WO2005036154A1 - Separation media including oxazoline polymers - Google Patents

Abstract

Separation media containing oxazoline polymers, and related systems and methods are disclosed.

Description

Separation Media Including Oxazoline Polymers

TECHNICAL FIELD This invention relates to separation media for use in the separation of components of a mixture and related systems and methods.

BACKGROUND Separations are typically performed within some structure that contains a separation medium. For example, electrophoresis separations are commonly performed within the internal bore of a capillary or along a channel of a microfluidic device. Fused silica is one of the most common materials for capillaries and microfluidic devices. Exemplary capillaries can be obtained from Polymicro Technologies of AZ (See, Polymicro Technologies, The Book on the Technologies of Polymicro, 1998, Polymicro Technologies). Polyimide-clad fused silica capillaries possess structural, electrical and optical properties that are suited for capillary zone electrophoresis ("CZE"). Single capillary and multiple capillary systems that employ fused silica as the capillary material are commercially available. The separations medium may support the separation of components of a mixture based at least in part on the size of the components.

SUMMARY This invention relates to separation media for use in the separation of components of a mixture and related systems and methods. In one aspect, the invention features a separation lane for separating a mixture of biological molecules. The separation lane includes an inner surface that defines an inner bore, the inner bore comprising an oxazoline polymer. In some embodiments, the separation lane includes an amount of the oxazoline polymer sufficient to support the separation of the biological molecules. Alternatively, or in addition, the oxazoline polymer in the separation lane may deactivate charges on the inner surface. In some embodiments, the inner bore of the separation lane further includes a wall coating (e.g., an oxazoline polymer, poly(acryloylaminopropanol), or a copolymer of acrylamide and allyl glycidyl ether) that deactivates charges on the inner surface. In some embodiments, the separation lane is an electrophoresis capillary. In other embodiments, the separation lane is a high pressure liquid chromatographic column. In another aspect, the invention features a method of separating a mixture of biological molecules. The method includes (1) introducing the biological molecules to a separation lane comprising an inner surface that defines an inner bore, the inner bore comprising an oxazoline polymer; and (2) subjecting the biological molecules to an electrical field sufficient for the migration and separation of the biological molecules along the separation lane. In some embodiments, the method includes, prior to the subjecting, introducing the oxazoline polymer to the separation lane as a solution of the oxazoline polymer and a buffer. In some embodiments, the biological molecules are peptides or proteins. In still another embodiment, the method includes, prior to the subjecting, denaturing and then labeling the peptides or proteins, e.g., labeling with a fluorescent tag. . In still another aspect, the invention features an electrophoresis system. The electrophoresis system includes (1) a separation lane that has an inner surface that defines an inner bore and has a detection zone, (2) a voltage source in electrical communication with the inner bore, (3) a light source that irradiates the detection zone, and (4) a detector that receives light from the detection zone. The inner bore includes an oxazoline polymer. In a further aspect, the invention features a separation medium for separating a mixture of biological molecules. The separation medium contains an oxazoline polymer and a buffer solution. One aspect of the invention relates to a method for analyzing DNA fragments. The fragments are subjected to electrophoresis along a separation lane including oxazoline polymer, e.g., an oxazoline polymer having a molecular weight more than 500,000 Daltons. The oxazoline polymer may be in combination with other constituents, such as a buffer (e.g., Tris-TAPS-EDTA), a solvent (e.g., DMSO, which may be present at about 5% v/v), a base (e.g., urea). The oxazoline polymer may be present at about 6 w/v%. The separation lane may include a wall coating, e.g., a high molecular weight polyvinylpyrrolidone (MW > 1.3xl0⁶) with or without about 50 μM cetyltrimethylammonium bromide or hexadecyltrimethylammonium bromide. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS Fig. 1 is an electrophoresis system including a separation lane. Fig. 2 is a separation lane. Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION Oxazoline polymers are used in the separation of biological molecules, e.g., in electrophoresis separations, liquid chromatographic separations, high pressure liquid chromatographic separations, gel permeation, or sedimentation separations. In some embodiments, the biological molecules include proteins, peptides, oligonucleotides,

RNAs, DNAs, or a combination thereof. The oxazoline polymer may be wholly or partially contained within a bore of a separation lane, such as a bore defined by a capillary, a two-dimensional slab, a liquid chromatographic column, or a channel of a microfluidic device. In some embodiments, the oxazoline polymer facilitates the separation of molecules based at least in part on a size difference between the molecules to be separated. For example, the oxazoline polymer may form a sieving matrix within a separation lane. Referring to Fig. 1, an electrophoresis system 100 includes a separation lane 10 (e.g., a capillary) and a voltage source 102, which is in electrical communication with an inner bore 11 of separation lane 10 via buffer reservoirs 104. Although system 100 shows a single separation lane 10, a plurality of separation lanes 10 (e.g., 64 separation lanes or 96 separation lanes) may be combined to form an array of separation lanes, such as a planar array. For example, electrophoresis systems may be operated with one or more capillaries, such as those discussed in U.S. Patent No. 6,352,633, the contents of which are incorporated herein in their entirety. System 100 is under the control of a processor 112, in communication with voltage source 102 and other components of the system. Referring tό^~Fϊg^".^*2 separation lane TO includes a wall 12 having an outer surface 14 and an inner surface 13. Outer surface 14 may include a coating, such as a protective coating, which may be opaque, e.g., a protective polyamide coating. Inner surface 13 defines an inner bore 11, which includes a first opening 16 and a second opening 18. Inner surface 13 may include a wall coating, such as a coating that reduces electroosmotic flow along the separation lane. Sample material to be subjected to separation, such as by electrophoresis, may be introduced to inner bore 11 via first opening 16. During separation, sample material migrates under the influence of an electric field along inner bore 11 toward second opening 18. The electric field strength can be, for example, between about 50 V/cm to about 300 V/cm (e.g., between about 100 V/cm and about 200 V/cm or between about 125 V/cm and about 175 V/cm). Separation lane 10 includes a detection zone 20 intermediate the first and second openings 16,18. Detection zone 20 is configured to allow at least one of (a) the introduction of excitation light into inner bore 11 through wall 12 and (b) the escape of light from inner bore 11 through wall 12. For example, the excitation light may be laser light directed through wall 12 into inner bore 11 and the escaping light may be fluorescence emitted by sample irradiated with the excitation light. The fluorescence is received and detected by a detector 110. ¹ In certain embodiments, separation lane 10 is flexible. For example, separation lane 10 can be bent without breaking at a temperature of 25 °C or less into a 360° loop having a radius of less than 30 cm, less than 15 cm, e.g., less than 5 cm. Separation lane 10 can have an outer diameter (not including any coating applied to its external surface) of less than 1000 microns, less than 750 microns, less than 500 microns, or less than 250 microns. Inner bore 11 of separation lane 10 can have a diameter greater than 1 micron, greater than 5 microns, e.g., greater than 25 microns. The diameter of the inner bore can be less than 500 microns, less than 250 microns, less than 125 microns, e.g., less than 75 microns. The diameter of inner bore 11 can be substantially less than its length, for example, the diameter of inner bore 11 can be less than l/500th, less than 1/lOOOth or less than l/5000th of its length. Inner bore 11 of separation lane 10 may be filled with a separation medium that supports and/or facilitates the components of a mixture, e.g., biological molecules of a mixture. Exemplary separation media include at least one oxazoline polymer or a copolymer including at least one oxazoline polymer. In some embodiments, the separation medium acts as a sieving medium that separates molecules, e.g., proteins or peptides, based at least in part on a size of the molecules. For example, the separation properties of the oxazoline polymer may be, at least in part, due to its entanglement structure in a solution. In certain embodiments, the oxazoline polymer is not cross-linked. The absence of cross-linking can provide a low viscosity when the oxazoline polymer is dissolved in a solution, which facilitates the introduction of the oxazoline polymer into the a separation lane. In certain embodiments, the oxazoline polymer has a viscosity sufficient to minimize or eliminate electroosmotic flow. In general, the oxazoline polymers have at least one monomer unit with the structure:

where each of R_{1 ?} R₂, R₃, and R₄, independently, is H or a hydrocarbon radical, and R₅ is H, OH, or a hydrocarbon radical. A hydrocarbon radical contains at least one carbon atom (e.g., one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms, six carbon atoms, seven carbon atoms, eight carbon atoms, etc.). A hydrocarbon radical can be saturated or unsaturated, substituted or unsubstituted, branched or straight chained, and/or cyclic or acyclic. Examples of substituted hydrocarbon radicals include halo-substituted hydrocarbon radicals, hydrocarbon radicals substituted with a nitrogen-containing group (e.g., amino), and hydrocarbon radicals substituted with a oxygen-containing group (e.g., hydroxy or carboxyl). Examples of hydrocarbon radicals include CH₃, CH₂CH₃, CH₂CH₂CH₃, CH₂CH₂CH₂CH₂CH₃, CH₂OH, CH₂CH₂OH, CH₂CH₂CH₂OH, or CH₂CH₂CH₂CH₂OH. Examples of monomer units include 2-oxazoline, 2-methyl-2-oxazoline, 2-efhyl- 2-oxazoline, 2-prδpyl-2-όxazolirϊe^ 2 buFyl-2- xazoline, 2^:hydrdxy-2-oxazoline, 2- hydroxymethyl-2-oxazoline, 2-hydroxyethyl-2-oxazoline, 2-hydroxypropyl-2-oxazoline, and 2-hydroxybutyl-2-oxazoline. In some embodiments, the oxazoline polymer is a homopolymer (all monomer units are the same). Examples of oxazoline homopolymers include poly(2-oxazoline), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), poly(2-propyl-2-oxazoline), poly(2-butyl-2-oxazoline), poly(2-hydroxy-2-oxazoline), poly(2-hydroxymethyl-2- oxazoline), poly(2-hydroxyethyl-2-oxazoline), poly(2-hydroxypropyl-2-oxazoline), and poly(2-hydroxybutyl-2-oxazoline). In certain embodiments, the oxazoline polymer is a copolymer (contain two or more different monomer units). In certain embodiments, the oxazoline polymer has a weight average molecular weight of at least about 50,000 Daltons, at least about 200,000 Daltons, at least about 500,000 Daltons, or at least about 1,000,000 Daltons. As used herein, weight average molecular weight is determined by gel permeation chromatography. Calibration curves for determining molecular weights can be generated using linear polystyrenes as molecular weight standards. In certain embodiments, the oxazoline polymer has an intrinsic viscosity at 25°C of at least about 50 centipoises, at least about 100 centipoises, at least about 1,000 centipoises, at least about 10,000 centipoises, at least about 100,000 centipoises, or at least about 500,000 centipoises. In certain embodiments, the oxazoline polymer has an intrinsic viscosity at 25°C of less than about 100,000 centipoises, less than about 10,000 centipoises, less than about 1,000 centipoises, less than about 500 centipoises, or less than about 250 centipoises. In certain embodiments, the oxazoline polymer can be prepared by a ring opening polymerization. For example, 2-ethyl-2-oxazoline (a monomer) can be polymerized by a cationic living ring-opening polymerization. Such a polymerization can be initiated by a strong electrophile (e.g., a cation). An electrophilic molecule first attacks the endocyclic tertiary nitrogen atom of a 2-ethyl-2-oxazoline molecule to form an oxazolinium ring. Another monomer molecule then attacks the carbon atom at the 5 position (i.e., C5) and the bond between C5 and O breaks to form a pendant carbonyl group. The polymer chain propagates by repeating this process. The tertiary nitrogen and carbons at 4 and 5 positions in me ring iorm me polymer backbone. As ^"a living polymerization, the polymer chain does not terminate under the polymerization conditions. One can introduce a strong nucleophile (e.g., piperidine) to terminate the polymerization. In some embodiments, an oxazoline copolymer can also be prepared by a cationic living polymerization. For example, one can introduce a nucleophile in the above- mentioned living polymerization to form end groups that can be polymerized by free- radicals. An acrylamide derivative or a derivative of vinyl pyrrolidone can be attached to both ends of the polymer to form a macromer when terminating the living polymerization. This macromer can then be co-polymerized with other monomers (e.g., acrylamide, N,N-dimethyl acrylamide, acryloylaminopropanol, or vinyl pyrrolidone) via a free-radical polymerization to form an oxazoline copolymer. In some embodiments, an oxazoline polymer can be prepared by sequentially adding other suitable monomers to the living polymerization mentioned above. Some oxazoline polymers can be purchased from a commercial source, e.g., from Sigma-Aldrich Co, St. Louis, MO. In some embodiments, the oxazoline polymer is dissolved in an aqueous buffer solution to form a separation medium for use in an electrophoresis. Buffers can be selected from typical electrophoresis buffers. The buffer used can depend on the materials to be separated. Buffer solutions are described, for example, in Andreas Chrambach, "The Practice of Quantitative Gel electrophoresis," VCH Publisher, Deerfield Beach, FL (1985), and U.K. Laem li, Nature, 227:680, (1970). Examples of such buffers include 4-(2-Hydroxyethyl)piperazine-l-ethanesulfonic acid ("HEPES"), tris(hydroxymethyl)aminomethane ("Tris")/ 3-[[tris(Hydroxymethyl)methyl]amino]- propanesulfonic acid ("TAPS"), and Tris/TAPS/ethylenediaminetetraacetic acid ("EDTA"). When the biological molecules to be separated include certain molecules, e.g., proteins or peptides, the separation medium can also include a certain concentration of a denaturing agent, e.g., sodium dodecyl sulfate ("SDS"). The buffer solution can have a pH greater than 2.0, greater than 3.0, greater than 4.0, or greater than 6.0. The concentration of the oxazoline polymer in the separation medium can be any such that effective separation of the biological molecules can be achieved based on the molecular sizes and/or charges. In some embodiments, the concentration of the oxazoline polymer in the separation medium is from about 0.01 to 10 weight percent (e.g., 8 weight percent). The concentration used depends on the parameters of the separation technique. For example, in capillary electrophoresis typical parameters include column configuration, diameter, and length, the molecular structure, intrinsic viscosity, the interactive character of the polymer itself, the range of and differences between the molecular weights of the biological molecules, and the effect of other factors influencing the separation, such as charge and electrophoretic mobility. One approach to determine a suitable concentration is to perform a separation at each of several different polymer concentrations. Based upon the separations, e.g., their resolution and efficiency, a suitable polymer concentration can be determined. In some embodiments, the separation lane contains a wall-coating. The wall coating can include an oxazoline polymer, or other suitable polymers, such as poly(acryloylaminoethanol), poly(acryloylaminopropanol), or a copolymer of, e.g., acylamide and allyl glycidyl ether. In certain embodiments, the copolymer of acylamide and allyl glycidyl ether is prepared by using an azo initiator (e.g., 2,2'- azobisisobutyronitrile or other suitable azonitrile initiators, such as DUPONT VAZO 44WSP). The wall-coating deactivates charges on the inner surface of a separation lane, thereby minimizing or eliminating electroosmotic flow. The wall-coating can also reduce the adsorption of biological molecules, e.g., proteins or peptides, to the inner wall of the separation lane. The wall-coating can be applied on the inner wall permanently via covalent modification. In certain embodiments, the wall-coating is a dynamic wall coating, which possesses self-coating properties and can be an additive to the separation medium. In certain embodiments, the oxazoline polymer is used as a sieving medium together with a wall coating (e.g., a permanent wall coating or a dynamic wall coating). The wall coating may or may not include the oxazoline polymer. In other embodiments, the oxazoline polymer can itself be used as a wall coating (e.g., a dynamic wall coating). In certain embodiments, the separation medium can also contain other additives, such as ethylene glycol. When separating proteins or peptides, they can be denatured and labeled before separation. In certain embodiments, the proteins or peptides are first denatured and then labeled to facilitate detection and/or separation of the molecules. For example, the proteins or peptides can be first denatured with SDS and then labeled with 5- furoylquinoline-3-carboxaldehyde ("FQ") and a cyanide salt (e.g., sodium cyanide or potassium cyanide).

EXAMPLE Below is an example of using an oxazoline polymer as a sieving medium in electrophoresis. An oxazoline polymer (e.g., poly(2-ethyl-2-oxazoline) having a weight average molecular weight of 500,000) is dissolved in an aqueous buffer solution (e.g., a HEPES buffer solution) that contains SDS as a denaturing agent to obtain a separation medium having a concentration of 8 w/v % of the polymer. The separation medium is filtered by a cellulose acetate filter with 8 micro pores. The separation medium is stable at room temperature without noticeable precipitation after three months. The separation medium thus obtained has a low viscosity and can be easily introduced (e.g., by a pump) into a separation lane (e.g., a capillary, column, or channel). A mixture of biological molecules (e.g., peptides or proteins) are added to the separation medium at one end of the separation lane. During separation, an electric field is applied to the separation medium along the separation lane to cause the biological molecules to migrate within the separation medium. U.S. provisional application no. 60/508,867 filed October 7, 2003, titled OXAZOLINE POLYMERS FOR ELECTROPHORESIS, by Richard Bernard et al. is incorporated herein by reference in its entirety. All publications cited herein are hereby incorporated by reference in their entirety.

OTHER EMBODIMENTS All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A separation lane for separating a mixture of biological molecules, the separation lane comprising an inner surface that defines_an jnner_bore, the inner bore comprising an oxazoline polymer.

2. The separation lane of claim 1, wherein the oxazoline polymer comprises at least one monomer unit having the structure: Ri R₃

-N-

R₂ R₄ C=0

R₅ wherein each of R_{l 5} R₂, R₃, and R , independently, is H or a hydrocarbon radical, and R₅ is H, OH, or a hydrocarbon radical.

3. The separation lane of claim 2, wherein R₅ is H, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH₂CH₂CH₂CH₂CH₃, OH, CH₂OH, CH₂CH₂OH, CH₂CH₂CH₂OH, or CH₂CH₂CH₂CH₂OH.

4. The separation lane of claim 3, wherein the oxazoline polymer is poly(2- ethyl-2-oxazoline).

5. The separation lane of claim 4, wherein the poly(2-ethyl-2-oxazoline) has a weight average molecular weight of at least about 500,000 Daltons.

6. The separation lane of claim 1, wherein the oxazoline polymer has an intrinsic viscosity of less than about 500 centipoises at 25°C.

7. The separation lane of claim 1, wherein the oxazoline polymer has an intrinsic viscosity of more than about 100,000 centipoises at 25°C.

8. The separation lane of claim 1, wherein the separation lane comprises an amount of the oxazoline polymer sufficient to support the separation of the biological molecules.

9. The separation lane of claim 1, wherein the oxazoline polymer deactivates charges on the inner surface.

10. The separation lane of claim 1, wherein the inner bore further comprises a wall coating that deactivates charges on the inner surface.

11. The separation lane of claim 10, wherein the wall coating comprises an oxazoline polymer.

12. The separation lane of claim 10, wherein the wall coating comprises poly(acryloylaminopropanol) or a copolymer of acrylamide and allyl glycidyl ether.

13. The separation lane of claim 1, wherein the separation lane is an electrophoresis capillary.

14. A method of separating a mixture of biological molecules, comprising introducing the biological molecules to a separation lane comprising an inner surface that defines an inner bore, the inner bore comprising an oxazoline polymer; and subjecting the biological molecules to an electrical field sufficient for the migration and separation of the biological molecules along the separation lane.

15. The method of claim 14, wherein the oxazoline polymer comprises at least one monomer unit having the structure: Ri R₃

— C C N—

R₂ R₄ C=0

R₅ wherein each of R_1; R₂, R₃, and R₄, independently, is H or a hydrocarbon radical, and R₅ is H, OH, or a hydrocarbon radical.

16. The method of claim 15, wherein the oxazoline polymer has a weight average molecule weight of at least about 500,000 Daltons.

17. The method of claim 14, wherein the oxazoline polymer has an intrinsic viscosity of less than about 500 centipoises at 25°C.

18. The method of claim 14, wherein the oxazoline polymer has an intrinsic viscosity of more than about 100,000 centipoises at 25°C.

19. The method of claim 14, further comprising, prior to the subjecting, introducing the oxazoline polymer to the separation lane as a solution of the oxazoline polymer and a buffer.

20. The method of claim 19, wherein the oxazoline polymer deactivates charges on the inner surface.

21. The method of claim 19, wherein the solution further comprises a wall coating that deactivates charges on the inner surface.

22. The method of claim 21, wherein the wall coating comprises poly(acryloylaminopropanol) or a copolymer of acrylamide and allyl glycidyl ether.

23. The method of claim 14, wherein the biological molecules are peptides or proteins.

24. The method of claim 23, further comprising, prior to the subjecting, denaturing and then labeling the peptides or proteins.

25. An electrophoresis system, comprising: a separation lane comprising an inner surface that defines an inner bore and having a detection zone, the inner bore comprising an oxazoline polymer; a voltage source in electrical communication with the inner bore; a light source that irradiates the detection zone; and a detector that receives light from the detection zone.

26. The electrophoresis system of claim 25, wherein the oxazoline polymer comprises at least one monomer unit having the structure: Ri R3

— C C N—

R₂ R₄ C O

R5 wherein each of R_l5 R₂, R₃, and R , independently, is H or a hydrocarbon radical, and R₅ is H, OH, or a hydrocarbon radical.