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WO2005036154A1 - Supports de separation comprenant des polymeres d'oxazoline - Google Patents

Supports de separation comprenant des polymeres d'oxazoline Download PDF

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Publication number
WO2005036154A1
WO2005036154A1 PCT/US2004/033288 US2004033288W WO2005036154A1 WO 2005036154 A1 WO2005036154 A1 WO 2005036154A1 US 2004033288 W US2004033288 W US 2004033288W WO 2005036154 A1 WO2005036154 A1 WO 2005036154A1
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WO
WIPO (PCT)
Prior art keywords
separation
oxazoline
oxazoline polymer
separation lane
lane
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2004/033288
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English (en)
Inventor
Richard Bernard
Gary W. Loge
Ksenia Krylova
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Spectrumedix Corp
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Spectrumedix Corp
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Filing date
Publication date
Application filed by Spectrumedix Corp filed Critical Spectrumedix Corp
Publication of WO2005036154A1 publication Critical patent/WO2005036154A1/fr
Anticipated expiration legal-status Critical
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • 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.
  • 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").
  • CZE capillary zone electrophoresis
  • 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.
  • 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.
  • the separation lane includes an amount of the oxazoline polymer sufficient to support the separation of the biological molecules.
  • the oxazoline polymer in the separation lane may deactivate charges on the inner surface.
  • 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.
  • a wall coating e.g., an oxazoline polymer, poly(acryloylaminopropanol), or a copolymer of acrylamide and allyl glycidyl ether
  • the separation lane is an electrophoresis capillary.
  • the separation lane is a high pressure liquid chromatographic column.
  • 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.
  • 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.
  • the biological molecules are peptides or proteins.
  • the method includes, prior to the subjecting, denaturing and then labeling the peptides or proteins, e.g., labeling with a fluorescent tag. .
  • 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.
  • 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.
  • 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).
  • 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 > 1.3xl0 6 ) with or without about 50 ⁇ M cetyltrimethylammonium bromide or hexadecyltrimethylammonium bromide.
  • a wall coating e.g., a high molecular weight polyvinylpyrrolidone (MW > 1.3xl0 6 ) with or without about 50 ⁇ M cetyltrimethylammonium bromide or hexadecyltrimethylammonium bromide.
  • 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.
  • 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.
  • the biological molecules include proteins, peptides, oligonucleotides,
  • RNAs, DNAs, or a combination thereof 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.
  • the oxazoline polymer facilitates the separation of molecules based at least in part on a size difference between the molecules to be separated.
  • the oxazoline polymer may form a sieving matrix within a separation lane. Referring to Fig.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • separation lane 10 is flexible.
  • 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.
  • 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.
  • the separation properties of the oxazoline polymer may be, at least in part, due to its entanglement structure in a solution.
  • 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.
  • the oxazoline polymer has a viscosity sufficient to minimize or eliminate electroosmotic flow.
  • the oxazoline polymers have at least one monomer unit with the structure:
  • each of R 1 ? R 2 , R 3 , and R 4 independently, is H or a hydrocarbon radical
  • R 5 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.
  • 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).
  • hydrocarbon radicals include CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH 2 CH 2 CH 2 CH 2 CH 3 , CH 2 OH, CH 2 CH 2 OH, CH 2 CH 2 CH 2 OH, or CH 2 CH 2 CH 2 CH 2 OH.
  • Examples of monomer units include 2-oxazoline, 2-methyl-2-oxazoline, 2-efhyl- 2-oxazoline, 2-pr ⁇ pyl-2- ⁇ xazolir ⁇ e ⁇ 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.
  • the oxazoline polymer is a homopolymer (all monomer units are the same).
  • oxazoline homopolymers examples 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).
  • the oxazoline polymer is a copolymer (contain two or more different monomer units).
  • 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.
  • 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.
  • 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.
  • the oxazoline polymer can be prepared by a ring opening polymerization.
  • 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.
  • a living polymerization the polymer chain does not terminate under the polymerization conditions.
  • an oxazoline copolymer can also be prepared by a cationic living polymerization.
  • 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.
  • 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.
  • 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).
  • buffers examples 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”).
  • HEPES 4-(2-Hydroxyethyl)piperazine-l-ethanesulfonic acid
  • Tris tris(hydroxymethyl)aminomethane
  • TAPS tris(tris(Hydroxymethyl)methyl]amino]- propanesulfonic acid
  • EDTA Tris/TAPS/ethylenediaminetetraacetic acid
  • the separation medium can also include a certain concentration of a denaturing agent, e.g., sodium dodecyl sulfate (“SDS").
  • SDS sodium dode
  • 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.
  • 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.
  • 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.
  • 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).
  • an azo initiator e.g., 2,2'- azobisisobutyronitrile or other suitable azonitrile initiators, such as DUPONT VAZO 44WSP.
  • 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.
  • the wall-coating is a dynamic wall coating, which possesses self-coating properties and can be an additive to the separation medium.
  • 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.
  • the oxazoline polymer can itself be used as a wall coating (e.g., a dynamic wall coating).
  • the separation medium can also contain other additives, such as ethylene glycol.
  • the proteins or peptides can be denatured and labeled before separation.
  • the proteins or peptides are first denatured and then labeled to facilitate detection and/or separation of the molecules.
  • 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).
  • FQ 5- furoylquinoline-3-carboxaldehyde
  • a cyanide salt e.g., sodium cyanide or potassium cyanide
  • 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
  • an aqueous buffer solution e.g., a HEPES buffer solution
  • SDS as a denaturing agent
  • 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
  • an electric field is applied to the separation medium along the separation lane to cause the biological molecules to migrate within the separation medium.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Electrochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
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  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des supports de séparation contenant des polymères d'oxazoline, ainsi que des systèmes et des méthodes associés.
PCT/US2004/033288 2003-10-07 2004-10-07 Supports de separation comprenant des polymeres d'oxazoline Ceased WO2005036154A1 (fr)

Applications Claiming Priority (2)

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US50886703P 2003-10-07 2003-10-07
US60/508,867 2003-10-07

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WO2005036154A1 true WO2005036154A1 (fr) 2005-04-21

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5338428A (en) * 1993-05-28 1994-08-16 California Institute Of Technology Poly(N-Acylalkylenimine) electrophoresis support media
WO2002000746A2 (fr) * 2000-06-30 2002-01-03 Institut Curie Solution de traitement de surface minimisant les phenomenes d'adsorption et/ou d'electroosmose

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5338428A (en) * 1993-05-28 1994-08-16 California Institute Of Technology Poly(N-Acylalkylenimine) electrophoresis support media
WO2002000746A2 (fr) * 2000-06-30 2002-01-03 Institut Curie Solution de traitement de surface minimisant les phenomenes d'adsorption et/ou d'electroosmose

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KAGUYA T. ET AL.: "Ring-openingpolymerization of 2-substituted 2-oxazolines", POLYMER LETTERS, vol. 4, 1966, pages 441 - 445, XP002984397 *
ZEWERT T. ET AL.: "Polyethyleneglycol methacrylate 200 as an electrophoresis matrix in hydroorganic solvents", ELECTROPHORESIS, vol. 13, 1992, pages 824 - 831, XP009040382 *

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