STABILIZED VESICLE-FUNCTIONALIZED MICROPARTICLES FOR CHEMICAL SEPARATIONS AND RAPID FORMATION OF POLYMER FRITS IN SILICA CAPILLARIES USING SPATIALLY-DEFINED THERMAL POLYMERIZATION

Abstract

Surface-modified silica microparticles that are functionalized with stabilized phospholipid vesicles are described herein. These stabilized vesicles can be functionalized with either transmembrane receptors or membrane associated receptors and used for affinity pull-down assays or other chromatographic separation modalities to provide affinity capture/concentration of low abundance ligands in complex mixtures with minimal sample preparation. Further described are methods and apparatus for forming polymer frits in a fused silica capillary. The capillary containing a monomer solution is placed between one or more heat sources connected to each other via a jig and operatively coupled to a temperature controller. The polymer frits are synthesized via thermal polymerization of the monomer solution using the heat sources, which allows for placement of the polymer frits at a spatially-defined location in the capillary.

Claims

1. An assay platform (10) for identifying a ligand (5), said assay platform (10) comprising: a. one or more microparticles (15); b. a plurality of lipid vesicles (20), wherein each vesicle (20) comprises a lipid bilayer (22), wherein the vesicles (20) are bonded to a surface (17) of each microparticle; and c. one or more target receptors (25) specific to the ligand (5), wherein the receptors (25) are embedded in the lipid bilayer (22) of the vesicle; wherein the assay platform (10) is mixed into a solution comprising the ligand (5) such that the ligand (5) binds to a receptor (25) of the one or more target receptors to form a ligand-bound assay platform, wherein the ligand-bound assay platform is removed from the solution, and detected via an analytical instrument, wherein when the ligand (5) is detected, the ligand (5) is identified by the receptor (25) that is specific to the ligand (5).

2-4. (canceled)

5. The assay platform (10) of claim 1, wherein the receptors (25) are membrane protein receptors or lipid-derived receptors.

6. (canceled)

7. The assay platform (10) of claim 1, wherein the lipid bilayer (22) comprises polymerizable lipid monomers and functionalized lipid monomers.

8. The assay platform (10) of claim 7, wherein the polymerizable lipid monomers are sorbyl- or dienoyl-containing lipid monomers.

9. (canceled)

10. The assay platform (10) of claim 7, wherein the functionalized lipid monomers are amine-functionalized lipid monomers.

11-12. (canceled)

13. The assay platform (10) of claim 1, wherein the lipid bilayer (22) comprises a plurality of non-polymerizable lipid monomers and a plurality of polymerized, hydrophobic non-lipid monomers.

14. The assay platform (10) of claim 13, wherein the lipid monomers are cell membrane fragments, 1,2-diphytanoyl-sn-glycero-3-phosphocholine monomers, naturally occurring lipids, or synthetic lipids.

15. The assay platform (10) of claim 13, wherein the plurality of polymerized, hydrophobic non-lipid monomers comprises a methacrylate and a cross-linking agent.

16-19. (canceled)

20. The assay platform (10) of claim 15, wherein the cross-linking agent is a dimethacrylate.

21. (canceled)

22. The assay platform (10) of claim 1, wherein the lipid bilayer (22) comprises naturally-occurring lipid membranes or synthetic lipid membranes.

23. (canceled)

24. The assay platform (10) of claim 1, wherein the surface (17) of each microparticle is modified to provide a covalent attachment point for the lipid bilayer (22) of each vesicle.

25-26. (canceled)

27. A method for identifying a ligand (5), said method comprising: a. providing the assay platform (10) of claim 1; b. mixing the assay platform (10) into a solution comprising the ligand (5), wherein the ligand (5) binds to the target receptors (25) to form a ligand-bound assay platform; c. removing the ligand-bound assay platform from the solution; and d. utilizing an analytical instrument to detect the ligand (5) bound to the receptor (25) of the assay platform (10), wherein when the ligand (5) is detected, the ligand (5) is identified by the receptor (25) that is specific to the ligand (5).

28. A method of preparing an assay platform (10) for identifying a ligand (5), said method comprising: a. providing a plurality of microparticles (15); b. depositing modifying molecules on a surface (17) of each microparticle to form a surface-modified microparticle; c. mixing a plurality of lipid monomers with one or more target receptors specific to the ligand to yield a monomer-receptor mixture; d. forming the monomer-receptor mixture into a plurality of lipid vesicles (20); e. polymerizing the lipid vesicles (20) such that the receptors (25) are embedded in a lipid bilayer (22) of each vesicle; and f. depositing the vesicles (20) on the surface of each surface-modified microparticle to form the assay platform (10), wherein the modifying molecules provide a covalent attachment point for the lipid bilayer (22) of each vesicle to attach to the surface-modified microparticle.

29-32. (canceled)

33. The method of claim 28, wherein the receptors (25) are membrane protein receptors or lipid-derived receptors.

34-35. (canceled)

36. The method of claim 28, wherein the lipid monomers comprise polymerizable lipid monomers and functionalized lipid monomers.

37. The method of claim 36, wherein the polymerizable lipid monomers are sorbyl- or dienoyl-containing lipid monomers.

38. (canceled)

39. The method of claim 36, wherein functionalized lipid monomers are amine-functionalized lipid monomers.

40-41. (canceled)

42. The method of claim 28 further comprising mixing a plurality of polymerizable, hydrophobic non-lipid monomers with the monomer-receptor mixture prior to polymerizing the mixture.

43. The method of claim 42, wherein the lipid monomers are cell membrane fragments, 1,2-diphytanoyl-sn-glycero-3-phosphocholine monomers, naturally occurring lipids, or synthetic lipids.

44. The method of claim 42, wherein the plurality of polymerizable, hydrophobic non-lipid monomers comprises a methacrylate and a cross-linking agent.

45-78. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0023] FIG. 1A shows a non-limiting example of some targets on a cell membrane for peptide/protein ligand screening.

[0024] FIG. 1B shows a schematic of the silica core-polymerized phospholipid vesicle shell particle according to an embodiment of the present invention.

[0025] FIG. 2 shows a schematic of the novel pull-down assay process according to an embodiment of the present invention.

[0026] FIG. 3 shows a non-limiting example of mass spectra of silica core-polybis-sorbPC/NH.sub.2/GM1 vesicle shell particles (top), and silica core-polybis-sorbPC/NH.sub.2 vesicle shell particles (bottom), after incubating with CTB and after washing.

[0027] FIG. 4 shows an exemplary structure of polymerizable lipid bis-sorbPC.

[0028] FIG. 5 shows a non-limiting example of a reaction scheme for the formation of receptor and NH.sub.2-functionalized, polymerized phospholipid vesicles.

[0029] FIG. 6 depicts sulfonate-modification of silica microsphere surface and immobilization of phospholipid vesicles to the modified surfaces according to an embodiment of the present invention.

[0030] FIG. 7 shows exemplary fluorescence images of bare and sulfonate-modified silica microspheres after incubating with fluorescein and amino-fluorescein.

[0031] FIG. 8 shows zeta potential measurements of silica microspheres at different modification steps (n=3) according to an embodiment of the present invention.

[0032] FIG. 9 shows non-limiting examples of fluorescence intensity measurements of sulfonate-modified silica microspheres with 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)/NH.sub.2 and polybis-sorbPC/NH.sub.2 vesicle coatings, without and with MeOH rinse, and stained with FM1-43 (measured by flow cytometry, n>5000).

[0033] FIG. 10A shows a schematic of silica core-shell particles with immobilized cell membrane vesicles containing membrane proteins used for high throughput ligand screening.

[0034] FIG. 10B shows NAN-190 fluorescence of silica core-shell particles with CHO-K1 vesicles containing overexpressed 5-HT1A receptors measured using flow cytometry in accordance with the embodiment shown in FIG. 10A.

[0035] FIGS. 11A-11B shows an exemplary reaction scheme for thermal frit polymerization within a fused silica capillary. FIG. 11A shows silanol on the capillary wall reacting with TMSPM to provide a methacrylate functionality on the capillary surface. FIG. 11B shows GMA (monomer), EGDMA (crosslinker), AIBN (thermal initiator) and decanol (not pictured) introduced into the capillary.

[0036] FIGS. 12A-12B show an exemplary setup for thermal polymerization using soldering irons, such as WELLER soldering irons, wherein WELLER is a registered trademark of Apex Tool Group, LLC. FIG. 12A depicts two soldering irons with 3 mm tips that are connected to variable alternating current (VAC) transformers for temperature control, such as VARIAC VAC transformers, wherein VARIAC is a registered trademark owned by Instrument Service & Equipment, Inc. A custom-built hollow aluminum alignment jig was used to stabilize the 3 mm tips of the soldering irons during polymerization. FIG. 12B shows VARIAC VAC transformers allowing for temperature control at 3 mm tips with linear regression y=3.24x21.4 R.sup.2=0.994.

[0037] FIG. 13 is a characterization of polymer frit length (mm) versus time (s) at four different polymerization temperatures; n=3. Error bars are standard deviations.

[0038] FIGS. 14A-14B shows deviation pressures for 8 mm frits. FIG. 14A shows a determination of frit deviation pressure by deviation in linearity of equilibration pressure versus flow rate indicated by vertical line. Solid linear regression: y=48.3x400 R.sup.2=0.99, dashed linear regression: y=88.5x886, R.sup.2=0.99. FIG. 14B is a comparison of frit deviation pressures for 8 mm frits polymerized using labeled conditions. Error bars are standard deviations: n=3.

[0039] FIG. 15 shows a chromatogram of FITC labeled aliphatic amines using C.sub.18 packed capillary with thermal initiated frits. Zonal chromatography of 125 nM FITC-labeled n-hexylamine (A) and n-octylamine (B) in ethanol with UV detection. Mobile phase: 45/55 ACN/H.sub.2O, 0.1% TFA (v/v), Range: 0.005, : 240 nm, 2.0 L min.sup.1.

[0040] FIG. 16 shows an 8 cm long thermally initiated polymer frit synthesized at 92 C. for 75 s. Inset is an SEM image of frit.

[0041] FIG. 17 shows a schematic of a setup used for cLC reverse phase separation using packed capillary. A) A section of 4.5 250 m i.d. PEEK tubing was connected to the outlet of the pump and connected to a 3.5 long 100 m i.d. capillary using a Microtight PEEK connector. B) Injection valve connected to 4 long 100 m i.d. capillary. C) Microtight union connected to the 30 cm long 100 m i.d. capillary with 17 cm packed and two 8 mm frits. D) Microtight union connected to 5.5 capillary with length to the detector of 8 cm.

[0042] FIGS. 18A-18B show exemplary set-ups for thermal polymerization using VARIAC VAC transformers and WELLER soldering irons. FIG. 18A shows two VARIAC VAC transformers connected to two soldering irons with 3 mm tips for temperature control. FIG. 18B shows a custom-built hollow aluminum alignment jig used to stabilize the 3 mm tips of the soldering irons during polymerization.

[0043] FIGS. 19A-19B show SEM Images of UV and Thermal Polymerized Frits. FIG. 19A shows a frit synthesized with soldering iron thermal polymerization at 92 C. for 75 s. FIG. 19B shows a frit synthesized with UV polymerization for 1 hr.

[0044] FIGS. 20A and 20B show exemplary set-ups and SEM images for thermal polymerization of the monomer solution to form polymer frits.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0045] Following is a list of elements corresponding to a particular element referred to herein:

TABLE-US-00001 5 ligand 10 assay platform 15 microparticle 17 microparticle surface 20 lipid vesicle 22 lipid bilayer 25 receptor 100 apparatus 105 capillary 107 capillary wall 109 capillary end 110 heating device 115 heating tip 120 temperature controlling device 130 jig 132 jig end

[0046] Stabilized Vesicle-Functionalized Microparticles

[0047] Referring now to FIG. 1-10B, the present invention features an assay platform (10) for identifying a ligand (5). In some embodiments, the assay platform (10) may comprise one or more microparticles (15), a plurality of lipid vesicles (20) each comprising a lipid bilayer (22), wherein the vesicles (20) are bonded to a surface (17) of each microparticle, and one or more target receptors (25) specific to the ligands (5). For example, the vesicles (20) may be covalently or non-covalently bonded to the surface (27) of microparticles. Preferably, the receptors (25) are embedded in the lipid bilayer (22) of the vesicle. The assay platform (10) can be mixed into a solution comprising the ligand (5) such that the ligand (5) binds to the target receptor (25) to form a ligand-bound assay platform. The ligand-bound assay platform is removed from the solution, and can be detected via an analytical instrument. When the ligand (5) is detected, the ligand (5) can be identified by the receptor (25) that is specific to the ligand (5). For example, the unknown ligand can be identified if it binds to the receptors since the identity of the receptors is known and the receptors are specific to the ligand.

[0048] In some embodiments, the microparticles (15) are silica particles. A diameter of each microparticle can range from between about 3 to 10 m. In other embodiments, the vesicles (20) may be spherical in shape and have a diameter of about 100-600 nm. In some embodiments, the receptors (25) can be membrane protein receptors or lipid-derived receptors.

[0049] In one embodiment, the lipid bilayer (22) may comprise polymerizable lipid monomers and functionalized lipid monomers. The lipid bilayer (22) of the vesicles (20) can be polymerized by thermal polymerization. The polymerizable lipid monomers may be sorbyl- or dienoyl-containing lipid monomers. For example, the dienoyl-containing lipid monomers are 1,2-bis[10-(2,4-hexadieoyloxy)decanoyl]-s-glycero-2-phosphocholine (bis-SorbPC) or 1,2-bis(octadeca-2,4-dienoyl)-sn-glycero-3-phosphocholine (bis-DenPC). In other embodiments, the functionalized lipid monomers are amine-functionalized lipid monomers. For example, the amine-functionalized lipid monomers may comprise an amino(polyethylene glycol) (NH.sub.2-PEG) component. Preferably, the amine functionality of the amine-functionalized lipid monomers is disposed outwardly and away from the vesicle (20), thereby making the microparticles (15) amine-reactive. In some embodiments, the mole ratio of the polymerizable lipid monomers, functionalized lipid monomers, and receptors may be about 95:5:1.

[0050] In other embodiments, the lipid bilayer (22) may comprise a plurality of non-polymerizable lipid monomers and a plurality of polymerized, hydrophobic non-lipid monomers. For example, the lipid monomers may be cell membrane fragments, 1,2-diphytanoyl-sn-glycero-3-phosphocholine monomers, naturally occurring lipids, or synthetic lipids. In some embodiments, the plurality of polymerized, hydrophobic non-lipid monomers may comprise a methacrylate and a cross-linking agent. The methacrylate may be an aliphatic methacrylate, such as an alkyl-substituted aliphatic methacrylate having an alkyl substitution of C4-C10, or an aromatic methacrylate, such as a benzyl methacrylate or a naphthyl methacrylate. The cross-linking agent can be a dimethacrylate, such as ethylene glycol dimethacrylate.

[0051] In still other embodiments, the lipid bilayer (22) can comprise naturally-occurring lipid membranes or synthetic lipid membranes. Preferably, these lipid membranes would not contain polymer scaffolds, and are therefore non-polymerizable.

[0052] In preferred embodiments, the surface (17) of each microparticle may be surface-modified, such as sulfonate-modification. Without wishing to limit the present invention to a particular theory or mechanism, the surface-modification can provide a covalent attachment point for the lipid bilayer (22) of each vesicle. For example, the surface (17) may be sulfonate modified such that the surface (17) comprises sulfonate molecules. Examples of sulfonates that may be used in accordance with the present invention include, but are not limited to, 2,2,2-trifluoroethanesulfonyl chloride, alkyl p-toluenesulfonates (tosylates) and related compounds. Sulfonate-modification is but one example of surface modification. It is to be understood that the surface of the microparticle may be modified with any suitable molecule that can provide a covalent attachment point for the lipid bilayer of each vesicle.

[0053] Another embodiment of the present invention features a method for identifying a ligand (5). The method may comprise providing any embodiment of the assay platform (10) described herein, mixing the assay platform (10) into a solution comprising the ligand (5) such that the ligand (5) binds to the target receptors (25) to form a ligand-bound assay platform, removing the ligand-bound assay platform from the solution, and utilizing an analytical instrument to detect the ligand bound to receptor of the assay platform. When the ligand (5) is detected, the ligand (5) is identified by the receptor (25) that is specific to the ligand (5).

[0054] A further embodiment of the present invention features a method of preparing an assay platform (10) for identifying a ligand (5). The method may comprise providing a plurality of microparticles (15), depositing modifying molecules on a surface (17) of each microparticle to form a surface-modified microparticle, mixing a plurality of lipid monomers with one or more target receptors specific to the ligand to yield a monomer-receptor mixture, forming the monomer-receptor mixture into a plurality of lipid vesicles (20) such that each lipid vesicle has a lipid bilayer (22), polymerizing the lipid vesicles (20) such that the receptors (25) are embedded in the lipid bilayer (22) of each vesicle, and depositing the vesicles (20) on the surface of each surface-modified microparticle to form the assay platform (10).

[0055] In one embodiment, the microparticles (15) are silica particles. A diameter of each microparticle can range from between about 3 to 10 m. In another embodiment, the vesicles (20) are polymerized by thermal polymerization. The vesicles can have a diameter of about 100-600 nm. In yet another embodiment, the monomer-receptor mixture is formed into a plurality of lipid vesicles (20). A non-limiting example of forming said lipid vesicles utilizes surfactant dialysis. The receptor is solubilized into a solution of surfactants that are subsequently removed by dialysis in the presence of excess lipid vesicles to localize the receptor into the lipid vesicle membrane.

[0056] In some embodiments, when the receptors (25) are membrane protein receptors, the method may further comprise reconstituting the membrane protein receptor with a surfactant prior to polymerizing the lipid vesicles. In alternate embodiments, the receptors (25) are lipid-derived receptors.

[0057] In some embodiments, the lipid monomers comprise polymerizable lipid monomers and functionalized lipid monomers. The polymerizable lipid monomers may be sorbyl- or dienoyl-containing lipid monomers. For example, the dienoyl-containing lipid monomers are 1,2-bis[10-(2,4-hexadieoyloxy)decanoyl]-sn-glycero-2-phosphocholine (bis-SorbPC) or 1,2-bis(octadeca-2,4-dienoyl)-sn-glycero-3-phosphocholine (bis-DenPC). In other embodiments, the functionalized lipid monomers are amine-functionalized lipid monomers. For example, the amine-functionalized lipid monomers may comprise an amino(polyethylene glycol) (NH.sub.2-PEG) component. Preferably, the amine functionality of the amine-functionalized lipid monomers is disposed outwardly and away from the vesicle (20), thereby making the microparticles amine-reactive. As a non-limiting example, a mole ratio of the polymerizable lipid monomers, functionalized lipid monomers, and receptors may be about 95:5:1.

[0058] In some embodiments, the method may further comprise mixing a plurality of polymerizable, hydrophobic non-lipid monomers with the monomer-receptor mixture prior to polymerizing the mixture. The lipid monomers may be cell membrane fragments, 1,2-diphytanoyl-sn-glycero-3-phosphocholine monomers, naturally occurring lipids, or synthetic lipids. The plurality of polymerizable, hydrophobic non-lipid monomers may comprise a methacrylate and a cross-linking agent. In one embodiment, the methacrylate is an aliphatic methacrylate, such as an alkyl-substituted aliphatic methacrylate having an alkyl substitution of C4-C10, or an aromatic methacrylate, such as benzyl methacrylate or a naphthyl methacrylate. In another embodiment, the cross-linking agent is a dimethacrylate, such as ethylene glycol dimethacrylate.

[0059] In other embodiments, the modifying molecules may be sulfonate molecules, which can provide covalent attachment points for the lipid bilayer of the vesicles to attach to the surface-modified microparticle. Examples of sulfonates that may be used in accordance with the present invention include, but are not limited to, 2,2,2-trifluoroethanesulfonyl chloride, tosylates, and related compounds. It is to be understood that the present invention is not limited to sulfonate modification, and that the surface of the microparticle may be modified with any suitable molecule that can provide covalent attachment points.

[0060] An exemplary embodiment of the present invention features a microparticle architecture that utilizes a silica core particle (15) that is functionalized with receptors within stabilized liposomes. This particle architecture was then used to perform pulldown assays in complex solutions with subsequent analysis by electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Recovery of serotonin via binding to 5-HT1A receptors within CHO-K1 cell membranes was evaluated. CHO-K1 cell membrane fractions were isolated through homogenization and centrifugation, and were extruded to form vesicles (20), which could be subsequently stabilized using polymer scaffold stabilization approaches. The vesicles (20) were then immobilized to the particle surface (17) to yield silica core-cell membrane vesicle shell particles. Particles were characterized using flow cytometry to verify attachment of cell membrane vesicles with and without 5-HT1A receptors to modified particles. Serotonin was incubated with the silica core-cell membrane vesicle shell particles containing the serotonin receptor, and centrifugation was used to pull down the particles. ESI-MS confirmed the pull down of the serotonin ligand.

Experimental Procedures

[0061] The following are exemplary embodiments of preparing silica core-vesicle shell particles and detecting peptide/protein ligands on specific targets embedded in the particles. It is understood that the present invention is not limited to the embodiments described herein.

[0062] Sulfonate modification of silica particles' Procedures are modified from Larson (Methods Enzymol. 1984, 104, 212-223) and Nilsson (Methods Enzymol. 1984, 104, 56-69).

[0063] Diol-Modification of Silica Particles:

[0064] About 40 mg of 5 m diameter silica particles was suspended in 20 mL of 5% HCl and stirred for 1 hour. The particles were then washed with nanopure water three times and with acetone three times sequentially by centrifugation. After washing, silica particles were re-suspended in a small amount of acetone and transferred into a 50 mL round bottom flask. The acetone was evaporated by a stream of N.sub.2, and the flask was connected to a vacuum and heated to 150 C., lasting for 4 hours. After 4 hours, heating was stopped. When the temperature dropped to between about 50-100 C., the vacuum was disconnected. A stir bar was placed into the flask. Then a mixture of 30 mL dry toluene, 0.6 mL 3-glycidyloxypropyltrimethoxysilane and 15.3 L triethylamine was added into the flask. The flask was connected to a condenser, which was sealed with a septum. The system was flushed with N.sub.2 three times and then an N.sub.2 balloon was attached to the top of the condenser. The mixture was heated to reflux, which lasted overnight. After overnight reflux, the particles were washed sequentially with toluene and acetone, and then dried with a stream of N.sub.2. Dried particles were re-suspended in 20-30 mL of 10 mM H.sub.2SO.sub.4 (by sonication) and then heated to 90 C. for 1 hour with stirring. After that, the particles were washed sequentially with water and acetone.

[0065] Sulfonate Modification of Diol-Silica Particles:

[0066] Diol-silica particles were washed with dry acetone three times. Then the particles were transferred into a cleaned, dried, 25 mL round bottom flask. The remaining dry acetone was evaporated with N.sub.2 stream and a stir bar was put inside the flask. The flask was sealed with a septum and flushed with N.sub.2 for several minutes. About 3 mL of dry acetone was added into the flask and stirring was started. Then 26 L dry pyridine and 18 L 2,2,2-trifluoroethanesulfonyl chloride were added sequentially. The mixture was stirred for 15-30 min at room temperature. After the reaction, sulfonate-silica particles obtained were washed with acetone, 1:1 (v:v) of acetone:5 mM HCl, 1 mM HCl, nanopure water and acetone sequentially. Lastly, the particles were dried with N.sub.2 stream and stored dry.

[0067] Aside from sulfonate modification, other surface modification chemistries can be used, such any appropriate modifications using covalent and non-covalent linkages of vesicles to the surface.

[0068] Formation and Thermal Polymerization of bis-sorbPC/NH.sub.2/GM1 Vesicles:

[0069] Bis-sorb PC was purified by HPLC as described in Gallagher (J. Chromatogr. A. 2015, 1385, 28-34). Procedures for polymerization of bis-sorbPC vesicles were modified from Sisson (Macromolecules. 1996, 29, 8321-8329). About 0.95 mg bis-sorbPC was mixed with 1,2-distearoyl-en-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG (2000)-NH.sub.2) and GM1 to make a mole ratio of about 95:5:1 of bis-sorbPC:GM1:DSPE-PEG (2000)-NH.sub.2. Azobisisobutyronitrile (AIBN) was dissolved in benzene to make fresh stock solution of 1 mg/ml. An appropriate amount of AIBN stock was added to the lipid mixture to make a mole ratio of 2.5:1 bis-sorbPC:AIBN. Organic solvents in the mixture were evaporated with an Ar stream and the mixture was further dried in a lyophilizer for at least 4 hours. After drying, the lipid mixture was re-hydrated in 200 L of degassed 20 mM phosphate, pH 7.4. The sample was warmed up in a 42 C. water bath and vortexed to re-suspend all the dried lipids. Then the sample went through 10 cycles of freeze (77 C.), thaw (42 C.), vortex, and was extruded through 2 stacked 0.2 m polycarbonate membrane filters by a mini extruder. The extrusion was carried out above the T.sub.m (29 C.) of bis-sorbPC. After extrusion, the vesicle solution was bubbled with a slight stream of Ar for 5 min and sealed with an Ar atmosphere. Then the vesicle solution was heated at 65 C. overnight for thermal polymerization. The handling of bis-sorbPC was done under yellow light before polymerization.

[0070] Formation of Silica Core-Vesicle Shell Particles:

[0071] About 200 L of 20 mM phosphate, pH 8.0 was added to 4 mg of sulfonate-silica particles. The mixture was sonicated to re-suspend the particles. The 200 L of polymerized vesicle solution was then added into the particle suspension. The total 400 L of sample was placed into 0.2 mL dome cap PCR tubes to completely fill the tube and cap space and eliminate air. The sample was incubated by slowly inverting up and down continuously on a mixing wheel for 3 hours. After incubation, samples were centrifuged at 7 G for 5 min and the supernatant was discarded. After tethering vesicles to the sulfonate-silica particles, washing of the particles by centrifugation needs to be done at low G forces to minimize loss of vesicle coating on the particle surface. About 400 L of 20 mM phosphate, 5 mM Tris, pH 8.0 was added to the particles, and the inverting incubation was continued for 1 hour to scavenge the sulfonate-silica surface that was not covered by vesicles. After surface scavenge, the particles were washed three times by 20 mM phosphate, pH 7.4 (7 G5 min each time).

[0072] Detection and Identification of CTB with MALDI-MS:

[0073] After the silica core-vesicle shell particles were washed by 20 mM phosphate, pH 7.4, the supernatant was discarded. Then about 114 L of 0.5 mg/mL CTB stock solution was added to the particles, and appropriate amount of 20 mM phosphate, pH 7.4 was added to the mixture to completely fill the 0.2 mL dome cap PCR tubes. The inverting incubation was carried out for 1 hour. Then the particles were washed three times by 20 mM phosphate, pH 7.4 (7 G5 min each time). Lastly, the particles were re-suspended in 200 L of 20 mM phosphate, pH 7.4. About 1 L from the particle suspension was mixed with 1 L of saturated CCA in water. The total 2 L of mixed sample was spotted on MALDI plate and dried at room temperature. MALDI-MS analysis was directly carried out on the dried particles. A Bruker UltraFlex III TOF-TOF mass spectrometer was operated in linear, positive ion mode.

[0074] Referring to FIGS. 10A and 10B, the following is a non-limiting example of preparing silica core-vesicle shell particles functionalized with transmembrane receptors and detecting peptide/protein ligands on specific targets embedded in the particles.

[0075] As shown in FIG. 10A, the buffers used were 50 mM Tris-HCl, pH 7.4, 10 mM MgSO.sub.4, 0.5 mM EDTA, 0.1% ascorbic acid (Buffer A), and 50 mM Tris-HCl, pH 7.4 (Buffer B). In one embodiment, 5 m silica particles were modified to obtain sulfonate surface modification. In another embodiment, the vesicles were CHO-K1 cell membranes with overexpressed 5-HT1A receptors extruded through 200 nm filters. About 0.5 mg CHO-K1 cell membranes with overexpressed 5-HT1A receptors. OE-CHO-K1 were attached to 0.5 mg of sulfonate modified particles through incubation for 3 hrs while spinning on a bloodwheel, thereby forming the silica core-vesicle shell particles. Then, 1% serum (final concentration) was added to both samples. Fluorescent 5-HT1A receptor antagonist, NAN-190 was added to sample 2 and incubated for 1 hour. Samples were washed 3 times with Buffer B. Two samples were prepared: Sample 1OE-CHO-K1 Membrane+1% FBS serum; and Sample 2OE CHO-K1 Membrane+1% FBS serum+NAN-190. 10,000 particles from each sample were analyzed using flow cytometry .sub.x:633 nm .sub.m:660/20 nm.

[0076] Referring to FIG. 10B, the silica core-shell particles with CHO-K1 cell membranes vesicles overexpressing the 5-HT1A receptor successfully bound NAN-190 in the presence of serum. Without wishing to limit the invention to a particular theory or mechanism, the G-protein coupled receptor is proven to have remained functional in this new platform due to the specific binding of NAN-190.

[0077] Results and Discussion

[0078] Referring to FIG. 3, cholera toxin binding unit (CTB) was successfully detected using ganglioside GM1 (CTB's membrane receptor) functionalized core-shell particles. The top mass spectrum shows CTB molecular ions (both singly charged and doubly charged) were successfully detected with MALDI-TOF-MS analysis on the silica core-polybis-sorbPC/NH.sub.2/GM1 vesicle shell particles, after incubating with CTB and after washing, because of the specific binding between GM1 and CTB. The bottom mass spectrum shows that no CTB was detected on the silica core-polybis-sorbPC/NH.sub.2 vesicle shell particles, after incubating with CTB and after washing, because of the lacking of GM1.

[0079] Referring to FIG. 7, the fluorescence image on the far right shows that the sulfonate-modified silica microspheres may be amine-reactive. As shown FIG. 8, the phospholipid vesicle coating may change the surface charge of the silica microspheres. A sulfonate-modified microsphere has a smaller negative zeta potential than that of a bare microsphere. Further still, a bis-sorbPC vesicle-modified microsphere has an even lower negative zeta potential than that of the sulfonate-modified microsphere or the bare microsphere. As shown in FIG. 9, after a MeOH rinse, polybis-sorbPC/NH.sub.2 vesicle coated microspheres have a normalized fluorescence intensity that is about 2.5 times greater than that of non-polymerizable DOPC/NH.sub.2 vesicle coated microspheres. This demonstrates that the polymerized vesicle coatings are more stable than non-polymerizable vesicle coatings on sulfonate-modified silica microspheres.

[0080] When synthesizing the silica core-polymerized phospholipid vesicle shell particles of the present invention, polymerization of the phospholipids was shown to improve vesicle coating stability. The present invention allows for peptides or protein ligands to be detected on specific targets, such as receptors, of the functionalized core-polymerized phospholipid vesicle shell particles. Examples include, but are not limited to, the detection of CTB on GM1-functionalized core-shell particles by MALDI-MS and the detection of serotonin via binding to 5-HT1A receptors. The silica core-shell particles with immobilized cell membranes vesicles containing membrane receptors of the present invention may be used as a simple and fast approach for high throughput ligand screening.

[0081] Rapid Formation of Polymer Frits in Capillaries

[0082] Referring now to FIG. 11-20B, the present invention features a method of forming polymer frits inside a capillary (105). In one embodiment, the method may comprise providing one or more heating devices (110) each having a heating tip (115), wherein the heating devices (110) are operatively connected to one or more temperature controlling devices (120), connecting each heating tip (115) together via a jig (130), rinsing the capillary (105) with a monomer solution such that at least a portion of the capillary is filled with the monomer solution, heating the heating tips (115) via the heating devices (110) to a polymerization temperature set by the temperature controlling devices (120), inserting the capillary (105) through the jig (130) such that the portion of the capillary containing the monomer solution is disposed between the heating tips (115), and heating the portion of the capillary via the heating tips (115) for a polymerization time to thermally polymerize the monomer solution, thereby forming the polymer frits, thereby forming the polymer frits.

[0083] Without wishing to limit the present invention to a particular theory or mechanism, the method can be effective for placement of the polymer frits at a spatially-defined location in the capillary (105), i.e. the heating tips can polymerize the monomer solution into polymer frits at precise locations along the capillary (105). Moreover, thermal polymerization can retain a capillary sheath of the capillary, thereby improving capillary stability, as opposed to other methods of polymerization where the capillary sheath is removed.

[0084] As used herein, the term frit refers to a porous material with pore sizes sufficiently small enough to retain particles, e.g. a fused or partially fused porous material.

[0085] As used herein, the term jig is defined as a device that is used to control a location and/or motion of other tools, or parts thereof.

[0086] In some embodiments, the heating devices (110) may comprise a soldering iron and the heating tip (115) is a soldering tip. In other embodiments, any appropriate heating device may be used to heat the capillary. In further embodiments, the temperature controlling devices (120) may comprise a variable alternating current transformer. However, it is understood that the temperature controlling devices (120) may also be any appropriate device that controls temperature. For example, the soldering iron may be an adjustable temperature soldering iron. In still other embodiments, the jig (130) is a metallic tubular jig. For example, the jig may be constructed from a metal such as aluminium, steel, iron, or any other suitable metal or metal alloy. Each heating tip (115) is positioned at a jig end (132) of the jig such that the heating tip is disposed in the jig. For instance, the heating tip is inserted into the jig end. Preferably, the capillary (105) is perpendicularly disposed through the jig (130).

[0087] In some embodiments, the polymerization temperature is about 92-140 C. For example, the polymerization temperature may be about 90-100 C. 100-120 C., 120-140 C., or greater than 140 C. In other embodiments, the polymerization time is about 10-110 seconds. For instance the polymerization time may be about 10-30 seconds, 30-60 seconds, 60-90 seconds, or 90-110 seconds. Preferably, the polymerization time is less than 2 minutes.

[0088] Embodiments of the present invention may utilize a fused silica capillary. The capillary (105) may modified prior to rinsing with the monomer solution. In exemplary embodiments, the step of modifying the capillary comprises rinsing the capillary (105) with a modifying solution comprising methacrylate under UV-free yellow light, wherein at least a portion of the capillary (105) is filled with the modifying solution, capping each capillary end (109) to seal the modifying solution within the capillary (105), heating the capillary (105) at a temperature of 60 C. for about 15 to 20 hours, and removing the modifying solution from the capillary (105). Preferably, the methacrylate can modify a capillary wall (107) to increase stability when the polymer frits are bonded to the capillary wall. The polymer frits may be secondary frits for retaining a packed-bed inside the capillary (105). In some embodiments, the capillary (105) may be dried after the modifying solution is removed. In other embodiments, each capillary end (109) may be capped.

[0089] In some embodiments, the methacrylate may be 3-trimethyoxysilylpropyl methacrylate (TMSPM). However, any suitable methacrylate may be used to modify the capillary wall. In other embodiments, the monomer solution may comprise glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EGDMA), and 2,2-azobisisobutyronitrile. An example of the volumetric ratio of GMA to EGDMA may be a 1:1 volumetric ratio. The monomer solution may also further comprise decanol.

[0090] Another embodiment of the present invention apparatus (100) for forming polymer frits inside a capillary (105). The apparatus may comprise one or more heating devices (110) each having a heating tip (115), wherein the heating devices (110) are operatively connected to one or more temperature controlling devices (120), and a tubular jig (130). In some embodiments, each heating tip (115) can be each positioned at a jig end (132) such that the heating tip (115) is disposed in the jig (130). The heating tips are heated via the heating devices to a polymerization temperature set by the temperature controlling. The capillary (105) may be perpendicularly disposed through the jig (130) such that the portion of the capillary (105) containing the monomer solution is positioned between the heating tips (115), which then thermally polymerizes the monomer solution to form the polymer frits. Preferably, the apparatus may be effective for placement of the polymer frits at a spatially-defined location in the capillary.

[0091] In some embodiments, the heating devices (100) may comprise a soldering iron and the heating tip (115) is a soldering tip. In other embodiments, any appropriate heating device may be used to heat the capillary. In further embodiments, the temperature controlling devices (120) may comprise a variable alternating current transformer. However, the temperature controlling devices may also be any appropriate device that controls temperature. For example, the soldering iron may be an adjustable temperature soldering iron. In still other embodiments, the jig (130) may be constructed from a metal, such as aluminium, steel, iron, or any other suitable metal or metal alloy.

[0092] In some embodiments, the polymerization temperature may be about 92-140 C. For example, the polymerization temperature may be about 90-100 C., 100-120 C., 120-140 C., or greater than 140 C. In other embodiments, the polymerization time is about 10-110 seconds. For instance the polymerization time may be about 10-30 seconds, 30-60 seconds, 60-90 seconds, or 90-110 seconds. Preferably, the polymerization time is less than 2 minutes.

[0093] Examples of capillaries (105) may be silica capillaries and fused silica capillaries. Preferably, a capillary wall (107) of the capillary is modified with a methacrylate to effect bonding of the polymer frits to the capillary wall for increased stability. Examples of the methacrylate include 3-trimethyoxysilylpropyl methacrylate (TMSPM) or any other suitable methacrylate.

[0094] In some embodiments, the monomer solution may comprise glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EGDMA), and 2,2-azobisisobutyronitrile. An example of the volumetric ratio of GMA to EGDMA may be a 1:1 volumetric ratio. In other embodiments, the monomer solution may further comprise decanol.

EXPERIMENTAL

[0095] Materials:

[0096] Fused silica capillary (100 m i.d., 360 m o.d.) was purchased from PolyMicroTechnologies. NaOH, HCl, acetone, decanol, HPLC grade methanol (99.9%), ethanol, and n-octylamine (98%) were purchased from Fisher Scientific. 3-trimethyoxysilylpropyl methacrylate (TMSPM), ethylene glycol dimethacrylate (98%) (EGDMA), 2-2-dimethoxy-2-phenylactetophenone (99%) (DAP), and 2,2-azobisisobutyronitrile (97%) (AIBN) were purchased from Sigma Aldrich. AIBN was recrystallized in methanol prior to use to remove impurities. Glycidyl methacrylate (98%) (GMA) was purchased from Alfa Aesar. Trifluoroacetic acid (TFA) and acetonitrile (ACN) were purchased from EMD Millipore. HxSil 5 m diameter C.sub.18-modified silica particles with 100 pores were purchased from Hamilton. A Cheminert injection valve was purchased from Valco. N-hexylamine (99%) was purchased from VWR International. Unless noted all chemicals are used as received. All H.sub.2O was purified to a resistivity of 18.3 M-cm using a Barnstead EASYpure UV/UF compact reagent grade water system.

[0097] Capillary Modification:

[0098] Sections of fused silica capillary (100 m i.d., 360 m o.d.) 30 cm long were rinsed at 0.5 mL min.sup.1 with 1M NaOH (5 min), 0.1M HCl (5 min), nanopure H.sub.2O (5 min), and acetone (10 min) using a syringe pump. Capillaries were dried using He for 30 min. A 50% (v/v) mixture of TMSPM and acetone was rinsed through capillaries under UV-free yellow light. Capillary ends were sealed with parafilm to withhold the solution and heated at 60 C. for 20 h. Capillaries were rinsed with methanol at 1 mL min.sup.1 for 10 min using a syringe pump. Capillaries were dried with He for 30 min and left to further air-dry overnight. This methacrylate modification allows for bonding of the frit to the capillary wall for increased stability.

[0099] Monomer Solution Preparation:

[0100] GMA and EGDMA were passed through an aluminum oxide column (0.253.0) prior to use to remove inhibitors. A monomer solution for thermal polymerization was prepared using 1.3 mg AIBN, 60 L GMA, 60 L EGDMA, and 280 L decanol. For UV polymerization, the thermal initiator AIBN was replaced with 2.5 mg DAP while maintaining the other reagents. The monomer solutions were sonicated for 10 min and degassed with N.sub.2 for 10 min.

[0101] Frit Synthesis:

[0102] Two soldering irons were connected to variable autotransformers to enable temperature control. In-house fabricated 3 mm soldering iron tips were attached to both soldering irons. The VARIAC VAC transformers were adjusted to achieve the desired polymerization temperature ranging from 92-140 C. and turned on 30 min prior to polymerization to allow the soldering irons to equilibrate to the set temperature. The monomer solution was rinsed through capillaries. Capillaries were placed in a custom-fabricated 7 mm long aluminum alignment jig and capillary ends were sealed with parafilm to contain the monomer solution within the capillary. Frits were synthesized by placing a soldering iron on either side of the capillary within the aluminum jig for the designated polymerization time ranging from 10-110 s. The reaction chemistry for frit polymerization is shown in FIGS. 11A and 11B. Capillaries were washed with methanol at 5 L min.sup.1 for 10 min to remove any unreacted components post polymerization using a syringe pumping system and dried overnight. Frits were imaged using a camera through the lens of a stereomicroscope and measured using Image J software.

[0103] UV initiated frits were polymerized in capillaries with 8 mm windows for 1 h using a Newport 100 W Mercury Arc lamp with H.sub.2O IR absorption filter and UV bandpass filter. All UV frits had an average length of 8.00.5 mm. UV initiated frits necessitated burning an 8 mm window within the capillary prior to capillary modification.

[0104] Frit Pressure Stability Studies:

[0105] The equilibration pressure of both thermal and UV initiated frits was monitored at various flow rates. The deviation pressure was defined as a deviation in linearity of pressure versus flow rate corresponding to the onset of frit compression or decomposition.

[0106] Packing Capillaries.

[0107] An air driven fluid pump was used to pack a slurry solution by pumping methanol at 500 psi. The slurry solution contained 5 m diameter C.sub.18 modified silica particles at a concentration of 6.4 mg particles/mL methanol. A 17 cm bed was packed. To synthesize the second retaining frit, the particle slurry solution was replaced with the monomer solution and pumped through the capillary at 500 psi for 10 min. The second retaining frit was polymerized by using soldering irons to apply heat at the edge of the packed bed or by positioning the previously burned capillary window in front of the lamp for UV irradiation. Packed capillaries were rinsed with methanol at 0.5 l min.sup.1 for 30 min and dried overnight.

[0108] Capillary Liquid Chromatography:

[0109] An EldexMicroPro pump equipped with 2 mL syringes was used with a Cheminert injection valve and 1.4 L injection loop (FIG. 17). A section of 4.5 250 m i.d. PEEK tubing was connected to the outlet of the pump and connected to a 3.5 long 100 m i.d. capillary using a Microtight PEEK connector from IDEX. The capillary was connected to the injection valve and a 4 long 100 m i.d. capillary. The 30 cm long 100 m i.d. capillary with a 17 cm packed bed and two 8 mm frits was connected using a Microtight union from Sigma Aldrich to a 5.5 capillary with length to the detector of 8 cm. A 3 mm detection window was used. 250 m i.d., 1/16 PEEK tubing was purchased from GRACE. Zonal chromatography was performed using 125 nM FITC labeled n-hexylamine and n-octylamine dissolved in ethanol. The 45/55 ACN/H.sub.2O 0.1% TFA (v/v) mobile phase was degassed using He for 30 min prior to use. The elution profile was monitored by UV absorbance detection at 240 nm. Signal from the detector was collected with an A/D converter and software written in Lab View, Sovitsky-Golay filtering was applied to the data using Origin.

[0110] Scanning Electron Microscopy:

[0111] An FEI Inspec-S SEM instrument was used to image both thermal and UV polymerized frits. Prior to imaging, capillaries were mounted vertically and a 4-5 mm gold coating was sputtered onto ends using the Hummer Sputter System. Samples were coated for 90 s, rotated and coated for an additional 90 s.

[0112] Safety Considerations:

[0113] When packing capillaries, the entire system should be contained to prevent injury if a fitting failed.

[0114] Results and Discussion

[0115] A new approach for thermal polymerization was developed to synthesize on-column polymer frits inside 100 m i.d. capillaries. This methodology uses two soldering irons attached to VARIAC VAC transformers for temperature control, as shown in FIG. 12A. An aluminum alignment jig provided greater stability and positional reproducibility of soldering irons during polymerization, aiding in reproducible frit synthesis. The capillary can be easily positioned within the aluminum alignment jig allowing for the synthesis of a second retaining frit directly at the end of the packed bed. Referring to FIG. 12B, VARIAC VAC transformers allow for precise temperature control at the soldering iron tips. Also, the polyimide capillary sheathing is retained during frit synthesis, maintaining a stable capillary compared to UV polymerization. Overall, this method for thermal polymerization is rapid, precise, cost-effective, and maintains capillary stability.

[0116] In developing a new frit fabrication method, it was necessary to determine how various conditions affect frit polymerization. Referring to FIG. 13, several polymerization times and temperatures were used to determine the effect on frit length. Frit length can be controlled via a combination of polymerization temperature and time. As the polymerization time increased, the frit length increased. Frit sizes were shown to exceed the size of the soldering iron tips. Heat conduction that occurs as the soldering iron tips heat the monomer solution within the capillary, creating a diffuse heating zone for frit polymerization. These different conditions demonstrate the frit size is tunable to the experimental application, thus the desired frit length is readily obtained using appropriate polymerization time and temperature. This thermal initiation approach allows for rapid polymerization times <2 min compared to UV polymerization methods that necessitate a minimum of 1 h. To minimize band broadening in a separation, a relatively short tilt with small deviation in frit size is desired. Frits <4 mm could be synthesized using smaller soldering iron tips.

[0117] In liquid chromatography, synthesized frits must withstand packing pressures and chromatographic backpressures without compressing or disintegrating. Pressure characterization was performed to compare the pressure stability of thermal and UV polymerized frits. The method uses a constant flow rate and monitored pressure for comparison of frit stability. The frit pressure stability was examined using Poiseuille's equation, which relates the linear velocity to the backpressure in the system (Equation 1):

[00001]$\begin{array}{cc}{}_0=\frac{r^2.Math..Math..Math.P}{8.Math..Math..Math.L_t}& \left(1\right)\end{array}$

where .sub.0 is the linear velocity of the solvent, r the internal radius of the capillary tube, P the applied pressure, L.sub.t is the total length of the capillary and the solvent viscosity. Based on Poiseuille's equation, the linear velocity and pressure within the system should exhibit a linear relationship.

[0118] Any deviation from linearity would suggest morphological change, either compression or disintegration, in the frit. This deviation was used to determine the frit deviation pressure, as shown in FIG. 14A. The tested thermal synthesis conditions for pressure stability studies were 92 C. or 124 C. for 75 or 15 s, respectively. Both conditions produced precisely sized and relatively small 8 mm frits (frits of the same size are necessary for pressure comparison). Thermal initiated frits synthesized at 92 C. over 75 s exhibited greater resistance to morphological change than frits synthesized at 124 C. over 15 s by a statistically value of 125 psi. A longer polymerization time allows for greater conversion of the monomer and cross-linker, and thus greater cross-linking of linear polymer chains. Referring to FIG. 14B, thermal initiated frits synthesized at 92 C. over 75 s showed pressure stability of 200 psi over the 8 mm frit length, which is comparable to 1 h UV polymerized frits.

[0119] Frit stability throughout column packing is the ultimate measure of frit strength. The frit and packed bed can have the same porosity; therefore, the 200 psi pressure drop is equivalent to 7500 psi over a 30 cm packed bed. When packing a capillary, the distance over which the pressure in the system can drop continually increases as packing continues, lowering the amount of pressure per unit distance. Therefore, the thermal frits would remain robust during packing and chromatography. Due to greater frit pressure stability, the 92 C. for 75 s synthesis condition was chosen for use in packed capillaries.

[0120] Packing a C.sub.18-modified silica particle column with thermal initiated frits allowed for assessment of thermal frit stability and chromatographic performance. Thermal initiated frits synthesized at 92 C. for 75 s remained stable during packing and allowed for formation of a 17 cm packed bed of 5 m diameter C.sub.18-modified silica particles. This demonstrates thermal frit stability at 500 psi. The second thermal retaining frit was successfully synthesized and the packed capillary was used in cLC. As shown in FIG. 15, the thermal frit packed capillary allowed for the separation of FITC-labeled n-hexylamine and n-octylamine in 35 min at a flow rate of 2.0 L min.sup.1.

[0121] Values of merit for reverse phase chromatography using a packed capillary with thermal frits are summarized in Table 1. This column allowed for resolution greater than baseline of the two analytes. Furthermore, the minimal error in retention time signifies the frits remain stable throughout multiple runs and allow for reproducible separations.

TABLE-US-00002 TABLE 1 Values of merit from cLC reverse phase separations of FITC labeled aliphatic amines. Analyte Retention Time (min) R.sub.s FITC-Hexylamine (n = 3) 12.95 0.04 3.39 0.09 FITC-Octylamine (n = 3) 30.86 0.09

[0122] The novel thermal polymerization method for preparing porous polymer frits has been developed and compared to an established UV polymerization method. The method allows for the synthesis of frits ranging from 4-10 mm depending on both the polymerization temperature and time, proving useful for tuning fit size to experimental application. Thermal frits exhibited sufficient porosity and robustness for pressure driven liquid chromatography. Additionally, the thermal initiated frits synthesized using the temperature-controlled polymerization allowed for reproducible separations of two aliphatic amines for use in a packed bed cLC.

[0123] This thermal polymerization method for polymer frits offers several advantages compared to existing methods. The soldering iron setup for thermal polymerization is much more affordable compared to expensive lamps needed for UV polymerization. The method allows for rapid polymerization times <2 min compared to 1 h for UV polymerization. Soldering irons allow for ease in placement of the second retaining frit with precise temperature control provided by VARIAC VAC transformers. The UV method necessitates removal of the capillary sheathing, thereby introducing fragility; whereas thermal polymerization allows for protection of the polyimide sheathing. Overall, this approach offers cost efficiency, fast polymerization times, simplicity, spatial precision and less fragile capillaries.

[0124] As used herein, the term about refers to plus or minus 10% of the referenced number.

[0125] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.

[0126] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. The reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase comprising includes embodiments that could be described as consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase consisting of is met.