CA 0226610~ 1999-03-17 SURFACE MODIFIED ELECTROPIIORETIC CIIAMBERS
This invention relates to electrophoresis.
Electrophoresis, in which entities are moved through a m~dhlm as a result of an 10 applied electric field, has become an increasingly indispensable tool in biotechnology and related fields. In electrophoresis, the electrophoretic medium through which the entities are moved is housed in an electrophoretic chamber. A variety of di~ei el.
chamber configurations find use, including slab gel holders, columns or tubes, microbore capillaries, grooves or ch~nnPI~ on a substrate surface elc., where 15 advantages and disadvantages are associated with each particular configuration.
The particular material from which an electrophoretic chamber is fabricated can have a significant impact on the results of the application in which the chamber is employed. Some materials, e.g, fused silica, have charged surfaces under conditions of electrophoresis which give rise to electroosmotic flow. The presence of electroosmotic 20 flow (EOF) can change the movement profile of the entities through the medillm during electrophoresis. Certain materials can also adsorb entities from the metli~lm~ such as proteins and other biomolecules, which can adversely affect the results of a particular application.
.. . .
CA 0226610~ 1999-03-17 wO 98/12~30 PCT/US97/17003 As knowledge of the effect of surface properties on the movement of entities through a me~ m in electrophoretic applications grows, there is an increasing interest in the development of methods to tailor the surface properties of the electrophoretic chamber to meet the needs of a particular application. For example, it may be desirable to have a surface modified to reduce or enhance EOF through the chamber, to reduce or Ánh~nre analyte adsorption to the walls of the chamber, to provide for stableatt~ÁhmÁnt of a gel network to the surface of the chamber, and the like.
Although fused silica has traditionally been the material of choice from which electrophoretic ~,ha~bel ~ are fabricated, of increasing interest as an alternative material are plastics. Various means have been developed for the surface modification of materials employed in electrophoretic applications. Surface modification techniques that have been employed include techniques based on the physical or chemical alteration of the material surface, e.g, etching, chemical modification, and coating a new material over the existing surface, e.g, solvent coating or thin film deposition by chemical or vapor deposition, radiation grafting, chemical grafting and RF-plasma. The particular surface modification means employed necessarily depends on the material to be modified. For methods specifically directed to the treatment of plastic surfaces of electrophoretic chambers, see EP 665 430 Al and EP 452 055 Bl.
Despite the availability of a number of different plastic surface modification techniques, there is a continued interest in the development of new surface modification procedures which would increase the number of di~t, t;ll~ available methods and provide for further surface property tailoring opportunities to best meet the needs of a particular application.
U.S. Patent No. 4,680,201 describes a method for covalently ~tt~ching a polyacrylamide surface layer to the inner surface of fused silica capillaries. U. S. Patent No. 5,433,898 describes a process for pl~?h~ g material for use in the construction of contact lenses comprising two or more polymers.
EP 665 430 Al and EP 452 055 Bl describe use of surface modified polymeric capillaries in electrophoresis.
Additional references describing electrophoresis in various surface modified capillaries include: Gilges et al., "Capillary Zone Electrophoresis Separations of Basic and Acidic Proteins Using Poly(vinyl alcohol) Coatings in Fused Silica Capillaries,"
CA 0226610~ 1999-03-17 wo 98/12~30 PCT/US97/17003 Anal. Chem. (1994) 66:2038-2046; Rohlicek e~ al., "Determination of the Isoelectric Point of the Capillary Wall in Capillary Electrophoresis, Application to PlasticCapillaries," J. Chrom. A. (1994) 662:369-373; Schiit~ner & Kenndler, "Electrophoresis in Synthetic Organic Polymer Capillaries: Variation of Electroosmotic 5 Velocity and ~ Potential with pH and Solvent Composition," Anal. Chem. (1992) 64:
~ 1991- 1995; Nielen, "Capillary Zone Electrophoresis Using a Hollow Polypropylene Fiber," J. High Resolution Chrom. (1993) 16:62-64; and Liu et al., "Polymeric Hollow Fibers for Capillary Electrophoresis," J. Microcol. Sep. (1993) 5:245-253; Hjertťn, "High Performance Electrophoresis F.limin~tion of Electroendosmosis and Solute Adsorption," J. Chromatogr. (1985) 347:191-198.
Encyclopedia of Polymer Science and Engineering, "Adhesion and Bonding,"
Vol. 1, pp. 476 e~ seq. (Wiley Interscience, 1985) describes reactive adhesives based on the surface interpenetration of reactive monomers.
A review of surface modification of polymer materials is provided in Ratner, Biosensors & Bioelectronics (1995) 10:797-804.
SUMMARY OF T~ INVENTION
Electrophoretic chambers having at least a region of surface modification, as well as methods for their fabrication, are provided. The subject devices find use in a wide variety of electrophoretic applications in which entities are moved through a me(lillm in response to an applied electric field.
In one aspect of the invention the region of surface modification includes an electrophoretic polymeric layer, which provides for the tailored surface properties in the modified region, stably bound to the polymeric material of the chamber through copolymerization with an anchoring polymeric layer that interpenetrates the surface of the chamber. The subject chambers are prepared by cont~cting the surface of the chamber with a first monomer capable of interpenetrating the surface. The resultant hl~ el1eLl~led surface is then contacted with a second monomer, followed by copolymerization of the first and second monoll.t;. ~.
In another aspect of the invention, the region of surface modification includes a polymeric layer which is bound to the interior surface of the polymeric material of the chamber wall by non-covalent molecular interactions without the aid of a second ..... ~ ... ...
CA 0226610~ 1999-03-17 anchoring polymeric layer. The noncovalently bound polymeric layer is applied to the surface by cont~cting a solution co~ g the dissolved polymer with the interior surface of the chamber, and allowing the polymer in solution to form molecular interactions with the surface to form the surface coating. The polymeric layer may be 5 immobilized at the surface by interpenetration of the polymeric material of the chamber wall, by ionic interactions, or by hydrophobic interactions.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a plot of measured electroosmotic force over time, showing changes in 10 EOF over the course of repeated electrophoresis runs in untreated PMMA capillaries (open triangles, -~-), in Poly(AMPS)-treated PMMA capillaries (filled diamonds, ----), and in Poly(DMA)-treated PMMA capillaries (filled squares, ----), as described in Example 5.
Fig. 2 is a plot of measured electroosmotic force over time, showing changes in EOF over the course of repeated electrophoresis runs in untreated PMMA Áh~nn~c (filled squares, ----) and in PMMA rh~nn~lc treated with PSSS (filled circles, ----), as described in Example 7.
Electrophoretic chambers having at least one region of surface modification, as well as methods for their fabrication, are provided.
In the region of surface modification, where the term region encompasses anywhere from the entire inner surface of the chambers to only a fraction thereof, the chambers comprise a rigid polymeric base material, an anchoring polymeric layer penetrating the surface of the base material and an electrophoretic polymeric layer, which provides the tailored surface properties, copolymerized with the anchoringpolymeric layer.
Alternatively, in the region of surface modification the chambers comprise a rigid polymeric base material and an electrophoretic polymeric layer which is 30 noncovalently bound on the surface material without the aid of a separate anchoring polymeric layer.
CA 0226610~ 1999-03-17 In further describing the subject invention, the chambers will first be described in greater detail followed by a rli~c~ iQn of the methods used to fabricate the subject chambers.
In the subject chambers, at least that portion of the chamber in the region of 5 surface modification will be fabricated from a solid, rigid polymeric material that is ~ insoluble in aqueous media. As the polymeric material is solid and rigid, it will have sllffir,ient strength to serve as a ."ech~ c~l support for an electrophoretic metli~lm, such as a buffer or gel. Although only the material in the region of surface modification will n.o.cess~rily be the rigid, solid polymeric base material, the entire chamber, e.g, capillary 10 or planar substrate having a microchannel on its surface, may be fabricated from the base polymeric material. Alternatively, the chamber may be fabricated from two or more different materials, so one has a cha..,ber fabricated from a composite material.
For example, in the walls of the chamber the base polymeric material can be present over a layer of another material, where the di~e. elll material may serve to modify the 1 S physical properties of the substrate. Where desired, the second material present in the composite substrate may be a heat di~sir~ting material which serves to absorb heat produced in the electrophoretic me~ m during electrophoresis. Materials that provide for heat absorption and dissipation and may be present in a composite substrate include glasses, ceramics, metals and the like. Specific heat absorbent materials of interest, 20 depending on the nature of the microchannel, include all-mimlm, copper, glass and the like. Where the base polymeric material is a composite, the layer of rigid polymeric material will be sufficiently thick so that, taken by itself, it can serve as a mechanical support and col~ nll.~ ,l means for the me~ium contained by it. The thickness ofthe base polymeric substrate will necessarily depend on the structural configuration of the 25 final device comprising the compositions, e.g, whether the device is a slab gel holder, capillary, microch~nnPl~ etc., as described in greater detail below, as well as the bulk properties of the base material, such as its tensile strength, brittlÁness~ flexural strength, and the like. Generally, the thickness ofthe substrate will be at least about 0.25 mm, more usually at least about 0.5 mm and will generally not exceed about 10 mm, and will 30 usually not exceed about 5 mm.
Polymeric materials suitable for use as the base material in at least the region of surface modification will be moldable and extrudable into a rigid objects that are CA 0226610~ 1999-03-17 2~;30 PCT/US97/17003 electrically non-conductive, have high resistivity to electric fields and are stable in the pl esence of a variety of electrophoretic media under electrophoretic conditions, incllltling aqueous solutions co~ ,l ishlg high salt concentrations and having pH ranges from 2 to 12. The polymeric material may comprise one or more di~elell~ polymers, 5 but will usually comprise no more than four di~rel enl polymers, more usually no more than two di~renl polymers. The polymers may be homo- or copolymeric, and be uncrosslinked or crosslinked. Polymers finding use will be synthetic, usually organic and may be addition or condton.c~tion polymers. Polymeric materials from which electrophoretic chambers have been fabricated and are amenable to surface 10 modification by the subject invention include: acrylics, e.g, polymethylmeth~crylate, polycarbonate; polyethylene terepthalate; polystyrene; polyethylene; polypropylene;
polyvinyl chloride; polyfluorocarbon; polybutylene terepthalate; polyvinyl alcohol;
polyetherether ketone; polyamides or nylons; phenyl silicones; polyurethanes;
acrylonitrile-styrene copolymers, copolymers of ethylmethacrylate and 15 methylmeth~crylate, and blends of polymethylmeth~rylate and polyethylmeth~.rylate, and the like.
Depending on the particular device, as well as the detection method and system in which it is employed, it may be desirable for the polymeric material to be optically transparent, where optically transparent means that the material allows light of20 wavelengths ranging from 180 to 1500 nm, usually from 220 to 800 nm, more usually from 250 to 800 nrn, to have low tr~n.~mi~ion losses. Such light tran.cmi~ive polymeric materials will be characterized by low crystallinity and include polycarbonate, polyethylene terepthalate, polystyrene, polymethylpentene, fluorocarbon copolymers, and the like, as well as the acrylic polymeric materials described in co-pending U. S.
25 Patent Applications Serial Nos. 08/627,484, filed April 4, 1996, and 08/878,437, filed June 18, 1997, the disclosures of which are hereby incorporated herein by I e~e~ ce, with polyacrylates, particularly polymethacrylates, and more particularly polymethylmethacrylate (PMMA) being prc~lled materials.
In some embo~limPntc, in the region of surface modification, interpenetrating 30 the internal surface of the electrophoretic chambers will be an anchoring polymeric layer. By intel ~e~ g is meant that the anchoring polymeric layer interdiffuses beneath the surface of the solid polymeric material. The interdiffused anchoring CA 02266l0~ l999-03-l7 WO 98/12530 . PCT/US97/17003 polymeric layer comprises linear polymeric strands, that may be either homopolymeric or copolymeric, exten~inE throughout the region of the base material adj~Á~nt to the surface. In other words, in the interdiffused region there are linear polymeric strands intertwined throughout the base polymeric network, with substantially no grafting of 5 the anchoring polymeric strands to the base polymeric strands. Since the method used to prepare the subject chambers requires a specific relationship between the base material and the monomer from which the anchoring polymeric layer is prepal ed (described in greater detail below), depending on the particular nature of the solid base polymeric material, the anchoring polymer may be polymerized from one or more of a 10 variety of dirrerel" monomers, where the monomers will generally be addition polymerizable ethylene cont~ininE monomers, usually vinylic, acrylic or pyrrolic, where the term acrylic inÁhldes methacrylic, where the acrylic monomers may be esters or amides. Specific anchoring polymers of interest are those polymerized from N-vinyl pyrrolidone, hydroxyethylmeth~r.rylate, dimethyl acrylamide, hydroxymethylacrylamide, 15 ethylene glycol ~limeth~r.rylate, glycerol methacrylate, glycidyl methacrylate, and the like, where polymers polymerized from dimethylacrylamide, N-vinyl pyrrolidone, hydroxymethylacrylamide and the like are pl ~rel able when the base polymeric material is polymethylmeth~Árylate. The dict~nre to which the interdiffi1sed portion of the base polymeric material extends beneath the surface of the base material will be a ~lict~nce 20 sufficient so that, when copolymerized with the surface electrophoretic layer, the inter~iffilsed strands of the anchoring polymeric layer stably secure the surface electrophoretic layer to the base polymeric material surface. The interdiffllced region of the base polymeric material will range in thickness to as much as about 1500 A, usually at least about 15 A; usually the interdiffused region is no thicker than about 700 A, 25 more usually no thicker than about 500 A. There will be no sharp delllalcalion at the border defininE the extent of the interdiffused anchoring polymeric layer.
The electrophoretic layer stably secured to the surface of the base material in the region of surface modification can serve to impart a number of di~l enl properties to the surface, inr.ludinE Áh~nEinE the inherent surface charge of the chamber, providing 30 for reactive functional groups, providing for an electrophoretic medium that substantially fills the inner volume of the chal"bel, and the like. Although theelectrophoretic layer may be polymerized from a variety of di~l e~.L monomeric CA 0226610~ 1999-03-17 compounds depending on the purpose of the layer, it will be polymerized from addition polymerizable monomers capable of copolymerization with the interpenetrated monomers of the anchoring layer.
By ch~n~inE the surface charge characteristics of the base polymeric material, 5 the electrophoretic layer can serve a variety of purposes, incl~lrlinE enh~n~inE or reducing the occurrence of EOF in the chamber, providing for enhancement, reduction or selectivity in entity adsorption to the surface of the chamber, e~c. For example, an electrophoretic layer polymerized from applopliate monomers can be employed in order to mask or cover any surface charge inherent in the solid polyrneric base material 10 under conditions of electrophoresis. By m~Cl~inE the surface charge, one can substantially reduce or elimin~te the occurrence of EOF during electrophoresis.
Electrophoretic layers which are suitable for at least reducing if not substantially elimin~tine the occurrence of EOF include those hydrophilic polymers having uncharged side groups, where the side groups may be amides, esters, pyrroles, 15 hydroxides and the like. Specific electrophoretic layers providing for reduced EOF
include: polyacrylamide and polymethacrylamide, polyhydro~yeLhyl.-leth~rylate, polyvinylpyrrolidone, polyhydroxymethylacrylamide and the like. One could also Ánh~nÁe the m~Enitude of the EOF, or reverse the direction of the EOF, by having an electrophoretic layer polymerized from monomers comprising charged groups of the20 same charge as the surface charge of the chamber. By selection of an electrophoretic layer polymerized from monomers having approp.iately charged groups, one can also provide for a reversal in the direction of EOF through the chamber. Charged groups of interest that may be present in the electrophoretic polymeric layer include carboxylic, sulfonic, phosphoryl, amine, and the like, where specific electrophoretic layers finding 25 use in the Pnh~nc.~m~nt or reversal of EOF include carboxylic, sulfonic, amine, and the like.
Instead of, or in addition to, Áh~nEinE the EOF in the chamber during electrophoresis, the electrophoretic layer can provide for a reduction, in~ tlinE a substantial ~limin~tion, of the adsorption of biomolecules to the surface of the chamber.
30 To accomplish a reduction in the adsorption of biomolecules to the chamber surface, the electrophoretic layer can be provided that comprises hydrophilic groups having no net electrical charge, where such groups include both neutral groups such as those CA 0226610~ 1999-03-17 Wo 98/12~30 PCT/US97/17003 described above, e.g, polyacrylamide, copolymers of polyethyleneglycol acrylates of molecular weight lower than 1000 dal, and polymers comprising zwitterionic groups, such as alanyl, betaine, sulfobetaine and choline derivatives, and the like. Alternatively, one could Ánh~nce the adsorption of entities to the surface of the charnber, as well as 5 provide for the selective adsorption of a particular class of entities, through the presence of the electrophoretic layer. Specific electrophoretic layers for reducing surface adsorption include polyacrylamide while specific electrophoretic layers for enh~n~ing surface adsorption include polyacrylamide-2-methylp~ opane sulfonic acid.
The electrophoretic layer may also provide for the presence of a variety of 10 reactive functional groups on the surface of the chamber in the region of modification, such as hydroxy, amino, epoxy, carboxy, amide, isocyanate, aldehyde, sulfonic and the like. By appropriate choice of monomer or monomers, the electrophoretic layer can provide for a single type of functional group or a plurality of di~el el" functional groups in the region of the surface modification. The presence of reactive functional groups on 15 the surface of the chamber can be useful where it is desired to covalently bond agents to the surface, e.g., enzymes, proteins, antibodies, dies, pH modifiers, complexing agents, e~c. For exŗmple, for the covalent att~hment of proteins comprising primary or secondary amino groups, an electrophoretic layer comprising epoxide and aldehydegroups will be of interest. Specific electrophoretic polymeric materials of interest 20 comprising reaction functional groups include: copolymers of glycidyl methacrylate and acrolein and the like.
The electrophoretic layer can also serve as an electrophoretic medium through which entities are moved in electrophoretic applications, where the electrophoretic layer is capable of providing for electrophoretic sieving as the entities move through the 25 mer~ m under the influence of the applied electric field. In this embodiment of the subject invention, the electrophoretic layer will substantially fill the entire inner volume of the electrophoretic chamber, at least in the volume bound by the region of surface modification, where the layer may comprise crosslinked and/or non-crosslinked polymers. Polymeric gel media suitable for use in electrophoresis are disclosed in 30 Barron & Blanch, Separation & Purification Methods, (1995) 24:1-1 18. Specific electrophoretic layers capable of serving as electrophoretic layers are those polymerized from addition polymerizable ethylene-cont~ining monomers, usually CA 0226610~ 1999-03-17 vinylic, acrylic or pyrrolic, with polyacrylamides being preferred. Also of interest in certain applications, e.g., prel)al~Li~re applications, are gels comprising reactive groups, such as arnino groups, sulfonic groups, and the like.
The subject electrophoretic chambers may be forrned in any of a variety of S di~relll configurations. Chambers having walls capable of being modified according to the subject invention include slab gel chambers, tubes, columns, as well as microchannel ~.h~mhP.rs, such as capillaries and ~lt;nches on the surface of planar polymeric substrate.
As tliccussed above, the entire inner surface of the chamber may be modified to comprise the electrophoretic layer, or only a region of the inner surface may be so 10 modified. In addition, the challlbel ~ may comprise one or more regions of surface modification, where when a plurality of regions of surface modification are provided, one has the opportunity to have a plurality of di~l ~ electrophoretic layers on the surface of the chamber, which increases the variety of different applications in which the chambers may be used. For example, a chamber could be prepared having a first 15 region in which the electrophoretic layer is a gel conl~h~inp an ionically charged group, e.g., carboxy, sulfonic, amino, etc., that provides for ion exchange. Dowll~llealll from the first region could be a second region colll~lishlg an enzyme that converts a sample conll)o~ L to a desired product. Downstream from the second region could then be a third region modified to comprise an electrophoretic sieving me(lil~m, e.g, cross linked 20 polyacrylarnide, in which the enzyme product is separated from the l e~ g sample components.
In some embodiments of particular interest the electrophoretic chamber is a microchannel. The microch~nn~lc may be open or closed, where by "open" is meant that the internal volume of the microchannel is not completely separated on at least one 25 lonEit-l~in~l side from the external environment, while by "closed" is meant that the internal volume of the channel is completely separated longit~lrlin~lly from the external en~hol~llel1t. Examples of open micro~h~nn~lc include troughs, trenches and the like, present on the surface of a planar substrate. Closed Áh~nnPlc are exemplified bycylinders, tubes, capillaries and the like; and by troughs, trenches and the like formed 30 on the surface of a planar substrate and enclosed by a suitable cover The subject micror.h~nn~ls will have micro scale cross-sectional inner dimensions, such that the inner cross-sectional dimensions of the micro~h~nnPlc will be greater than I llm and CA 0226610~ 1999-03-17 less than 1000 llm. Generally, the cross-sectional inner ~lim~n~iQn(s) of the microch~nn~l, i.e. width, depth or diameter depending on the particular nature of the ch~nn~l, will generally range from about I to 200 ~lm, usually from about 10 to 150 llm, more usually from about 20 to 100 ~,lm, with the total inner cross sectional area of the microchannel providing for capillary flow through the ch~nn~l, and ranging from about 100 to 40000 ~lm2, usually from about 400 to 25,000 ,uml. The inner cross-sectional shape of the microchannel may vary among a number of dirrelelll configurations, inr.l~lrling rect~n~ r, square, rhombic, triangular or V-shaped, circular, semicircular, ellipsoid and the like. The length of the microchannel will necess~, ily depend on the specific nature of the vessel as well as the electrophoretic device in which it is to be employed. For example, where the microchannel is a trough or trench in a substrate, the length of the microchannel may range from about 0.1 to 100 cm, and will generally range from about 1 to 20 cm, usually from about 1 to 10 cm, and more usually from about 5 to 10 cm, while for capillaries the length will generally range from about 10 to 100 cm, usually from about 10 to 75 cm, more usually from about 20 to 50 cm. Where the subject microvessel is contained within a capillary, the thickness of the wall ofthe capillary may range from about 50 to 1000 ~,lm, usually from about 100 to ~ 500 llm, more usually from about 100 to 150 ~lm, to provide a capillary with an outer di~meter ranging from about 100 to 2000 llm, usually from about 150 to 400 ~,lm.Where the microchannel is a trench or trough e~ct~nlling downward from the surface of a substrate, conveniently a groove in the substrate, the substrate may be square, rect~n~ r, circular and the like, and will have dimensions which will vary considerably depending on the intended use of the microch~nn~l Where the substrate has card-like or substantially regular parallelepiped dimensions, the length of the substrate will typically range from about 2 to 200 mm, the width of the substrate will typically range from about 2 to 200 mm, while the thickness of the substrate will typically range from about 0.1 to 10 mm. One or more, usually at least 2 and up to 100 or more, microrh~nn~l~ may be present on or at the surface of the substrate, where when a plurality of microch~nnel~ are present at the substrate surface, the possibility exists to have a number of di~ elll electrophoretic applications running at the same time on a single substrate. The micro~.h~nnel(s) present in the substrate surface can be linear, branched or in some other convenient configuration. With branched CA 0226610~ 1999-03-17 micronh~nn~l.c or trenches, the possibility exists to have a first trench or channel e.~e~i~ed by one or more side ch~nn~lc, where the side ch~nn~lc may intersect the main channel at any convenient angle. In branched configurations, the chamber will comprise a main microchannel in intersecting relationship with at least one secondary 5 microrh~nnel, where at least one pair of electrodes will be associated with each microÁh~nnel, with one member of the pair being positioned at either of the termini of the Áh~nnel, in order to apply an electric field to the me~ m in the microch~nn~l See, e.g., U.S. Patent No. 5,126,022, and U.S. Patent Applications Nos [08/###,###], filed July 30, 1997 [A-62855-1/RFT/BKl, the disclosure of which is hereby incorporatedl 0 herein by reference.
As the microch~nn~l(s) present on the substrate surface may be open, it may be desirable to separate the internal volume of the Áh~nnel, and thereby the mÁdil~m housed in the channel, from the external environment. In such inct~nce~ a cover plate can be employed which rests on the surface of the substrate and thereby separates the 15 internal volume of the channel from the envh onlllelll. The cover plate may be fabricated from any of a number of dirrelenL materials, inclllding fused silica, acrylic polymeric materials, and the like. Where necessary and desirable, one or more of the cover plate surfaces may be treated to control (that is, to reduce or augm~nt or the change the direction of) any EOF that may arise during electrophoresis. A number of di~,e.lL
20 methods are known which reduce or elimin~te EOF. Alternatively, where the cover plate is a rigid polymeric material, the method of the sub~ect invention can be employed to applopliately modify the surface. As with the substrate, the coverplate may be fabricated from a single type of material or be a composite of one or more, usually two, materials. See, e.g, U S. Patent Application Serial No. 08/878,437, filed June 18, 1997, 25 the disclosure of which is hereby incorporated herein by rerelellce.
Usually, either the cover plate or the substrate will be provided with one or more apertures or wells for introduction of sample or solvents or buffers and the like into the microchannel structure. The walls of such apertures or wells can if desired be treated according to the invention to alter the mobility of fluids or entities in the fluids 30 into or out from the aperture or well, or to anchor particular l~agellL~ on the walls of such structures.
CA 0226610~ 1999-03-17 Wo 98/12530 PCT/US97/17003 The thickness ofthe cover plate will usually range from about 0.01 to 10 mm, more usually from about 0.1 to 1.0 mm, where the length and width of the cover plate may be similar to, or di~. ~l-L from, the length and width of the substrate, but will usually be substantially the same as those of the substrate. The cover plate may have 5 substantially smooth, planar, flat surfaces, or optionally may be a mirror image of the substrate. Although not necessary, the cover plate will generally be sealed to the substrate. The cover plate and substrate may be sealed using any convenient means, such as ultrasonic welding, pressure, thermoprocessing, adhesives, se~nts7 physical conformance and the like. See, e.g, U.S. Patent Application Serial No. 08/878,437, I 0 supra.
The electrophoretic chal.lbel ~ can be used in any of a variety of electrophoretic devices. Numerous electrophoretic devices are known in the art, and include devices which require manual operation as well as automated devices requiring a minimal amount of operator interaction. The electrophoretic chambers of any of these devices 15 can be substituted with the subject electrophoretic chambers of analogous configuration.
In prepalillg the subject electrophoretic chambers, the base polymeric layer is contacted sequentially with first and second monomer compositions which are thensubsequently copolymerized to produce the region of surface modification.
20 Copolymerization with the first and second monomers will be through addition polymerization, with the first and second monomers cont~ining polymerizable ethylene groups, usually vinylic monomers, where at least the second monomer will be different from the monomer(s) from which the base polymeric material is polymerized, where the first and second monomers may be the same or di~renL and are usually di~erenL, so 25 that at least the electrophoretic layer differs from the rigid polymeric base material. In those select situations where the first and second monomers are the same, the two step process of the subject invention allows in the first step for deep interpenetration of the monomer in a solvent selected to promote interpenetration by effective swelling of the polymer substrate. In the second step a second solvent, suitable for polymerization, 30 carries the monomer and other polymerization components to form the electrophoretic - layer effectively anchored to the solid polymeric substrate surface. As the first and second monomers are copolymerized and are usually dirre.enl, the kinetic CA 0226610~ 1999-03-17 WO 98/12530 rCT/US97/17003 copolymerization relationship between the first and second monomers will lie between ideal and altell,aling, i.e. 0 < r,r2 <1, where the relationship will be closer to ideal, with rlr2 usually being from 0 to 2, more usually between 0 and 1.
The first step is to contact the region to be modified with a first monomer 5 capable of interpenetrating the surface. In interpenetrating the surface of the base polymeric material, the first monomer swells the surface of the base polymeric material and incorporates or becomes embedded beneath the surface of the material, where it positions itself among the polyrneric strands of the base material. The distance to which the first monomer penetrates below the surface of the base material will be at least 10 about l5A, in some embodiments at least about 30A and may be as great as about 1500A or greater, but will usually not exceed about 500A. Although the first monomer swells the surface of the polymeric material through interpenetration, because the inter-liffilsed region extends only to at most a few nanometers below the surface of the layer, the bulk properties of the material, such as water solubility or rigidity, will not be 15 changed as a result of interpenetration.
Because the first monomer must be capable of penetrating the surface of the base polymeric material, the first monomer employed will be chosen in view of several difrele..L considerations, including: (a) the particular chemical structure and physical morphology of the polymeric base material; (b) the similarity in the solubility 20 parameters between the first monomer and the base polymeric material; (c) the nature of the electrophoretic layer with which it is to be copolymerized; and the like. Usually, small molecular systems are employed, the monomers inclutling ethylene polymerizable groups, and p~rel~bly cont~ining O or N, where the O or N may in some embodiments be part of a cyclic structure, and where the N can be mono- or di-substituted. For 25 acrylic polymeric base materials, e.g., polymethacrylates, first monomers that find use include addition polymerizable ethylene cont~ining monomers, usually vinylic, acrylic or pyrrolic monol"e, ~. Substituent~, when present on the N, will generally be lower alkyls, usually C4 or lower, more usually C2 or lower, particularly methyl, with acrylic and pyrrolic monomers being of interest, with specific monomers of interest being 30 dimethylacrylamide, N-vinyl pyrrolidone, methyl methacrylate and the like.
~ n cons~cting the first monomer with the base polymeric material, the first monomer may be present as a pure liquid or in a solvent, where the solvent pr~re,~bly CA 0226610~ 1999-03-17 WO 98/12530 PCT/US97tl7003 promotes the swelling of the surface of the base material and the intel l,elle~ ion of the first monomer. Thus, the solvent will also generally have a similar solubility parameter to that of the base material. For acrylic base materials, solvents of interest include lower alkanols, such as methanol, isoplopanol and the like. Where the first monomer is5 present in a solvent, the first monomer will typically be present in an amount ranging ~ from about 1 to 100 % by volume, usually from about 3 to 75 % by volume, and more usually from about 3 to 50 % by volume. Contact may be accomplished under dynamic or static conditions, as is convenient. Under dynarnic conditions, the first monomer or solution thereof will be moved through the chamber at a flow rate that ranges from 10 about 10 ~ll/min to 5 ml/min, more usually from about 25 ~,lUmin to 3 ml/min.The pal ~nl~lers of the cont~cting step will be selected to achieve the desired level of surface swelling and interpenetration of the first monomer without comprising the bulk mech~ni~l properties of the base polymeric material. Parameters that will be chosen accordingly include duration of contact, nature of solvent, concentration of 15 monomer in solvent, temperature and the like. Contact will generally be m~int~ined for a period oftime ranging from about .25 to 4 hr, usually from about .5 to 2 hr, and more usually from about .5 to I hr. After sufflcient time has elapsed for the first monomer to interpenetrate the polymeric surface, excess first monomer will be removed from the surface. The excess first monomer may be removed using any convenient means, such 20 as wiping, washing, flushing nitrogen or air under pressure and the like.
The next step in the subject method is to contact the intel~,ell~llaled or interdiffused surface of the base material with a second monomer composition. The second monomer will be copolymerizerable with the first monomer through additionpolymerization, and will usually be vinylic. The vinylic second monomer will comprise a 25 moiety which imparts the particular surface modification characteristics to the electrophoretic layer into which it is polymerized. For electrophoretic layers that modify the inherent surface charge of the base material under conditions of electrophoresis, the second monomer will be hydrophilic and can comprise neutral or charged groups, depending on the purpose of the electrophoretic layer. Where the30 electrophoretic layer is to reduce or elimin~te the occurrence of EOF through the chamber during electrophoresis, second monomers that find use include those monomers having neutral hydrophilic groups, such as carbonyls, in~ ing acrylic and .. . .
CA 0226610~ 1999-03-17 pyrrolic monomers, where acrylic monomers may be esters or amides. Specific second monomers of interest for use in the reduction of EOF inc}ude: acrylamide, hydroxyethylmeth~rrylate, vinyl pyrrolidone, end-capped polyethylene glycol acrylates of molecular weight lower than 1000, and zwitterionic monomers such as the betaine derivatives, and the like. For ~nh~ncing the m~gnitude of, or reversing the direction of, EOF through the chamber, the second monomer can be a monomer comprising a charged group, where the charged group can be negative or positive, where negatively charged groups include carboxylic groups, sulfonic groups, phosphoryl groups, and the like, as found in monomers such as vinylic acids, e.g, acrylic acid, methacrylic acid, and the like, while positively charged groups include amino, and the like, as found in 2-(dimethylamino)ethyl acrylate, 2-(diethylamino)ethyl ethacrylate and the like.
Where the electrophoretic layer is introduced to reduce the adsorption of entities to the surface of the chamber, second monomers of interest include: the neutral group comprising hydrophilic monomers listed above, e.g, acrylamide, hydroxyethylmethacrylate, dimethylacrylamide, vinyl pyrrolidone, low molecular weight (less than 1000 dal) polyethylene glycol acrylates, and the like; zwitterionic groups having an overall net charge of zero, such as N-(3-sulfopropyl)-N-methacryloxyethyl-N,N-dimethyl al"ll,onium betaine, and the like; as well as polyethylene glycol acrylates of low molecular weight, and the like.
For electrophoretic layers comprising reactive functional groups, the second monomer will comprise a moiety which is the functional group. For the immobilization of an affinity agent to the surface of the chamber, reactive functional groups that provide covalent bonding to the affinity agent are of interest. Various techniques employing a variety of di~l elll functional reactive groups for the immobilization of affinity agents to the surface of polymeric substrates are known. See, e.g., Trevan, "Irnmobilized Enzymes, An Introduction and Applications in Biotechnology," (J. Wiley & Sons, 1980), Taylor & Dekker, eds., Protein Immobilization, Fundamentals and Applications (1991); and Walsh & Headon, "Protein Biotechnology" (J. Wiley & Sons 1994), the pertinent disclosures of which are herein incorporated by reference. Reactive 30 functional groups of interest which can either react directly with an affinity agent or be - treated to provide for groups capable of directly reacting with afflnity agents include CA 0226610~ 1999-03-17 WO 98/12530 PC'r/US97/17003 hydroxy, arnino, epoxy, carboxy, amide, isocyanate, aldehyde and the like. Specific second monomers of interest include glycidyl methacrylate, acrolein and the like.
Where the surface electrophoretic layer is to substantially fill the inner volume of the chamber to provide for an electrophoretic merli11m where the met?illm may5 comprise linear or crosslinked polymeric networks, second monomers of interest~ include acrylarnide, dimethylacrylamide, other monosubstituted and disubstituted acrylamides, and the like.
The second monomer will be present in a solution, where any of a variety of solvent systems may be employed, inc1l1tling co-solvent systems. Solvent systems of 10 interest include pure water and water/lower alkanol mixtures, where the lower alkanol will typically be a C4 or lower alkanol, such as ethanol, propanol, isopropyl alcohol and the like. Instead of, or in addition to, a lower alkanol, other polar organic solvents may be employed as co-solvents, such as dimethylformamide, dimethylsulfoxide and the like.
The volume percent of the water in the solvent system will range from 10 to 100 %.
15 The volume percent of the co-solvent in the system, when present, will not exceed 90 %, and will usually not exceed 50 %. Non-aqueous solvent systems may also be employed, where the non-aqueous solvents may be selected from any convenient ~ organic solvent, such as those listed above. The volume percent of second monomer in the solvent will generally range from about 3 to 20 %, usually from about 3 to 12 %
20 and more usually from about 3 to 8 %.
In addition, the second monomer solution may further comprise various agents necessary and/or desirable for the polymerization, where such agents include those agents useful in physical and chemical initiation. Chemical initiators include:
persulphate + 3-dimethylaminopropionitrile (DMPAN), persulphate +
25 tetramethylethylene~i~mine (TEMED), persulphate, persulphate + thiosulfate, persulphate + bisulfite, persulphate + diethylmethyl~minerli~mine (DEMED), H2O2 +
Fe2+, benzoyl peroxide, lauroyl peroxide, tetralin peroxide, actyl peroxide, caproyl peroxide, t-butyl hydlop~-oxide, t-butyl ~I.en7oate, t-butyl diperphth~l~te, cumene hydroperoxide, 2-butanone peroxide, azoinitiators, e.g, azodiisobutyronitrile and 30 azodicarbonamide, riboflavin, methylene blue + a redox couple, and the like. Preferably a chemical polymerization initiator such as persulphate will be employed.
CA 0226610~ 1999-03-17 wo 98/12530 PCT/US97/17003 For the pl epa-~ion of cross-linked electrophoretic layers, the second monomer composition may further comprise various cross linking agents, which will be selected depending on the nature of the second monomer. For example, with acrylic monomers, e.g., acrylamide, cross linking agents of interest include: N,N'-ethylene bisacrylamide 5 (Bis); ethylene diacylate (EDIA); N,N'-diallyltartardiamide (DATD); N,N'-bis acrylyl cystamine (BAC); N,N'-(1,2-dihydroxyethylene)bisacrylamide (D~BA); and the like.Other agents of interest that may be present for a variety of reasons in the composition, e.g, to provide for suitable electrophoretic sieving through the layer polymerized from the composition, include various salts, particularly buffering salts, where the 10 concentration of the buffering salts will vary from 0.01 to 0. 5 M, more usually from 0.01 to 0.1 M. The salts may include Tris, phosphate, EDTA, MOPS, and the like.
Denaturing agents may also be present in the aqueous phase, incl~l~ing urea, SDS, foll--~ ide, methylmercuric hydroxide, alkali, and the like, where the concentration will vary depending on the particular denaturing agent, e.g, for urea, the concentration will 15 range from about 0.1 to 9.0 M.
Following contact of the second monomer composition with the interpenetrated surface, the first and second monomers will be copolymerized. Depending on the particular second monomer composition, polymerization may already have been initiated upon p-epdlalion ofthe second monomer composition, e.g, where a chemical 20 initiator such as pers--lph~te is employed. Where polymerization is not initi~ted upon pre~a.~lion of the second monomer composition, pol~ e~ lion may then be initiated once contact is made using any convenient means, inchl~insg heat, electron beam,photopolymerization, gamma radiation, microwave radiation, and the like. However, the particular pol~ e-i~lion technique employed will be chosen so that little or no 25 grafting of the base polymeric material occurs during copolymerization of the first and second monomers.
Upon initiation of the polymerization in the second monomer composition, as the polymer chain extends through the second monomer composition, those second monomers near the interpenetrated surface react with the first monomers embedded in 30 the material near the surface, which then react with first monomers further below the surface, whereby the growing polymer chain continues to extend below the surface CA 0226610~ 1999-03-17 through the interdiffused region of the polymer, adding embedded first monomers to the growing chain.
Contact of the second monomer with the surface may be either static or dynamic, depending on the desired properties of the electrophoretic layer. For example, static conditions will be employed to obtain a thick electrophoretic layer, such as those ~ electrophoretic layers that are to serve as a gel medium Alternatively, dynamic conditions can be employed to achieve a thinner electrophoretic layer having a more uniform surface comprising lower molecular weight networks, which may be desirable for those applications where the electrophoretic layer is to alter the inherent surface charge of the material or to provide for the introduction of certain functional groups on the surface of the material in the region of modification. Where dynamic conditions are employed for contact, the flow rate of the second monomer composition through the chamber will typically range from 10 Ill/min to 5 ml/min, usually from about 25 IlL/min to 3 ml/min, and more usually from about 50 !ll/min to 3 ml/min.
Polymerization will be allowed to continue for sufficient time for an electrophoretic layer of desired properties to be produced, and will generally be allowed to proceed to completion. Although the exact time will vary depending on the particular nature of the system employed, usually poly",t~ alion will proceed from about .25 to 4 hr, usually from about .5 to 2 hr, and more usually from about .5 to 1 hr.
Following polymerization, the surface modified chamber may be further treated as necessary, depending on the electrophoretic application in which it is to be employed. For example, where the electrophoretic surface is a gel medium, the fluid phase of the gel medium may be replaced with a running buffer. Alternatively, where the electrophoretic layer comprises functional groups for covalent att~Áhment of affinity agents such as ligand or receptors, the modified surface may be contacted with such affinity agents, and then washed to remove any unbound agents.
In other embodiments of the invention, an electrophoretic polymeric layer is coated on the surface of the base material without the aid of an anchoring polymeric layer. In these embodimentc~ the polymer comprising the polymeric coating layer is first dissolved in a suitable solvent, and then contacted with the surface of the base material.
During this cont~cting step, dissolved polymer is selectively adsorbed or absorbed at the surface of the base material, and forms a surface coating. The composition of the CA 0226610~ 1999-03-17 polymer co",l lising the coating layer may be selected such that it has a solubility parameter similar to that of the base material, and such that it interpenetrates the surface of the base material. Such intel l,eneL. ~Lion will be promoted if the solvent carrying the polymer also has a solubility parameter similar to that of the base material, 5 and promotes the swelling of the base material. Because the molecular weight of the polymer will be much larger than that of the monomers used in forming anchoring layers in the embodiment described earlier in this application, the interpenetration of the surface layer will not be as significant as in this earlier embodiment. However, sufficient interpenetration can be achieved to provide a stable electrophoretic polymeric layer.
Alternatively, the composition ofthe polyrner comprising the coating layer may be selected such that it does not interpenetrate the surface of the base material, but instead forms molecular interactions with the surface of the base material. These interactions may be ionic, hydrogen bonding, or dispersive. Because each polymermolecule in the coating layer can interact with the surface at multiple locations, the 15 additive effect of even weak interactions between repeating units on the polymer and moieties at the surface of the base material will often be sufficient to hold the electrophoretic polymer layer at the surface. In some cases, the electrophoreticpolymeric layer formed in this manner may slowly dissolve into solution during electrophoretic analysis. However, the surface properties will remain relatively stable 20 over several analyses before such slow dissolution alters these properties significantly.
Cationic and anionic polymers are suitable for deposition onto the inner walls of plastic microch~nnel~ to control electroendoosmotic effects. Examples of anionicionomers and polyelectrolytes suitable for this type of treatment are: Nafion~) (perfluorinated ionomeric membrane with perfluorinated propylene grafted chains with 25 an ending sulfonic group), polystyrenesulfonic acid and corresponding salts, poly(vinylsulfonic acid) and its salts, poly(styrenesulfonic acid-co-maleic acid), poly(acrylic acid), poly(methacrylic acid), poly(acrylic-co-methylmeth~crylate),poly(acrylamide-co-acrylic acid), poly(2-acrylamido-2-methyl-1-propanesulfonic acid) and poly(ethylene-co-acrylic acid). The presence of sulfonic groups is plt;~lled to 30 carboxylic groups for EOF effects independent of pH. A balance of carboxylic and sulfonic groups may be desirable to control flow with pH variations. Cationic polymers suitable for the build up of positive charges on the microÁh~nn~l~ surface are CA 0226610~ 1999-03-17 poly(diallyldimethylammonium chloride), poly(acrylamide-co-diallyldimethylammonium chloride), and polymer with amino groups in the backbone or as part of the side groups.
The subject electrophoretic chambers find use in a variety of electrophoretic applications, where by electrophoretic applications is meant that charged entities are moved through a me~linm housed in a chamber under the influence of an applied electric field, where movement of the entities may be the result of either an inherent electrical charge of the entities or bulk fluid flow through the chamber. Illustrative applications are reviewed in Andrews, Electrophoresis (1990); Barron & Blanch, Separation&PurificationMethods(1995)24:1-118 andU.S. PatentNo. 5,126,022, the disclosures of which are hereby incorporated herein by reference. Illustrative applications include methods based on sample component separation and identification, e.g, sequencinE~ sample component purification, synthesis applications, sample preparation and the like.
The following examples are offered by way of illustration and not by way of limitation.
Example 1. P~ )al~lion of Cross-linked Polyacrylarnide Gel Bound Between Two Polymethacrylate Plates and Its Use in the Electrophoretic Separation of a ~X174/HaeIII DNA Ladder Flat polymethylmethacrylate (PMMA) plates 160 x 180 mm were cut from an Acrylite OP4 acrylic sheet (Cyro Industries). The plates were stringently wiped with meth~nol to ensure that no particles or residues were left on the surface of the plates.
10 ml of 10 % dimethylacrylamide (DMA) in methanol were pipetted onto the surface of each plate. The DMA solution was gently spread using a lab paper tissue to cover the entire surface of the plates. The DMA solution was allowed to penetrate the surface ofthe plates for 20 mimlt~ After 20 minllte~, the DMA solution was wiped clean using a lab paper tissue and lightly flushed with nitrogen gas. The surface was then CA 0226610~ 1999-03-17 wiped with a lab paper tissue wetted with methanol, resulting in a clear and spotless surface.
21.5 g of T5C3 ( 1 g of acrylamide, 35 ~,lg of bisacrylamide and 20.5 g water) was combined with 1.0 g of 10 x TBE (0.89 M tris(hydroxymethyl)aminomethane, 0.89 M boric acid and 0.05 M ethylene~i~minetetraacetic acid) and 2.2 !11 of 25 mM
ethitlinm bromide solution. 150 ~ll of 10 % ammonium persulfate solution and 75 ~11 N,N,N,N-t~ e~}-ylenediamine (TEMED) were added to the above solution and mixed gently. The resultant solution was poured between the two treated PMMA
surfaces separated by 75 ~,lm spacers and allowed to polymerize for about two hours. A
well forming comb was introduced between the plates to form sample wells for electrophoresis .
The resultant cross-linked polyacrylamide gels had good adhesion to the PMMA plates as a result of the surface treatment. In contrast, cross-linked gels poured between two PMMA plates having untreated surfaces slid out of the plates upon removal from the casting stand.
Electrophoresis of q)X1 74/HaeIII DNA fragments was pe,~l".ed using standard procedures with a run voltage of 7.4 V/cm for 2.25 hours. The results were vi.cu~li7ed on a standard W im~gin~ system.
The non-denaturant separation of (i>X1 74/HaeIII DNA fr~gm~nts under these conditions resolved 8 bands of the 1 1 possible fr~gments resolvable under more ideal conditions. The experiment was designed to demonstrate the resolving capacity of the gel while being ~tt~h~d to the surface of the PMMA plates using the described method.
The results demonstrate that the surface interpenetration method according to the subject. invention can be used to stably secure an electrophoretic gel me~ m to the surface of a polymethylmethacrylate plate and that such a secured gel can be used succ~s~fi-lly in electrophoretic separation.
Example 2. Preparation of Pol~ elhyl~ rylate Capillaries Having an Inner Linear Polyacrylamide Coating The inner surface of a cleaned microbore polymethylmeth~crylate (PMMA) capillary is contacted with a 10 % solution of dimethylacrylamide (DMA) in mPth~nol CA 0226610~ 1999-03-17 for 20 minutes under static conditions. After 20 min~ltes, the excess DMA solution is rinsed from the internal capillary surface and a fresh solution of 3 % acrylamide in water with ammonium persulfate and N,N,N,N-t~ lneLllylÁne~ mine (TEMED) is introduced into the internal volume of the capillary. The 3 % acrylamide solution is ",~ L~ined in the capillary for two hours at room temperature under static conditions, allowing for copol~ l iGa~ion of the linear acrylamide with DMA monomers that penetrated the surface of the capillary wall. Since the copol~ aLion is carried out under static conditions, the linear polyacrylamide strands grow into a thick intertwining network that ess~nti~lly fills the entire volume of the capillary. The resultant linear acrylamide filled polymethylmeth~c~late capillary can be used in electrophoreticseparation applica~ions.
The above procedure is also carried out with the variation that the acrylamide solution is introduced into the capillary under dynamic conditions, with a flow rate of 3 ml/min. Introducing the acrylamide solution under dynamic conditions results in formation of a thin linear acrylamide layer covering the surface of the capillary.
Example 3 . Preparation of Polymethylmeth~srylate Capillaries Having an Inner Surface Comprising Epoxy Functional Groups.
A PMMA capillary having a DMA interpenetrated surface is contacted with a solution of glycidyl methacrylate comprising t-butyl peroxypyvalate (TBPP) underdynamic conditions, e.g, the glycidyl methacrylate is flowed through the chamber at a rate of 50 ~ll/min at 40 ~C. Copolymerization results in the presence of a thin, uniform surface polymeric layer co~ g epoxy groups. The epoxy groups can then be converted to other functional groups, as may be appropriate depending on the use of the functional group, e.g, to hydroxy groups through acid hydrolysis.
Example 4. Plt;par~lion of Polymethylmeth~c.rylate Capillaries Comprising Ion Co,~ ;"illg Cross-linked Gels ~. Capillaries Filled with Amine Cont~ining Gel Structures A polymethylme~h~srylate capillary is filled with a 20 % solution of methylmethacrylate in methanol under static conditions for 30 min. The capillary is then rinsed and a fresh 15 % solution of dimethylaminoethylacrylate in meth~nQI co~ g CA 0226610~ 1999-03-17 5 % ethylene glycol diacrylate and 1 % t-butyl peroxypyvalate (TBBP) is introduced into the capillary. Polymerization is allowed to proceed for 2 hr at 40 ~C. The resultant capillary is sub~LallLially filled with an amino cont~inin~ gel structure which finds use in anion exchange applications, e.g, ion removal in high ionic strength samples.
B. Capillaries Filled with Sulfonic Acid Cont~inin~ Gel Structures An MMA surface interpenetrated PMMA capillary as prepared in Example 4.A.
above is contacted with a 20 % solution of 2-acrylamido-2-methylpropanesulfonic acid comprising 5 % N,N'-ethylene bisacrylamide and 0.15 % of persulfate/bisulfate in water cont~inine 10 % sodium hydroxide (p~I adjusted to 7-8) under static conditions.
Polymerization is allowed to proceed for 1 hr at 55 ~C with careful control ofthe temperature of the bath. The resultant capillary comprises a sulfonic acid cont~inin~ gel structure which finds use in cation exchange applications, e.g. ion removal in high ionic strength samples.
15 Example 5. Alteration of EOF by Surface Tl e~L~I~ellL of Polymethylmethacrylate Capillaries with an Uncharged Polymer, Poly (DMA), and with a Negatively Charged Polymer, Poly(AMPS).
A. Surface Treatment Protocol 1. Poly (AMPS). A PMMA capillary (inner ~i~meter 75 ~lm, outer (li~meter 375 I,lm, 3 ft. total length) was flushed for 10 min with a solution of 50 %
(wt/wt) N N-dimethylacrylamide (DMA) in methanol at room temperature under nitrogen gas at 100 p.s.i. Next, the capillary was flushed for 10 min with nitrogen gas at 100 p.s.i. The capillary was then flushed with a freshly prepared solution of 20 %
(wt/wt) 2-acrylamido-2-methyl propanes.llfonic acid (AMPS) in water con~ e 0.5 % ammonium persulfate (APS) and 0.2 % sodium metabisulfite (Na2S205) at 40 ~C
under nitrogen gas at 100 p.s.i. The pressure chamber and capillary were both irnmersed in a water bath which was heated to 40 ~C. Flow through the capillary ceased as polymerization of AMPS formed a gel. Finally, the capillary was flushed for about 1 hour with water at room tel~ ure under nitrogen gas at 100 p.s.i.
2. Poly (DMA). A PMMA capillary as in Example 5.A. I . above was flushed for 10 min with a solution of 50 % (wt/wt) N,N-dimethylacrylamide (DMA) in methanol at room temperature under nitrogen gas at 100 p.s.i. Next, the CA 02266l0~ l999-03-l7 capillary was flushed for 10 min with nitrogen gas at 100 p.s.i. The capillary was then flushed with a freshly prepared solution of 20 % DMA in water (wt/wt) cont~ining0.06 % APS (v/v) and 0.13 % TEMED (v/v) at room te~ elal-lre under nitrogen at 100 p.s.i. The flow through the capillary ceased over the course of an hour as 5 polymerization of DMA formed a gel. Finally the capillary was flushed with water for about 1 hour under nitrogen at 100 p.s.i.
B. EOF Measurements Measurements of EOF were made with a Beckman PIACE using the neutral marker mesityl oxide. Electropherograms were measured in 40 mM TAPS, 25 mM
10 AMPD; pH 8.3 at a field of 230 V/cm (capillary Lt=26 cm, Ld=19 cm). Prior to EOF
measurements, each capillary was subjected to preelectrophoresis for 60 min at 230 V/cm in TAPS/AMPD buffer. Electropherograms were 30 min in duration for the Poly(AMPS)-treated capillary and 60 min in duration for the Poly(DMA)-treated capillary. Mesityl oxide (l llg/ml in TAPS/AMPD buffer) was injected for 4 sec under 15 hydrostatic pressure. EOF values were calculated from the peak migration times.
Fig. 1 shows the results for EOF measurements in PMMA capillaries that are either untreated and that are treated by the protocols described in Example 5.A. above, which are ~l~cigned to attach either of two polymeric coatings to the surface of the capillaries: an uncharged polymer, Poly(DMA); or a negatively charged polymer, 20 Poly(AMPS). Only one series of measurements is shown for each of the treated capillaries. EOF values are plotted vs. electrophoresis time, rather than vs. trial number, to emphasize the effect of the time of successive electropherograms on the change in the measured EOF value.
As appears in Fig. l, the capillary treated with Poly(AMPS) exhibited an initial25 EOF of about 20 x 10-5 cm2/Vs. The EOF decreased modestly, but steadily, during the successive 17 runs (30 min per run). A~er 9.5 hours of electrophoresis, the EOF had decreased by about 6 %.
The capillary treated with Poly(DMA) exhibited a much greater change over the same duration of EOF measurement. Initially, the EOF was very low, but it increased 30 dramatically (~ 260 %) over the first 10 runs (60 min per run) before reaching a plateau at about 13 x 10-5 cm2/Vs.
. . _ CA 02266l0~ l999-03-l7 W O 98/12530 PCT~US97tl7003 The change in the measured EOF values for both treated capillaries over the course of s~lcceccive electrophoretic measule~ sugeestc that the coatings are progressively removed as a result. These initial results suggest that the Poly(DMA) coating is removed much more quickly than the Poly(AMPS) coating. In the 5 Poly(DMA) treated capillaries, the pH 8.6 buffer employed during measurement of EOF may have caused hydrolysis of amide groups to carboxylic groups on the Poly(DMA) coating, adding negative charges at the surface and ~nh~n~ing the EOF
values as a result. If degradation were limited to progressive ~Lli~pi~lg away of the applied coating layer, then the EOF measurements would be expected to approach 10 those of the untreated polymer surface.
Example 6. Alteration of EOF by Surface Deposition of Nafion~ onto a Polymethylmethacrylate Electrofluidic Channel of 400 ~12 Cross-section.
Microchannel surfaces are modified as follows. Pre-treat the surface of the 15 microch~nn~lc by flushing isoplopanol for 2 minutes through the wells acc~ssing the rnicrochannels in the microfluidic device. Fill the microch~nn~lc with a 0.5 % solution of Nafion~ perfluorinated ion-exchange powder, diluted with isopropanol from a collllllel cially available solution of Nafion(~ in water-alcohol mixtures (Aldrich, Catalog No 27,470-4). Nafion(~ is a perfluorinated ion-exchange material, see Aldrichimica 20 Acta, 19(3):76 (1986); J. Electrochem. Soc., 140:2279 (1993). Careful filling is required to avoid bubble formation within the channels. Allow the solution to stabilize within the channel for 5-10 minntec Using a vacuum pump pull the solution from the sh~nnelc slowly. Total time for this process varies depending on the channel pattern on the microfluidic device. The process will extract the solvent from the sh~nnelc leaving 25 behind a layer of Nafion~ ionomer non-covalently bound to the acrylic surface of the ch~nnel, uniformly applied over the surface of the channel. This procedure provides surface modified ~.h~nn~lc with an EOF highly independent of pH and about 30 %
lower than those measured with standard uncoated fused silica capillaries of equivalent cross-section. Microchannels coated using this procedure are stable for about 2 hours 30 of analytical work.
CA 02266l0~ l999-03-l7 Example 7. Alteration of EOF by Surface Deposition of Poly(sodium 4-styrenesulfonate) (PSSS) on to a polymethylmethacrylate electrofluidic channel of 400 ~m2 cross-section Microchannel surfaces are modified as follows. Flush the microrh~nnÁlc with a 5 3.3 % solution of PSSS in water for up to 16 hours. Using a vacuum pump, pull the solution from the ch~nnel.c slowly. Continue the evacuation of the Áh~nnelc for about 30 minntes to completely dry and stabilize the layer of PSSS polymer bound to the acrylic surface. This procedure provides an EOF of the order of 5.8 10'4 cm2s lV-l measured at pH 8.6 of 50 mM TRIS buffer. The coatings are stable for about 15 runs during 2 10 hours of analysis.
Example 8. Alternative Process for EOF Modification by Surface Deposition of Poly(sodium 4-styrenesulfonate) (PSSS) on to a Polymethylmeth~rrylate Electrofluidic Channel of 400 ~lm2 Cross-l 5 section Microchannel surfaces are modified as follows. Pre-treat the surface of the microGh~nn~lc by flushing isopropanol for 2 minutes through the wells accessing the microch~nn~lc in the microfluidic device. Flush the microch~nn~lc with a 3. % solution of PSSS in water for up to 16 hours. Bubble formation should be avoided during the filling by sparging the solution with helium gas after the dissolution of the polymer in water. Using a vacuum pump, pull the solution from the r.h~nnelc slowly. Continue the evacuation of the channels for about 30 minutes to completely dry and stabilize the layer of PSSS polymer bound to the acrylic surface. This procedure provides an EOF
ofthe order of 5.8 10'4 cm2s lV I measured at pH 8.6 of 50 mM TRIS buffer. The coatings are stable for about 15 runs during 2 hours of analysis.
Example 9. Improved Process for Alteration of EOF by Surface Deposition of Nafion~ onto a Polymethylmeth~crylate Electrofluidic Channel of 400 ~lm2 Cross-section Microchannel surfaces are modified as follows. Fill the microch~nnÁlc with a 0.5 % solution of sodium equilibrated NafÓon~, diluted with isoplopallol. This solution is plepaled by titration with sodium hydroxide to pH 7 of a commercially available CA 0226610~ 1999-03-17 Wo 98/1~530 PCT/U$97/17003 solution of Nafion~) in water-alcohol mixtures (Aldrich, Cat. No. 27,470-4). Careful filling is required to avoid bubble formation within the ch~nnPlc. Equilibrate for 5 - 10 minlltes. Pump out the solution from the ch~nn~l~ slowly. Repeat the process in five more cycles of filling and evacuation of the channels with 0. 5 % solution of sodium 5 equilibrated Nafion~. Bake the coated ch~nn~l~ for 30 minutes at 104 ~C. This process provides surface modified Áh~nnÁl.c with an EOF highly independent of pH, and more stable than that observed when surface modifying as in Example 6.
It is evident from the above results and discussion that the subject invention 10 provides a powerful methodology for the tailoring of the surface p- c,pe, Lies of an electrophoretic chamber to best suit the needs of a particular application. With the subject invention, electrophoretic mediums stably secured to the surface of the chamber material can be fabricated. Furthermore, one can readily control the nature of the functional groups introduced on the surface, providing for a homogenous surface 15 where desired. With the subject methodology one can also provide for a tailored surfaces having a plurality of regions of di~erenl modification, which greatly increases the number and nature of applications in which the subject chambers can be employed.
All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or 20 patent application was specifically and individually indicated to be incorporated by reference.
The invention now being fully described, it will be apparent to one of ordinary slcill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.