 of the channel substrate 101 between the channel substrate 101 and the capping layer 104. In some embodiments, the one or more porous polymer monoliths 103 may be mechanically anchored to walls of the one or more channels 102 of the channel substrate 101. In some embodiments, the one or more porous polymer monoliths 103 may be mechanically anchored to the capping layer 104. In some non-limiting embodiments, the one or more porous polymer monoliths 103 may be anchored directly to walls of the one or more channels 102, and the one or more porous polymer monoliths 103 are not within one or more capillaries having outer surfaces covalently attached to walls of the one or more channels 102.

[0081] FIGS. 1C and 1D illustrate perspectives of the porous polymer monolith 103. In some non-limiting embodiments, as illustrated in FIGS. 1A and 1B, the one or more channels 102 may have a trapezoidal cross-section, and, as illustrated in FIGS. 1B-1D, the one or more porous polymer monoliths 103 may have a trapezoidal cross-section. In some non-limiting embodiments, trapezoidal channels 102 and/or the trapezoidal monoliths 103 may have edges slanted at an angle (e.g., 45) from the surface normal. However, a trapezoidal cross-section is not required, and, in some alternative embodiments, the one or more channels 102 and the one or more porous polymer monoliths 103 may have different cross-sectional shapes, such as, for example and without limitation, rectangular, triangular, or rounded (e.g., U-shaped) cross-sections. For example, FIGS. 2A-2D illustrate a non-limiting alternative embodiment of the fluidic chip 100 where the one or more channels 102 and the one or more porous polymer monoliths 103 have a triangular cross-section. FIGS. 2A-2D correspond to FIGS. 1A-1D, respectively, except for the cross-sectional shape of the one or more channels 102 and the one or more porous polymer monoliths 103.

[0082] FIG. 3 is a flowchart illustrating a process 300 for incorporating one or more porous polymer monoliths 103 into a fluidic chip 100 embodying aspects of the present invention. In some embodiments, the process 300 may include a step 301 for ex situ fabrication of one or more bare porous polymer monoliths 103. In some embodiments, the process 300 may include a step 302 for ex situ functionalization of one or more bare porous polymer monoliths 103. In some embodiments, the process 300 may include a step 303 for integration of the monolith of one or more bare porous polymer monoliths 103 into one or more channels 102 of a channel substrate 101 of the fluidic chip 100. In some embodiments, the integration step 303 may anchor and fluidically seal of the one or more integrated monoliths 103 in the one or more channels 102.

[0083] FIG. 4 is a flowchart illustrating a non-limiting process 400 for ex situ fabrication of one or more discrete bare porous polymer monoliths 103. In some non-limiting embodiments, the process 400 may be performed in step 301 of the process 300 for incorporating one or more porous polymer monoliths 103 into a fluidic chip 100.

[0084] In some embodiments, the process 400 may include a step 401 of adding a pre-monolith solution to a mold. As illustrated with reference to FIG. 5, step 401 may include adding a pre-monolith solution 504 to a mold 505. In some embodiments, the mold 505 may include a molding substrate 506 and a mold capping layer 507. In some non-limiting embodiments, the channel substrate 101 may comprise a thermoplastic, such as, for example and without limitation, COC. In some embodiments, the molding substrate 506 may include one or more molding channels 508. In some non-limiting embodiments, the one or more molding channels 508 may be one or more open trenches in a surface of the molding substrate 506. In some embodiments, the one or more molding channels 508 may have dimensions (e.g., cross-sectional dimensions) larger (e.g., 5-10% larger) than the dimensions of the one or more channels 102 of the channel substrate 101 of the fluidic chip 100.

[0085] In some embodiments, the mold capping layer 507 may temporarily seal the one or more molding channels 508 of the molding substrate 506. In some non-limiting embodiments, the mold capping layer 507 may comprise PDMS or electrical tape. In some embodiments, the pre-monolith solution 504 may be added to the one or more temporarily-sealed molding channels 508 of the molding substrate 506. In some non-limiting embodiments, an insertion device 509 (e.g., a syringe or pipette) may add the pre-monolith solution 508 to the one or more molding channels 508. In some non-limiting embodiments, the pre-monolith solution 504 may fill the one or more molding channels 508. In some non-limiting embodiments, the pre-monolith solution 504 may be added to the one or more temporarily-sealed molding channels 508 via one or more access holes. In some non-limiting embodiments, the one or more access holes may be sealed (e.g., with electrical tape) after the pre-monolith solution 504 is added to the one or more molding channels 508.

[0086] In some embodiments, measures to promote attachment to the walls of the molding channels 508 are omitted to ensure that the one or monoliths 103 may be removed from the molding channels 508 following photopolymerization. In some embodiments, the pre-monolith solution 504 may comprise GMA. In some non-limiting embodiments, the pre-monolith solution 504 may comprise GMA and one or more of ethoxylated trimethylolpropane triacrylate (SR454), cyclohexanol, and methanol. In one non-limiting embodiment, the pre-monolith solution 504 may comprise 24% GMA, 16% SR454, 50% cyclohexanol, and 10% methanol (by weight). In some non-limiting embodiments, a photoinitiator, such as, for example and without limitation, 2,2-dimethoxy-2-phenylacetophenone (DMPA), equaling 1% of the combined weight of the GMA and SR454 may be added to the pre-monolith solution 504. However, this specific solution is not required, and some alternative embodiments may comprise another pre-monolith solution 504, such as, for example and without limitation, a solution comprising 1.4% GMA, 14.2% SR-454, 53.4% Cyclohexanol, 10.7% methanol, and 0.4% DMPA (by weight).

[0087] In addition, it is not necessary that the pre-monolith solution 504 comprise GMA, and, in some alternative embodiments, the pre-monolith solution 504 may comprise another monomer, such as, for example and without limitation, BMA. In some non-limiting alternative embodiments, the pre-monolith solution may comprise BMA and one or more of ethylenedimethacrylate (EDMA), 1,4butanediol, 1-propanol, and photoinitiator (e.g., DMPA). In one non-limiting alternative embodiment, the pre-monolith solution 504 may comprise 23.5% BMA, 15.5% EDMA, 34% 1,4butanediol, 26% 1-propanol and 1% DMPA (by weight).

[0088] In some embodiments, the process 400 may include a step 402 of photopolymerizing the pre-monolith solution 504 to form one or more bare porous polymer monoliths 103. In some embodiments, photopolymerization of the monoliths 103 may be accomplished using a UV light source outputting UV light for a period of time. In some non-limiting embodiments, the photopolymerization may be accomplished by a UV light source outputting at a surface power density (e.g., 22 mW/cm.sup.2 or 39 mW/cm.sup.2) for a period of time (e.g., 600 s).

[0089] In some embodiments, the process 400 may include a step 403 of removing one or more bare porous polymer monoliths 103 from the mold 505. As illustrated with reference to FIG. 6, de-molding step 403 may include removing the mold capping layer 507 (e.g., peeling away a layer of electrical tape) and removing the one or more bare porous polymer monoliths 103 from the one or more molding channels 508 of the molding substrate 506 of the mold 505. In some non-limiting embodiments, the mold capping layer 507 may be removed from the molding substrate 506 with tweezers.

[0090] In some non-limiting embodiments, the process 400 may include cleaving or cutting the one or more bare porous polymer monoliths 103 to a prescribed length. In some non-limiting embodiments, the process 400 may include soaking the one or more bare porous polymer monoliths 103 in methanol followed by a methanol in water solution (e.g., 20% methanol in water) under gentle agitation (e.g., on a laboratory shaker) to remove solvent and any unreacted prepolymer. In some non-limiting embodiments, the process 400 may include one or more final rinse steps in small glass vials with gentle agitation (e.g., manual agitation). In some embodiments, the cleanup steps (e.g., soaking and/or rinse steps) may be part of a batch cleanup process in which multiple bare porous polymer monoliths 103 are cleaned up at the same time.

[0091] FIG. 7 is an optical micrograph of a trapezoidal porous polymer monolith 103 that may be formed by some embodiments of the ex situ monolith fabrication of step 301, which may be carried out, for example, by performing the ex situ monolith fabrication process 400 illustrated in FIG. 4. The trapezoidal porous polymer monolith 103 illustrated in FIG. 7 has a maximum width of approximately 200 m. The trapezoidal porous polymer monolith 103 illustrated in FIG. 7 is being manipulated by a fine tip paint brush 710.

[0092] In some embodiments, as illustrated with reference to FIGS. 8A and 8B, the ex situ functionalization step 302 of the monolith incorporation process 300 may include immersing one or more bare porous polymer monoliths 103 in one or more solutions (e.g., solutions 811a and 811b) to functionalize the one or more bare porous polymer monoliths 103. In some embodiments, the one or more solutions 811a and 811b may each be held in a container (e.g., containers 812a and 812b, respectively). In some non-limiting embodiments, the functionalization step 302 may be a batch functionalization process in which multiple bare porous polymer monoliths 103 are functionalized at the same time (e.g., in the same container). In some non-limiting embodiments, as shown in FIGS. 8A and 8B, two or more different functionalizations may be performed in parallel. That is, in some non-limiting embodiments, a first set of one or more bare porous polymer monoliths 103a may be functionalized in a first solution 811a to have a first function, and, at the same time, a second set of one or more bare porous polymer monoliths 103c may be functionalized in a second solution 811b to have a second function that is different than the first function.

[0093] In some non-limiting embodiments, the ex situ functionalization step 302 may include chemically functionalizing one or more bare porous polymer monoliths 103. In some non-limiting embodiments, chemically functionalizing one or more bare porous polymer monoliths 103 may include immobilizing capture probes on the one or more porous polymer monoliths. In some non-limiting embodiments, the capture probes may be antibodies, proteins, aptamers, amino acids, peptides, or synthetic capture probes. In some non-limiting embodiments, the capture probes may be labeled with a fluorescent markers (e.g., rhodamine, FITC, or quantum dot). In some non-limiting embodiments, the capture probes may be capable of electrostatically driven, pH modulated nucleic acid capture (e.g., chitosan).

[0094] In some non-limiting embodiments, chemically functionalizing one or more bare porous polymer monoliths 103 may be accomplished using a multi-step reaction process. In some non-limiting embodiments, the multi-step reaction process may involve one or more crosslinkers (e.g., thiol groups and/or N-[-maleimidobutyryloxy]succinimide ester (GMBS)). In one none-limiting embodiment, the multi-step reaction process to functionalize the one or more bare porous polymer monoliths 103 may comprise one or more of (i) immersing the one or more monoliths 103 in a sodium hydrosulfide solution (e.g., a 2M sodium hydrosulfide solution created from a mixture of 20% methanol and 80% 0.1 M sodium phosphate dibasic at pH 8.15) to convert epoxide groups to thiols, (ii) eliminating (e.g., hydrolyzing) remaining epoxide groups by treatment (e.g., overnight treatment) of the one or more monoliths 103 with sulfuric acid (e.g., 0.5M sulfuric acid), (iii) incubating the one or more monoliths 103 in a GMBS solution (e.g., a 2 mM solution of GMBS in ethanol), and (iv) reacting the one or more monoliths 103 with the desired capture probes (e.g., the desired protein) diluted in a buffer solution (e.g., phosphate-buffered saline (PBS)) over a concentration range (e.g., 50-500 g/mL) for a period of time (e.g., one hour).

[0095] However, a multi-step reaction process (e.g., involving one or more cross-linkers) is not necessary for chemically functionalizing one or more bare porous polymer monoliths 103, and, in some alternative embodiments, chemically functionalizing one or more bare porous polymer monoliths 103 may instead be accomplished using a multi-step reaction process through a direction reaction of the desired capture probes (e.g., the desired protein) with the one or more bare porous polymer monoliths 103.

[0096] In some embodiments, the ex situ functionalization step 302 may be optional. In other words, some embodiments incorporate one or more bare preformed porous polymer monoliths 103 that have not been subjected functionalization, and the ex situ functionalization step 302 is not performed for any such monoliths 103.

[0097] FIG. 9 is a flowchart illustrating a non-limiting process 900 for integration of one or more bare porous polymer monoliths 103 into one or more channels 102 of a channel substrate 101 of the fluidic chip 100. In some non-limiting embodiments, the process 900 may be performed in step 303 of the process 300 for incorporating one or more porous polymer monoliths 103 into a fluidic chip 100. In some embodiments, the integration process 900 may anchor and fluidically seal of the one or more monoliths 103 in the one or more channels 102. In some non-limiting embodiments, the integration process 900 may be a solvent bonding process that mechanically anchors the one or more monoliths 103 to the walls of one or more channels 102 (as opposed to chemically attaching the one or more monoliths 103 to the channel walls).

[0098] In some embodiments, the process 900 may include a step 901 of temporarily softening the capping layer 104 and one or more channels 102 of the channel substrate 101 of the fluidic chip 100. In some embodiments, temporarily softening the capping layer 104 comprises exposing the capping layer 104 to a solvent. See, e.g., T. I. Wallow et al., Low-distortion, high-strength bonding of thermoplastic microfluidic devices employing case-II diffusion-mediated permeant activation, Lab on a chip, vol. 7, no. 12, pp. 1825-31, December 2007. In some embodiments, temporarily softening one or more channels 102 comprises exposing at least a portion of the one or more channels (e.g., at least the portion of the one or more channels 102 where the one or more monoliths 103 will be placed) to the solvent. In some non-limiting embodiments, the solvent may comprise decahydronaphthalene (decalin). In some non-limiting embodiments, the solvent may comprise a solution of decalin in ethanol (e.g., a solution of 20 to 25% (by volume) decalin in ethanol). In some non-limiting embodiments, a small volume (e.g, 1-5 l) of the solvent solution is pipetted into the region of the one or more channels 102 where the one or more monoliths 103 will be placed. In some non-limiting embodiments, the solvent solution is effective to achieve full sealing between the surfaces of the one or more monoliths 103, the one or more channels 102 of the channel substrate 101, and the capping layer 104 while preventing distortion of the one or more channels 102 during bonding. In some embodiments, the step 901 may include subsequently (e.g., after 10 minutes of solvent solution exposure) washing the one or more channels 102 of the channel substrate 101 and the capping layer 104 (e.g., in 100% ethanol) and quickly drying the channel substrate 101 and the capping layer 104 (e.g., using a stream of nitrogen) to prevent further solvent uptake.

[0099] In some embodiments, as illustrated with reference to FIG. 10, the process 900 may include a step 902 of inserting one or more bare preformed porous polymer monoliths 103 into one or more channels 102 of the channel substrate 101 of the fluidic chip 100. In some non-limiting embodiments, the one or more bare preformed porous polymer monoliths 103 are manually positioned at desired locations in the one or more channels 102 (e.g., using the fine-tipped brush 710 illustrated in FIG. 7). In some embodiments, as illustrated in FIG. 11, the insertion step 902 may insert two or more bare preformed porous polymer monoliths 103 (e.g., monoliths 103a and 103c) into a channel 102 of the channel substrate 101, and the two or more bare preformed porous polymer monoliths 103 may have different functionalizations.

[0100] In some embodiments, the as illustrated with reference to FIG. 12, the process 900 may include a step 903 of bonding/sealing a capping layer 104 to the channel substrate 101 of the fluidic chip 100. In some embodiments, bonding the capping layer 104 to the channel substrate 101 may seal the one or more inserted bare porous polymer monoliths 103 in the one or more channels 102 of the channel substrate 101 of the fluidic chip 100. In some non-limiting embodiments, the capping layer 104 may be bonded to the channel substrate 101 substrate using a pressure (e.g., in a range of 2 to 4 MPa, such as, for example and without limitation, approximately 3.5 MPa or 2.1 MPa) for a period of time (e.g., in a range of 10 to 20 minutes) within a temperature range (e.g., approximately 60 C.).

[0101] In some embodiments, to account for monolith shrinkage during processing, the one or more channels 102 receiving the one or more monoliths 103 may be fabricated with dimensions (e.g., cross-sectional dimensions) smaller (e.g., 5-10% smaller) than the dimensions of the one or more molding channels 508 to ensure full sealing at the periphery of the one or more monoliths 103. That is, in some embodiments, the one or more monoliths 103 may be oversized relative to the one or more channels 102.

[0102] In some embodiments, the bonding step 903 forces one or more bare porous polymer monoliths 103 into one or more solvent-softened channels 102 and results in an intimate seal between the material of the one or more monoliths 103 and the material(s) of the one or more channels 102 and the capping layer 104.

[0103] Although in some embodiments the one or more channels 102 and the one or more monoliths 103 have rectangular cross-sections, embodiments in which the one or more channels 102 and the one or more monoliths 103 have trapezoidal, triangular, or U-shaped cross-sections may have advantageous over the rectangular cross-section embodiments. For example, in rectangular cross-section embodiments, during insertion of a rectangular monolith into a rectangular channel, vertical alignment must be maintained to avoid collision of the rectangular monolith with either side-wall of the rectangular channel. In addition, because the rectangular monolith may be slightly oversized to ensure a good seal with the rectangular channel walls, high forces are required to insert the oversized rectangular monolith into the rectangular channel, which can result in monolith fracture. As a result, yield for intact monoliths integrated into rectangular cross-section channels may be poor. Monoliths having trapezoidal, triangular, or U-shaped cross-sections may overcome these issues.

[0104] In some trapezoidal, triangular, or U-shaped embodiments, the trapezoidal, triangular, or U-shaped channel cross-section may greatly simplify the alignment and insertion of monoliths with nearly-matched cross-sections during the initial steps of reintegration. In some trapezoidal, triangular, or U-shaped embodiments, the insertion step 902 (see FIG. 9) may effectively be one of self-assembly, with each monolith 103 readily seating itself into the receiving channel 102 after being manually positioned into the general target region. In these embodiments, careful alignment of the monolith 103 with the channel 102 may not be needed.

[0105] FIG. 13A-13D illustrate an example of monolith self-assembly process, which may be performed in the insertion step 902, for insertion of a bare preformed porous polymer monolith 103 into a channel 102 of a fluidic chip 100. Although a trapezoidal channel 102 and trapezoidal monolith 103 is illustrated in FIG. 13A-13D, the monolith self-assembly process is applicable to other channel and monolith cross-sectional shapes, such as, for example, channels and monoliths having triangular and U-shaped cross-sections.

[0106] In some embodiments, the monolith self-assembly process may include suspending a bare porous polymer monolith 103 in water, and drawing the suspended monolith 103 into a pipette. In some embodiments, as illustrated in FIG. 13A, the monolith self-assembly process may include depositing the monolith 103 within a droplet 1314 of water onto the channel substrate 101 from the pipette. In some embodiments, as illustrated in FIG. 13B, the monolith self-assembly process may include seating the deposited monolith 103 into a channel 102 of the channel substrate 101. In some non-limiting embodiments, the monolith 103 may be seated in the mating channel 102 through gentle agitation together with capillary forces to move the monolith 103 until it enters the mating channel 102 to maximize surface area contact with the channel substrate 101. Although the angled sidewalls of the channel 102 and monolith 103 encourage seating of the monolith 103 into the desired configuration, a monolith 103 may enter the channel 102 in an improper orientation. In this event, the droplet 1314 may be retracted by pipette before repeating the process. In some embodiments, the monolith self-assembly process may include, as illustrated in FIG. 13C, removing the water droplet 1314 from the channel substrate 101 (e.g., using the pipette) and/or, as illustrated in FIG. 13D, drying the channel substrate 101 prior to the bonding step 903.

[0107] In some embodiments, in addition to supporting effective self-assembly of the one or more monoliths 103 into their mating channels 102 during the insertion step 902, the slanted sidewalls may ensure that each surface experiences a normal force during the bonding step 903 that serves to embed the one or more monoliths 103 within the channel walls of the channel substrate 101 during the bonding.

[0108] FIG. 14 is a scanning electron microscope image of a non-limiting example of a trapezoidal porous polymer monolith 103 integrated in a channel 102 of a fluidic chip 100 according to some embodiments. FIG. 15 is a scanning electron microscope image of mechanical interlocking of a porous polymer monolith 103 with a wall of a channel 102 of a channel substrate 101 in a non-limiting example of a fluidic chip 100 according to some embodiments. In some embodiments, as illustrated in FIGS. 14 and 15, the integration of one or more ex situ fabricated monoliths 103 may result in anchoring of the one or more inserted monoliths 103 to walls of the one or more channels 102. In some non-limiting embodiments, as illustrated in FIG. 15, the anchoring may be mechanical interlocking of the one or more inserted monoliths 103 and the walls of the one or more channels 102. In some non-limiting embodiments, the anchoring does not result in covalent attachment of the one or more inserted monoliths 103 and the walls of the one or more channels 102.

[0109] Due to their controllable porosity and high surface area, porous polymer monoliths 103 may be able to eliminate voids and achieve high bond strength between the channel walls and monolith surfaces and, thus, ensure predictable and uniform flow through the porous monolith structures. Compared with traditional in situ monolith fabrication, the solvent-assisted integration of one or more ex situ fabricated monoliths 103 provides excellent monolith to channel substrate 101 and capping layer 104 anchoring (e.g., monolith-COC anchoring) due to mechanical interlocking between the materials. The example of the interface between the monolith 103 and channel wall following bonding illustrated in FIG. 15 reveals the morphology of the interlocking materials.

[0110] To evaluate the quality of the interface, a dilute fluorescein solution was pumped at 2 l/min for 5 min to 10 min through an ex parte fabricated integrated trapezoidal monolith 103 as well as a trapezoidal monolith formed in situ within an identical channel. Care was taken to produce monoliths of similar lengths (approximately 2 mm) for each case. FIGS. 16A-16C are fluorescence images of the fluorescein solution being pumped through an ex situ fabricated bare trapezoidal porous polymer monolith 103 integrated in a trapezoidal channel 102 of a fluidic chip 100 at different times. FIG. 16D is a graph illustrating fluorescence intensity profiles at the different times for the ex situ fabricated bare porous polymer monolith 103 integrated in the channel 102. FIGS. 17A-17C are fluorescence images of a fluorescein solution being pumped through a trapezoidal porous polymer monolith formed in situ in a trapezoidal channel of a fluidic chip. FIG. 17D is a graph illustrating fluorescence intensity profiles at the different times for the porous polymer monolith formed in situ in the channel.

[0111] As shown in FIGS. 16A-16D, flow through the ex situ fabricated integrated monolith 103 occurs within the bulk porous matrix, with uniform fluorescence intensity that matches well with the trapezoidal cross-section of the monolith 103. In contrast, as shown in FIGS. 17A-17D, flow through the in situ monolith occurs primarily at the monolith-microchannel interface, indicating the presence of extensive voids between the materials that result in a non-uniform flow profile and prevent significant flow through the core of the porous structure. The ex situ integration technique in which one or more ex situ fabricated monoliths 103 are mechanically attached to channel walls of a fluidic device 100 allows for very short lengths of large cross-section monolith with excellent sealing at the interface between the monolith 103 and the walls of the channel 102, eliminating voids which would otherwise allow fluidic bypass and sample loss.

[0112] One application of microfluidic monoliths that holds particular promise is in the area of optofluidic sensing, where the porous monolith matrix may serve as a functionalized volumetric detection zone capable of enhancing local analyte concentration and detection sensitivity. See, e.g., K. Jiang, A. Sposito, J. Liu et al., Microfluidic synthesis of macroporous polymer immunobeads, Polymer, vol. 53, no. 24, pp. 5469-5475, November 2012. In some non-limiting embodiments, the process 300 (see FIG. 3) for incorporating one or more ex situ fabricated porous polymer monoliths 103 into a fluidic chip 100 may be used to create optofluidic sensor, such as, for example, a fluorescent immonosensor.

[0113] In some non-limiting embodiments, the process 300 (see FIG. 3) for incorporating one or more ex situ fabricated porous polymer monoliths 103 into a fluidic chip 100 may incorporate an array of porous polymer monoliths 103 with different chemical or biochemical functionalities within a single fluidic chip 100, which may enable multiplexed detection of different analytes within a single sample. Known strategies for the integration of arrays of porous or 3-dimensional detection elements into microfluidic chips including (i) integration of discrete functionalized capillary segments, see, e.g., H. Hisamoto et al., Capillary-assembled microchip for universal integration of various chemical functions onto a single microfluidic device, Analytical chemistry, vol. 76, no. 11, pp. 3222-8, June 2004; (ii) patterning of hydrogel micropatches, see, e.g., J. Heo and R. M. Crooks, Microfluidic biosensor based on an array of hydrogel-entrapped enzymes, Analytical chemistry, vol. 77, no. 21, pp. 6843-51, November 2005; and (iii) 1-dimensional microbead arrays. See, e.g., H. Zhang et al., Detection of single-base mutations using 1-D microfluidic beads array, Electrophoresis, vol. 28, no. 24, pp. 4668-78, December 2007. For the case of porous monoliths, the in situ formation of a multifunctional array within a sealed microchannel is possible, but this approach would require a laborious sequential fabrication process (see K. W. Ro, R. Nayak, and D. R. Knapp, Monolithic media in microfluidic devices for proteomics, Electrophoresis, vol. 27, no. 18, pp. 3547-58, September 2006) together with the need for complex flow control to deliver separate functionalization reagents to each array element.

[0114] The process 300 (see FIG. 3) may be used to integrate an array of fully functionalized ex situ fabricated monoliths 103 and can avoid the limitations of in situ formation of a multifunctional array, allowing facile construction of arrays of sealed porous polymer monoliths 103 with different functionalities within a single continuous channel 102 or fluidic network. In some non-limiting embodiments that use decalin for solvent bonding, the low volatility of decalin relative to other commonly used solvents may allow the surface (e.g., the COC surface) of the channel substrate 102 to remain softened for up to several minutes following solvent exposure, providing ample time for integration of multiple bare preformed porous polymer monoliths 103 to enable the creation of closely spaced arrays. However, decalin is not required to create monolith arrays, and some alternative embodiments may use other solvents capable of softening the channel substrate surface for a sufficient amount of time. An illustrative example of this capability is presented in FIG. 18, where bare preformed porous polymer monoliths 103a and 103c batch-functionalized with two different fluorescent markers (e.g., a covalently attached rhodamine and a covalently attached IgG-FITC) are placed immediately adjacent to each other within a single channel 102 of a channel substrate 101, with no overlap and minimal buffer space between the monoliths 103a and 103c.

[0115] In some non-limiting embodiments, the process 300 (see FIG. 3) may be used to create an immunosensor 1915 capable of realizing a simple flow-through immunoassay. A non-limiting example of the immunosensor 1915 is illustrated in FIGS. 19A and 19B. In some embodiments, the immunosensor 1915 may include a detection element 1918. In some embodiments, the immunosensor 1915 may include one or more of an integrated positive control 1920 and an integrated negative control 1916.

[0116] In some non-limiting embodiments, the detection element 1918 may be for detection, for example and without limitation, FITC-IgG, which may be from human serum. In some non-limiting embodiments, the detection element 1918 may be a bare porous polymer monolith 103 functionalized with covalently-attached immunoglobin-binding protein (e.g., covalently-attached protein G). In some non-limiting embodiments, the negative control 1916 may be an un-functionalized bare porous polymer monolith 103 that is incubated with a protein concentration standard, such as, for example and without limitation, bovine serum albium (BSA). In some embodiments, the negative control 1916 may indicate the extent of non-specific analyte binding. In some non-limiting embodiments, the positive control 1920 may be a bare porous polymer monolith 103 functionalized with covalently-attached conjugate of a fluorescent marker and an immunoglobin (e.g., FITC-IgG). In some embodiments, the positive control 1920 may provide a positive control fluorescence standard against which the fluorescence of detection element 1918 may be compared for quantitative readout.

[0117] FIG. 19A illustrates the immunosensor 1915 before analyte exposure, and FIG. 19B illustrates the immunosensor 1915 after analyte exposure. As illustrated in FIG. 19A, before the analyte, in this case FITC-IgG, is perfused through the channel 102, only the positive control 1920 is visible under fluorescence microscopy using a FITC filter set. As illustrated in FIG. 19B, after a solution of 100 g/ml FITC-IgG in PBS is perfused through the series of monoliths at 5 l/min for 20 min, followed by an equal flow rate and volume of rinse buffer, the fluorescence intensity in the negative control 1916 and the detection element 1918 increases. FIG. 19C is a graph illustrating the fluorescence intensity as a function of position in the channel 102 of the immunosensor 1915 (averaged across the channel width) before and after analyte exposure. In some additional embodiments, this concept may be readily extended to include one or more additional porous polymer monoliths 103 functionalized with other capture probes to realize higher levels of multiplexing in a simple flow-through assay.

[0118] In some non-limiting embodiments, the process 300 (see FIG. 3) for incorporating one or more ex situ fabricated porous polymer monoliths 103 into a fluidic chip 100 may be used to create a wettability-based bubble separator 2022. A non-limiting example of the wettability-based bubble separator 2022 is illustrated in FIGS. 20A-20F. The formation of unwanted air bubbles is a common challenge for many microfluidic systems, and the process 300 may be used to integrate multiple porous polymer monoliths 103 with different chemistries into a bubble separator 2022 capable of removing trapped air from a channel based on the differential wettability.

[0119] In some embodiments, the wettability-based bubble separator 2022 may comprise a T-junction formed by channels 102 of a channel substrate 101. In some embodiments, the channels 102 of the channel substrate 101 may comprise an inlet 2024 of the T-junction, a first downstream branch 2026 of the T-junction, and a second downstream branch 2028 of the T-junction. In some embodiments, the wettability-based bubble separator 2022 may comprise a first monolith 2030 and a second monolith 2032. In some embodiments, the first monolith 2030 may be in the first downstream branch 2026 of the T-junction, may be adjacent to the inlet 2024 of the T-junction, and may comprise a bare porous polymer monolith 103 having a hydrophobic monolith chemistry (e.g., comprising BMA). In some embodiments, the second monolith 2032 may be in the second downstream branch 2028 of the T-junction, may be adjacent to the inlet 2024 of the T-junction, and may comprise a bare porous polymer monolith 103 having a hydrophilic monolith chemistry (e.g., comprising GMA). In some non-limiting embodiments, the first and second monoliths 2030 and 2032 may have approximately the same dimensions (e.g., approximately 1 mm long, 1 mm wide, and 350 m deep). In some embodiments, the first monolith 2030 may completely fill the first downstream branch 2026 of the T-junction, and the second monolith 2032 may completely fill the second downstream branch 2028 of the T-junction. In some embodiments, the wettability-based bubble separator 2022 may include an air outlet 2034 at an end of the first downstream branch 2026 and a water outlet 2036 at an end of the second downstream branch 2028.

[0120] At low inlet pressures, the pores of the hydrophobic first monolith 2030 remain air filled and present a low resistance to gas flow. Likewise, the pores of the hydrophilic second monolith 2032 wet readily and spontaneously fill with water by capillary flow. To test the separator 2022, water was pumped at 5 l/min through the inlet channel 2024, with air bubbles 2038 introduced using an off-chip flow splitter. One full cycle of water-air-water injection is presented in FIGS. 20A-20F, revealing complete removal of the air bubble 2038 through the hydrophobic first monolith 2030 and an isolated flow of water achieved through the hydrophilic second monolith 2032.

[0121] As illustrated in FIG. 20A, an air bubble 2038 may enter the inlet 2024 of the separator 2022, displacing the remaining water at the channel intersection through the hydrophilic second monolith 2032 until, as illustrated in FIG. 20B, the front of the bubble 2038 reaches the hydrophilic second monolith 2032. As illustrated in FIG. 20C, the air bubble 2038 traverses the hydrophobic first monolith 2030 until, as illustrated in FIG. 20D, the trailing edge of the bubble 2038 clears the gap between the first and second monoliths 2030 and 2032. As illustrated in FIGS. 20E and 20F, water 2040 behind the trailing edge of the bubble 2038 re-establishes flow through the hydrophilic second monolith 2032 as the remainder of the air bubble 2038 enters the hydrophobic first monolith 2030.

[0122] In the wettability-based bubble separator 2022 example, two different monolith chemistries were used, unlike the multiplexed immunosensor 1915 example, which took advantage of different functionalization paths with a single monolith chemistry. Indeed, driven largely by their use increasing in chromatographic separations, an exceptionally wide range of demonstrated polymer monolith chemistries beyond GMA and BMA, such as those discussed in F. Svec, Porous polymer monoliths: amazingly wide variety of techniques enabling their preparation, Journal of chromatography. A, vol. 1217, no. 6, pp. 902-24, February 2010, can be readily adapted to the solvent-assisted integration process. Similarly, a vast array of inorganic metal and oxide monolith chemistries, such as those discussed in Z. Walsh, B. Paull, and M. Macka, Inorganic monoliths in separation science: a review, Analytica chimica acta, vol. 750, pp. 28-47, October 2012, many of which require high temperature synthesis that prohibits their integration into thermoplastic microfluidics by in situ fabrication, may be integrated via the solvent-assisted process. Also, the choice of monolith material may not be limited by sidewall anchoring requirements because the solvent-assisted process may employ mechanical interlocking rather than chemistry-specific covalent attachment.

[0123] The integration of pre-fabricated monoliths, which may also be pre-functionalized, into microfluidic devices by the solvent-assisted process has the potential to greatly simplify the preparation of a wide variety of fluidic devices. The ex situ integration process may allow porous, high surface area, and both chemically- and functionally-diverse monolith structures to be prepared off chip in a highly parallel batch process, followed by post-synthesis insertion into fully sealed fluidic channels without concern for traditional limits on monolith homogeneity, resolution, and chemical compatibility with the fluidic substrate. The ex situ integration process may allow multiple monolith elements with different surface functionalities or bulk polymer chemistries to be used within a single device, and the concept may be further extended to other monolith materials including inorganic oxides or metals, significantly widening the range of porous materials that may be integrated into thermoplastic microfluidics. While the method has been demonstrated here for immunosensing, bubble removal, and sample purification, the technique can offer wide utility for applications including molecular separation and solid phase extraction, filtration, biosensing, microreaction, and beyond.

[0124] In some non-limiting embodiments, the process 300 (see FIG. 3) for incorporating one or more ex situ fabricated porous polymer monoliths 103 into a fluidic chip 100 may be used to create a fluidic chip 2100 capable of pH modulated nucleic acid capture and purification using one or more chitosan functionalized porous polymer monoliths 103. A non-limiting example of the fluidic chip 2100 is illustrated in FIGS. 21A and 21B. FIG. 21A is flat side view of the chitosan functionalized triangular porous polymer monolith 103 in the triangular channel 102 of the channel substrate 101. FIG. 21B is a scanning electron microsope cross-section image of a chitosan functionalized triangular porous polymer monolith 103 in a triangular channel 102 of a channel substrate 101.

[0125] In some embodiments, the fluidic chip 2100 may include one or more bare porous polymer monoliths 103. In some embodiments, the one or more bare porous polymer monoliths 103 of the fluidic chip 2100 may be fabricated ex situ in step 301 of the process 300 illustrated in FIG. 3. In some non-limiting embodiments, the one or more bare porous polymer monoliths 103 of fluidic chip 2100 may be fabricated ex situ according to the process 400 illustrated in FIG. 4. In some non-limiting embodiments, the one or more bare porous polymer monoliths 103 of fluidic chip 2100 may comprise GMA.

[0126] In some embodiments, one or more bare porous polymer monoliths 103 may be functionalized ex situ in step 302 of the process 300 illustrated in FIG. 3. In some embodiments, the one or more bare porous polymer monoliths 103 may be functionalized by anchoring a molecule (e.g., chitosan) capable of electrostatically driven, pH modulated nucleic acid capture on the one or more monoliths 103.

[0127] Some embodiments may employ one of two different approaches for functionalization of porous polymer monoliths 103 with chitosan. In the first approach, a bifunctional cross-linker (e.g., GMBS) may be used to couple amines from the chitosan polymer with epoxy groups on the polymer monolith 103. In some non-limiting embodiments, the bifunctional cross-linker GMBS was used to attach chitosan to the GMA monoliths using an adaptation of a known antibody anchoring procedure. See J. Liu et al., Flow-through immunosensors using antibody-immobilized polymer monoliths, Biosens. Bioelectron., vol. 26, no. 1, pp. 182-8, September 2010. The cross-linker approach is effective in generating a dense layer of chitosan on the monolith surface, as determined by nucleic acid capture. However, the cross-linker approach requires a complex multistep reaction process.

[0128] In the second approach, chitosan may be anchored to the porous polymer monolith 103 through a direct reaction of the chitosan with the porous polymer monolith 103. The direct attachment approach may avoid the complex multistep reaction process constraint but requires long incubation times. To overcome the limitation imposed by long incubation times required for direct chitosan attachment, large numbers of monolith elements may be manufactured in parallel, functionalized off-chip (i.e., ex situ) in bulk solution, and individually integrated into microfluidic chips. Due to the parallel and bulk nature of this process, the long chitosan incubation time may have little impact on the overall efficiency of the fabrication method. Furthermore, compared with on-chip (i.e., in situ) chitosan attachment using both the cross-linking and direct attachment routes, this off-chip functionalization strategy may avoid clogging of the monolith 103, which was commonly observed during on-chip attachment due to excessive chitosan build-up during active perfusion through the monolith.

[0129] In some embodiments, reaction conditions for direct attachment of chitosan may be optimized by varying both reaction time and temperature. In some embodiments, because chitosan has limited solubility at pH 8, the direct attachment method may be performed in an unbuffered solution. In some non-limiting embodiments, a low chitosan concentration (e.g., 1%) in water may be used because higher chitosan concentrations may result in excessive solution viscosity. In some non-limiting direct chitosan attachment embodiments, direct chitosan functionalization of one or more bare porous polymer monoliths 103 was achieved by soaking newly formed, washed monoliths 103 in a 1% solution of chitosan in water and controlling temperature over long reaction times by placing a vial of the monoliths 103 and solution in an oven. FIG. 22 illustrates a direct reaction between a GMA monolith and chitosan.

[0130] Testing found DNA capture and elution performance of monoliths functionalized using on-chip crosslinking and off-chip direct reaction to be similar.

[0131] In some non-limiting embodiments, one or more chitosan functionalized bare preformed porous polymer monoliths may be integrated in one or more channels 102 of the fluidic chip 2100 in step 303 of the process 300 illustrated in FIG. 3. In some non-limiting embodiments, the one or more chitosan functionalized bare preformed porous polymer monoliths may be integrated according to the process 900 illustrated in FIG. 9.

[0132] In some embodiments, one or more chitosan functionalized monoliths 103 were integrated into channels 102 of a channel substrate 101 of the fluidic chip 2100 and used for DNA capture and recovery. FIG. 23 is a graph illustrating experimental average DNA concentration data in eluent at an outlet of the fluidic chip 2100 during loading (first data point), washing (second data point), and elution (following data points). In the experiment, 40 l of 1 ng/l DNA solution was loaded, followed by 30 l of wash buffer and 60 l of elution buffer, all at 5 l/min. As shown in FIG. 23, DNA concentrations at the outlet were negligible during the loading and washing steps, and DNA concentration may reached a peak after approximately 10 microliters of elution buffer have been flushed through the system. Because of the strong pH dependence of Picogreen fluorescence, two separate serial dilutions were prepared to allow quantitation of DNA at pH 5 and at pH 10. The transitional first fraction after switching from pH 5 wash buffer to pH 10 elution buffer represents a potential undercounting of DNA. Using data collected from 3 separate batches of fabricated chips 2100, the chitosan-functionalized monoliths 103 were effective at capturing and releasing DNA, with an average capture efficiency is greater than 99% and an average recovery efficiency of 54.214.2%.

[0133] To ensure that DNA capture results from the chitosan modified monolith surface, rather than physical or chemical interactions with the GMA monolith itself, native monoliths without surface modification were used as controls. A particular concern was that DNA might become trapped between small submicron gaps formed at the interface between adjacent polymer nodules within the porous monolith matrix, resulting in physical retention of DNA within the matrix. FIG. 24 is a graph showing a comparison between outlet DNA concentration for a control monolith 103 and chitosan coated monolith 103 during the capture, wash and elution of 40 ng of DNA. As shown in FIG. 24, approximately 10% of the DNA was retained in the control monolith 103 during loading and washing at pH 5. All of this DNA is recovered in the elution buffer, confirming that the captured DNA is not physically trapped within the porous matrix. Loading DNA into a control monolith at high pH resulted in no measureable retention. Thus, while bare, unfuctionalized GMA monolith does exhibit weak pH dependent DNA capture capability, these tests demonstrate that the addition of a layer of chitosan is desirable for capturing a significant percentage of the DNA in solution.

[0134] The maximum DNA loading capacity of a non-limiting sample of a chitosan functionalized monolith 103 having a length of 1 mm and occupying 300 nl of channel volume was determined by pumping a solution of 1 ng/l DNA at pH 5 through the sample monolith at 5 l/min while collecting the eluent in 10 microliter fractions. FIG. 25 is a graph showing DNA capture capacity measurement of the non-limiting sample of the chitosan functionalized monolith 103. The DNA capture capacity was tested by loading 160 l of 1 ng/l DNA solution, followed by 30 l of washing buffer, and 70l elution buffer, all at a rate of 5 l/min. The capture capacity of the sample monolith 103 was reached at approximately 110 ng, and the concentration of DNA in the eluent rose to match the inlet concentration. After washing, approximately 40% of the captured DNA was eluted in a sharp peak with an elevated concentration.

[0135] As shown in FIG. 25, the concentration of DNA in the eluent remained negligible until the capture capacity of the sample monolith 103 was reached, after which it rose to match the inlet concentration over the course of several fractions. A total of 110 ng DNA is captured before unbound analyte was observed to pass through the monolith during further loading. Of the DNA bound to the monolith, approximately 40% was recovered after switching to the elution buffer. The eluted DNA was amplifiable by PCR in all cases and quantitative PCR measurements agreed well with fluorescence assays.

[0136] Any DNA not accounted for in the eluent is assumed to remain within the monolith, bound to the chitosan coated surface, where it may be visualized with a DNA intercalating dye as shown in FIG. 26. Reversal of flow through a monolith after completion of the initial elution phase yielded negligible free DNA, implying that the remaining DNA is not merely physically trapped at the upstream end of the monolith, but rather tightly bound to the surface even after charge switching.

[0137] The test results shown in FIGS. 23-25 validate a simple, direct chitosan attachment approach to charge-switch based nucleic acid capture and purification using discrete integrated porous monoliths that can be batch functionalized off-chip, followed by integrating individual monoliths into channels (e.g., thermoplastic microchannels) using standard device bonding protocols based on solvent bonding. The high surface area to volume ratio and large cross-sectional area to length ratio of the chitosan coated porous polymer monoliths 103 allows for exceptionally high DNA capture capacity and efficient release in a small on-chip footprint and without introducing significant hydrodynamic resistance for effective flow-through operation. Because the total active surface area is defined by the volume of monolith used, this technique can be tailored to extract and release specific quantities of nucleic acid from a microfluidic fluid stream by defining only the length of monolith inserted. The flexibility and simplicity offered by the monolith-enabled method may realize effective nucleic acid capture, purification, and controlled release in, for example, thermoplastic micro total analysis systems using a highly manufacturable process. The direct chitosan attachment approach also avoids the increased complexity and cost of the overall fabrication process associated with conventional methods for in-line nucleic acid cleanup in fluidic systems that require on-chip functionalization of the capture surfaces.

[0138] Embodiments of the present invention have been fully described above with reference to the drawing figures. Although the invention has been described based upon these preferred embodiments, it would be apparent to those of skill in the art that certain modifications, variations, and alternative constructions could be made to the described embodiments within the spirit and scope of the invention.