Patent Application
20080017512
These and various other features and advantages of embodiments of the present invention will be apparent upon reading the following detailed description in conjunction with the accompanying drawings and the appended claims provided below, where:
The following definitions are provided for illustration and are not intended to limit the teaching of the invention:
An “analyte” refers to any organic or inorganic molecule. Non-limiting examples of analytes that can be detected include proteins, oligopeptides and peptides, derivatives and analogs, including proteins containing non-naturally occurring amino acids and amino acid analogs. Other examples include, but are not limited to: carbohydrates, polysaccharides, glycoproteins, whole cells, cellular constituents such as enriched organellar fractions, bacteria, viruses, metabolites, cofactors, nucleotides, polynucleotides, transition state analogs, inhibitors, drugs, nutrients, electrolytes, hormones, growth factors and other biomolecules as well as non-biomolecules, as well as fragments and combinations of all the forgoing. An analyte can be a specific molecule one is interested in studying or it can be used to detect another molecule.
“Adsorbed” refers to the process of accumulation of a gas, liquid, or solid on the surface of a solid, as in a film or a coating. Often, but not always, the adsorbed material is a polymer.
“Block” refers to a portion of a macromolecule, comprising many constitutional units, that has at least one feature which is not present in the adjacent portions.
“Block copolymer” refers a copolymer that is a block polymer. In a block copolymer, adjacent blocks are constitutionally different, i.e., each of these blocks comprises constitutional units derived from different characteristic species of monomer 10 or with different composition or sequence distribution of constitutional units.
“Capillary” refers to any small bore tube. Capillary usually refers to an interior channel and the wall or surface that defines it. There is no limit on the exterior configuration. A capillary could therefore be contained within a ‘chip’, slide, or other device containing one or more tubes. The term capillary may also refer to a capillary channel in a chip or chip-like configuration. Cross sections of a capillary are usually circular but can be any shape including but not limited to oblong, triangular, rectangular, square, polygonal, or irregular.
“Capture” refers to the adherence of an analyte to a substrate, via formation of a chemical bond, such as but not necessarily, a covalent bond, ionic bond, or complex, and the like.
“Coating” refers to any film or layer of material on a capillary wall. The composition and thickness of the film is not limited in any way.
“Capture moiety” refers to any of a wide variety of reactive chemical groups suitable for linking, covalently or otherwise, two molecules together. Suitable chemistries include, but are not limited to, photoreactive groups, chemical reactive groups, and thermoreactive groups.
“Copolymer” refers to a polymer derived from more than one species of monomer.
“Covalent bonding” refers to an intramolecular form of chemical bonding characterized by the sharing of one or more pairs of electrons between two components, producing a mutual attraction that holds the resultant molecule together.
“Graft copolymer” refers to a copolymer containing adjacent blocks that are constitutionally different. Each of these blocks comprises constitutional units derived from a different characteristic species of monomer or from species with different compositions or sequence distributions of constitutional units.
The current invention relates generally to methods, devices, systems, and the use of adsorbed and/or covalently attached coatings that contain analyte capture moieties. Upon application of these coatings to a glass or plastic surface they form a film that can mask the properties of the surface thereby introducing a new behavior to the surface. Embodiments of the inventive coatings comprise activatable or triggerable chemistries that can be used to capture molecules of interest such as biomolecules.
The use of adsorbed and/or covalently attached coatings of the invention alleviates the need for multi-step surface functionalization which is typical for the prior art preparation of glass substrates with covalently attached polymer layers (Doherty E.A.S., et. al., 2003). The prior art procedures often require a pretreatment of silica substrates with reactive silanes followed by the deposition of functional polymer. The latter polymers do not have the capability to be induced to capture the electrophoretically separated analytes. In contrast, coatings according to some embodiments of the present invention can be produced in one step by contacting a solution of a premade polymer with the substrate. Unlike other prior art describing coatings used in electrophoretic separations, the embodiments of the present invention provide triggerable analyte capture moieties.
This provides advantages, for example by allowing for polymers produced as coatings to be synthesized outside of the capillaries prior to deposition according to one embodiment of the present invention. This is in contrast to prior art techniques practiced in US2006/0029978A1 which result in uncertain compositions that are poorly reproduced. Polymers synthesized outside the capillaries according to embodiments of the present invention can be produced in large batches providing a sufficient amount of material for purification and characterization. Of further advantage, characterizing the polymers prior to coating as enabled by embodiments of the present invention promotes batch to batch reproducibility. This allows use of well characterized polymeric materials for coating, instead of a mixture of unidentified molecules produced inside the capillaries upon their covalent functionalization as required by prior art techniques.
In another aspect, the inventors have discovered that adsorbed coatings according to embodiments of the present invention have proven to be an adequate replacement for covalently modified glass capillaries in terms of electroosmotic flow (EOF) suppression. Further, adsorbed coatings of embodiments of the present invention provide the separation characteristics which meet the highest standards of modern capillary electrophoresis (CE) (such as standards described in Doherty E.A.S., et. al., 2003).
Embodiments of the present invention are now described in greater detail. In one aspect, adsorbed coatings used in CE for the effective separation of biomolecules are provided, comprising: a hydrophilic polymer, said polymer further comprising functional groups configured to achieve any one or more of: adheance to a glass surface, control of EOF, and inhibition of interactions between an analyte and the glass surface More specifically, in some embodiments polymer coatings of the present invention comprise (1) hydrophilic portions that provide an environment conducive to the separation of biomolecules and; (2) a sufficient amount of functional groups that adhere to the glass surface, preferably suppressing EOF and shielding the glass surface from interaction with biomolecules and; (3) functional groups that, when induced, will capture analytes of interest. There are a limited number of polymeric coatings that satisfy the first two requirements due to the difficulty of precisely balancing the ratio of hydrophilic and hydrophobic groups incorporated into a polymeric network.
For instance, adsorbing dimethylacrylamide polymer or polyvinyl pyrrolidone to a glass surface efficiently suppresses EOF by minimizing the interaction between solutes and the surface (Doherty E.A.S., et. al., 2003). A certain degree of hydrophobicity is required to adsorb a polymeric film to a glass substrate and provide for a stable coating. On the other hand, the hydrophobic properties of dimethylacrylamide polymer and its polyvinyl pyrrolidone analog make them less suitable for electrophoretic separations where good resolution of biomolecules is required (Doherty E.A.S., et.al., 2003). According to embodiments of the present invention, the novel developed polyvinyl alcohol grafted polymers represent materials with well balanced hydrophobic and hydrophilic properties. The hydrophobic fragments incorporated into the polymeric network contribute to strong adsorption to the glass surface and efficient capture of biomolecules. At the same time, the surface of the developed polymeric film remains predominantly hydrophilic, providing good resolution of proteins during isoelectric focusing.
In alternative embodiments of the present invention, covalently attached coatings used in CE for the effective separation of biomolecules are provided. In an exemplary embodiment, one or more polymers are synthesized outside of a microfluidic device. The polymer(s) are then introduced into the device after the polymerization process is completed or partially completed. The polymer then forms a covalent attachment to the surface of the device to form a coating therein. The covalent linkage with the surface may be achieved in a number of ways, such as but not limited to: radical based chemistry, alkylation, acylation, condensation reactions and also chemically, catalytically, enzymatically, electrochemically, photochemically or by thermally initiated processes.
Additional embodiments of the present invention describe a novel methodology for producing or making polymers for absorbtive coatings. In addition to forming the structural fragments that provide a polymer with good adhesion and separation characteristics, such as peak resolution, embodiments of the inventive method also allows for the incorporation of photochemically active moieties into the same polymer without having a negative effect on features (1) and (2) above. In an exemplary embodiment,
In this embodiment a main function of the polymeric backbone of block A is to provide sufficient adsorbtion of the polymer to a surface, such as a capillary or channel wall. In some embodiments, polymeric backbone A has a molecular weight in the range of about 100 to 3,000,000 Da preferably within a range of about 2,000-300,000 Da, and more preferably within a range of about 20,000-100,000 Da. Polymeric backbone A can be made prior to its grafting with groups X and Y or synthesized in situ during grafting. Polymeric backbone A is comprised of any type of linear non-cross-linked or partially cross-linked polymer which has the ability to adhere to the glass surface. In one embodiment, polymeric backbone A is comprised of a hydroxyl-containing polymer such as polyethylene glycol, polypropylene glycol, partially or fully hydrolyzed polyvinyl alcohol or a carbohydrate-based polymer such as one based on cellulose, dextran, chitosan or cyclodextrin, or other types of hydroxyl-containing polymers produced via polymerization of the hydroxyl-containing monomers or oligomers or monomers or oligomers that carry the functional groups that can be converted into the hydroxyl groups. Examples of such monomers include, but are not limited to: acrylates, methacrylates, and other vinyl- or allyl-containing compounds with hydroxyl, ester or epoxy groups being a part of their structure. In one embodiment, polymeric backbone A comprises ionic groups such as cation-exchange groups—sulfonic acids, carboxylic acids, phenolics, phosphonic acids, or silanol groups or anion-exchange groups—primary amines, secondary amines, tertiary amines or quaternary amines or other groups that can be converted into the ionic groups. Polymerization of these classes of compounds is well understood by those skilled in the art.
The polymer coatings described in this invention can be applied to a variety of different surfaces made of different materials. Examples of these materials include but are not limited to: glass, silicon oxide, aluminum oxide, zirconium oxide, titanium oxide, any type of polymer or plastic composition, metal, silicon, carbon, glassy carbon or combinations of the above. The wall or surfaces of the separation channel can be used in raw form or can be treated with chemical reagents prior to coating. A surface treatment may be used to improve adhesion to the surface or to suppress undesirable characteristics such as electro-osmotic flow. For instance, the glass capillaries can be treated with alkaline and/or acidic solutions of various compositions, which may also include peroxides or other oxidizing agents.
As one of many options for surface treatment prior to coating according to embodiments of the present invention, one may treat glass capillaries with various silane reagents. Hydrophobic silane reagents attached to the glass surface may improve the adhesion to hydrophobic groups incorporated into an adsorbing polymer. Examples of hydrophobic silane reagents include but are not limited to: alkyl or aryl silanes containing the reactive functionality in the form of chloro, alkoxy, amino silane or silanol groups. The reactive functionality forms a covalent Si—O—Si bond with the glass surface via interaction with surface silanol groups. The number of reactive groups in one silane molecule that form covalent bonds with the glass surface may vary depending on the purpose of the surface treatment. For instance, when a hydrolytically stable surface is required, dialkoxy, trialkoxy or bridged polyfunctional silanes including silicon polymers can be used to form more than one covalent bond between a silane molecule and the substrate surface prior to its coating with an adsorbtive polymer. The reactive silanes can also be used to make the surface more hydrophilic prior to coating. In the latter case, the reactive silanes, preferably alkoxy silanes containing various hydrophilic moieties can be utilized. Examples of these hydrophilic moieties include but are not limited to: alcohols, epoxy groups, amides, sulfonamides, carbamates, urea, ethers, esters, polyethylene oxide groups, carboxylic acids, sulfonic or phosphonic acids, amines, secondary amines, tertiary amines, or quaternary amines, and also the groups that can be converted to these moieties, or combinations of the above.
Another embodiment for surface treatment prior to coating according to the present invention is the formation of C—Si bonds between a glass surface and an undercoating molecule. The latter can carry either hydrophobic or hydrophilic groups. This particular type of surface treatment can be performed with organometallic reagents as described in US00514753A.
Additionally, another embodiment of the present invention provides for surface treatment prior to applying a coating of the present invention by depositing a layer of an adsorbtive or covalently attached polymer onto the surface, providing better adhesion to the second adsorbtive polymer that contains activatible groups Examples of the polymers that can be used for surface treatment prior to coating with an adsorbtive activatible layer include but are not limited to: polyethylene glycol polymers, polypropylene glycol polymers, polybutadiene polymers, polyurethane polymers or polymers produced from any type of acrylic, methacrylic, styrenic, allylic, or vinyl monomers or combinations thereof. The polymers that can be used for surface treatment prior to coating with an adsorptive activatible layer may contain a variety of functional groups that may facilitate the adsorption of the activatible polymer to the surface of the separation channel or surface via physical adsorption, via physical adsorption followed by covalent bonding, via metal chelation, coordination to metals, hydrogen bonding, hydrophobic interactions, hydrophilic interactions, electrostatic interactions, π-π interactions or any combination of the above.
According to some embodiments of the present invention, the groups X provide a hydrophilic functionality which is compatible with aqueous media. The final product—polymer may comprise multiple functional groups X of the same type or a combination of different X moieties. Examples of functional groups X include, but are not limited to: amides, sulfonamides, ethers, esters, thiols, carbamates, urea, alcohols, sulfonic acids, carboxylic acids, phenolic, phosphonic acids, silanol groups, amines, secondary amines, tertiary amines, or quaternary amines.
According to some embodiments of the present invention, the groups Y can be comprised of photochemically activatible moieties such as alkyl or aryl halides, azo or peroxy groups, diazirines, azide groups, acetophenone, benzophenone, or anthraquinone derivatives.
Although an exemplary synthesis of polymer (as shown in example 3) describes the incorporation of photochemically activatible group Y for the purpose of capturing the analytes, other activation mechanisms besides photocapture can be utilized to trigger the formation of a covalent bond or an interaction between analytes and the surface of the separation channel or surface so that the analytes adhere to the surface as a result of such interaction. Thus, an activatible group Y is not limited to the photoactive functionalities, and can be represented by any other activatible groups such as thermoactivatible groups, or by groups that can be activated for capture in the presence of a chemical reagent or catalyst. For instance, in addition to the examples of photoactivatible groups mentioned earlier, the groups Y can comprise epoxy groups, alcohols, esters, thiols, aldehyde groups, enoles, phospho-organic, boroorganic, metal organic functionalities and others.
The functionalization of polymeric backbone A with groups X and Y can be carried out via various synthetic routes. In one embodiment, the polymeric backbone A has one or more reactive sites of the same or different nature, which is grafted with the polymeric chains independently comprising the groups X or Y or both. These polymer chains can be synthesized prior to grafting, and can be further attached covalently to the polymeric backbone of A. The groups X and Y may also serve as attachment points connecting the polymeric chains to the polymer A. Moreover, polymeric backbone A may also incorporate the groups X and Y containing polymer strands via various interactions between these strands and polymeric backbone A. In this latter case, there is no covalent connection between the polymeric backbone A and the groups X and/or Y containing polymer strands. The interactions between A and the X and/or Y containing polymer strands can be independently hydrophobic, hydrophilic, hydrogen bonding, metal ion chelation and coordination, electrostatic interactions, π-π interactions and others. Some of these interactions can lead to an entanglement, formation of complexes or tertiary structures between A and the X and Y containing polymer strands, which hold the functional elements A, X, and Y together.
As a preferable way of combining the elements A, X, and Y in a single material, one skilled in the art may consider the covalent attachment of the elements A, X, and Y by grafting a polymeric backbone A using one or several individual monomers carrying the groups X and Y. Examples of relevant grafting processes can be found in (Jalal Zohuriaan-Mehr, M., 2005). The mechanisms of grafting include radical polymerization via proton abstraction, radiation-initiated polymerization, chemically-initiated polymerization, ring-opening polymerization, and anion or cation-initiated polymerization.
In other embodiments, a polymer coating is synthesized by grafting a pre-made polymeric backbone A with two individual monomers providing the polymer of the invention with functional groups X and Y, respectively, where both X and Y are attached covalently to the polymeric backbone of A. In one embodiment, the polymeric backbone A comprises one, two or more hydroxyl groups or other groups that can be converted into hydroxyl groups either during grafting or during their further chemical modification. Polymeric backbone A may also comprise a mixture of different polymers of various nature and various molecular weights. In one embodiment, polymeric backbone A is comprised of polyvinyl alcohol with a molecular weight in a range of about 2,000 Da to 300,000 Da.
In an illustrative embodiment as shown in
In another embodiment, a hydroxyl containing polymeric backbone A is grafted with polymer strands produced from X and Y containing monomers using Ce4+ as an initiator. First, Ce4+ forms radicals on the alpha carbon atoms of polymeric backbone A, which are next to the hydroxyl groups of A. Reference Jalal Zohuriaan-Mehr, M., 2005 which refers to an initiation process that may be used. The radicals formed on the backbone of A start the polymerization of X and Y containing monomers which are both present in the reaction mixture. Thus, the functional groups X and Y become incorporated into the polymeric chains that are covalently attached to polymeric backbone A.
Example 1 below describes an exemplary method for the preparation of polymer via the grafting of polymeric backbone A with X- and Y-containing monomers, according to one embodiment of the present invention. The exemplary structures of the X and Y containing monomers are given in
In other embodiments, Y containing monomer is comprised of: (1) one or more photochemically reactive groups, for example but not limited to: alkyl or aryl halides, azo or peroxy groups, diazirines, azide groups, acetophenone, benzophenone (shown as element 22 on
The benzophenone groups undergo conversion into long-lived triplet diradicals upon UV irradiation, which further form covalent bonds with different fragments of biomolecules via proton abstraction. An example of mechanisms of photocapture and a comprehensive list of other organic reagents that may be used as capture sites for biomolecules upon their photochemical activation are described in Fleming S. A., 1995 which is hereby incorporated by reference; which given the teaching of the present invention may then be employed in embodiments of methods and systems of the present invention.
As mentioned above, the hydrophilicity/hydrophobicity balance and photochemical reactivity parameters of the polymer of the invention are important, and this balance is achieved in some embodiments of the present invention by making certain structural modifications of Y-containing monomers. These modifications include, but are not limited to, adding a particular substituent to aromatic fragments of the benzophenone molecule and/or varying the length and/or nature of the linker connecting the photoactive and polymerizable groups of Y-containing monomer.
In one embodiment, a polymerizable group of Y-containing monomer is a double bond which can be acrylic 21, methacrylic, styrenic, allylic, vinyl and others. The incorporation of Y into the polymer of the invention can also be performed by the insertion of radicals, carbenes or nitrenes formed from Y during its photochemical activation. In this latter case, Y may comprise a group that is able to form a radical, carbene or nitrene, and is not represented by the acrylic, methacrylic, styrenic, allylic, or vinyl group.
The nature and chain length of the linker connecting sites 21 and 22 of Y described above can be adjusted in accordance with solubility requirements and hydrophilicity/hydrophobicity characteristics of the polymer of the invention. In one embodiment, Y-containing monomer has a —CONH(CH2CH2O)nNHCO— linker connecting the polymerizable acrylic double bond 21 to the capture moiety benzophenone 22 (as shown in
Although a linker connecting groups 21 and groups 22 comprise preferably neutral hydrophilic groups such as polyethylene glycol fragments, amide groups, hydroxyl groups and others, it may also comprise hydrophobic moieties such as hydrocarbon fragments, aromatic rings and fluorinated hydrocarbon groups, or ionic groups such as carboxylic acids, sulfonic acids, phosphonic acids, amino groups, secondary amino groups, tertiary amino groups or quaternary amino groups, depending on particular requirements for the separation and capture of biomolecules using the separation channels or surfaces coated with polymers of the invention.
Embodiments of the present invention provide for covalently attached coatings capable of being triggered to capture analytes. Referring to
In some embodiments, the surface of a microfluidic channel is comprised of a raw substrate such as glass, polymer, carbon, glassy carbon, metal, metal oxide, silicon, silicone, and the like. In other embodiments, the surface of the separation channel is comprised of raw substrate that is chemically or physically modified prior to covalent attachment of the functional polymer. The surface may be modified by a number of techniques, including for example, but not limited to: coating, grafting, corona discharge, vapor deposition, plasma vapor deposition, and attachment of a variety of functional molecules such as polymers, reactive silanes, thiols, and other small molecules which provide the surface of the device with the ability to further form a permanent attachment, or covalent bond with the polymer capable of being triggered to capture an analyte.
Examples of surfaces that can be covalently functionalized with pre-made polymers (sometimes referred to as an “undercoat”) according to embodiments of the invention are illustrated in
In some embodiments, functional polymers of the present invention further comprise one or more activatible chemical groups Y as shown in
Examples of photoactivatible groups include, but are not limited to: a benzophenone, an anthraquinone, or an acetophenone derivatives; and also azides, alkyl and aryl halides, azo compounds and the like. The nature and morphology of the functional polymer of the invention can vary depending on a particular application. Thus, functional polymer can be comprised of a co-polymer, grafted polymer, brush polymer, dendrimer, gel, particles, foam, block co-polymer and the like. In some embodiments, functional polymer is comprised of polyvinyl alcohol, polybutadiene, polystyrene, polyacrylamide, polymethacrylamide, polyvinylpyrrolidone, polyethylene glycol, polypropylene glycol, polyethylene imine, polysiloxane, polyacrylonitrile fragments or other functionalities and heteroatoms.
As illustrated in
Referring again to
In another embodiment the raw glass is chemically derivatized prior to addition of the polymer of the invention that is capable of forming a covalent linkage. These derivatized surfaces, also called undercoated surfaces, do not contain triggerable analyte capture moieties. Rather, they introduce characteristics that enable electrophoresis or provide a substrate for the activatable polymer to form a covalent linkage to. An exemplary structure of such an undercoating is shown in polymeric structure 62 of
The following examples are provided for illustration purposes only, and are not intended to limit the scope of the invention in any way. The identity of all synthesized small organic molecules was confirmed by NMR spectroscopy and mass spectrometry methods.
A suspension of 1.38 grams of 1-[(4-benzoylbenzoyl)oxy]-2,5-pyrrolidinedione 33 (C. Thiele and F. Fahrenholz, 1993) in 30 mL of acetonitrile was treated with 1.79 mL (3 eq) of triethylamine and a solution of 1.59 grams (1.5 eq) of [2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamic acid, 1,1-dimethylethyl ester 32 (R. N. Zuckermann, et. al., 1994) in 5 mL of acetonitrile added in one portion. The solution became homogeneous over the next two minutes. The reaction was stirred at room temperature for 3 hr, and the solution was concentrated to give a residue, which was partitioned between 100 mL of ethyl acetate and 50 mL of water. The organics were washed with another 100 mL of water, and then dried over sodium sulfate. Filtration and concentration gave a light orange oil, which was subjected to flash column chromatography on silica gel first with 1%, and then with 2% methanol in dichloromethane. The appropriate fractions were combined and concentrated to give 1.91 g of the desired product 34. This was used in the next reaction without further purification.
A solution of the BOC-protected benzophenone derivative 34 in 10 mL of dichloromethane was treated with 6 mL of trifluoroacetic acid, and the reaction was stirred at room temperature for 2 hr, at which time the reaction was complete (TLC, silica gel, 5% ethyl acetate in dichloromethane). The reaction was concentrated to give an oil, which was reconcentrated with 25 mL of hexane and 25 mL of ethyl acetate to give a yellow residue. This material was used in the next step without further purification.
A solution of the above amine in 20 mL of dichloromethane was treated with 2.3 mL (4 eq) of triethylamine, followed by the drop-wise addition of 500 μL (1.5 eq) of acryloyl chloride. The reaction was stirred at room temperature for 3 hr, and then the solution was concentrated to give an orange semisolid. This was partitioned between 100 mL of ethyl acetate and 50 mL of water. The organics were washed with another 50 mL of water, then with 25 mL of saturated aqueous NaCl. Filtration and concentration gave an orange oil, which was subjected to flash column chromatography on silica gel with 4% methanol in ethyl acetate. The appropriate fractions were combined and concentrated to give 2.32 grams of a very light-yellow oil. This was taken up in 10 mL of ethyl acetate to give a slightly cloudy solution, which was filtered through celite, using ethyl acetate to wash the celite. The solution was concentrated to a volume of about 10 mL, and 5 mL of hexane was added. The solution became cloudy and an oil began to separate. A crystal of authentic product was added, and the mixture was stirred while a crystalline solid began to form. After stirring for 1 hr, the solid was isolated by filtration, washed with 10 mL of 50% ethyl acetate in hexane, and then air-dried to give 855 mg (51%) of the desired product 35 as a white solid.
0.15 g of polyvinyl alcohol was dissolved in 1.5 ml of hot water and cooled to 25° C. Then, 15 g of acrylamide X (
1N HCl was passed through 100 μID fused-silica capillaries (Polymicro Technologies Phoenix, Ariz., USA) over 45 min. time period. The capillaries (1 m segments) were flushed with a solution of 75 mg of the polymer produced in example 3 (suspended in 3 ml of DI water) for 3 hr using 15 psi pressure from an argon cylinder to drive the liquid through the capillary. The capillaries were washed with DI water for 15 min, and dried with argon.
A solution of 4% (w/v) acrylamide (Sigma A3553) and 4 mol % of the acrylamide benzophenone monomer 35 was prepared in a total volume of 1 mL of DMSO (dimethylsulfoxide, EMD MX1456-6) in a 4 mL autosampler vial. The vial was capped, vortexed, and inverted several times to dissolve the materials. A 21 gauge needle was connected to the argon line and the free end of the line inserted into the open vial. The mixture was degassed with argon for a minimum of 20 minutes. 4 μL of ammonium persulfate solution (50 mg Aldrich A7460 dissolved in 200 μL H2O) and 1 μL TEMED (Aldrich T-7024) were added to the mixture, which was then mixed by degassing for another 30 seconds. The reaction mixture was left to polymerize overnight. The polymerization mixture was pumped through the capillaries according to the procedure from example 4.
100μ ID fused-silica capillaries functionalized with vinyl groups which were directly attached to the Si atom according to US00514753A (Polymicro Technologies Phoenix, Ariz., USA), were flushed with the DMSO solution of the polymer.
The procedure from example 2 was used to coat 100μ ID fused-silica capillaries functionalized with phenyl groups directly attached to Si atoms of the glass capillaries according to US00514753A (Polymicro Technologies Phoenix, Ariz., USA).
The procedure from example 2 was used to coat 100μ ID fused-silica capillaries functionalized with ethyl groups directly attached to Si atoms of the glass capillaries according to US00514753A (Polymicro Technologies Phoenix, Ariz., USA).
The procedure from example 2 was used to coat 100μ ID fused-silica capillaries (Polymicro Technologies Phoenix, Ariz., USA) functionalized with ((chloromethyl)phenylethyl)-dimethylchlorosilane. The functionalization of raw glass capillaries with ((chloromethyl)phenylethyl)-dimethylchlorosilane was carried out as follows. A solution of ((chloromethyl)phenylethyl)-dimethylchlorosilane in toluene (1/1, v/v) was used to fill dry capillaries (1 m segments) previously treated in sequential manner with 1 M NaOH, 1M HCl, and water. The ends of the capillaries were sealed with a torch, and the capillaries were placed into a 100° C. oven overnight. The toluene solution was removed with dichloromethane pumped into the capillaries at 15 psi using argon. Capillaries were dried with argon.
The following references are provided in the specification of the subject application and are incorporated by reference herein.
Dolnik, V. 2005. Capillary electrophoresis of proteins 2003-2005. Electrophoresis, Vol 26, pp 1-16;
Doherty, E. A. S., R. J. Meagher, M. N. Albarghouthi, and A. E. Barron. 2003. Microchannel wall coatings for protein separations by capillary and chip electrophoresis. Electrophoresis, Vol 24, pp 34-54;
Horvath, J., V. Dolnik. 2001. Polymer wall coatings for capillary electrophoresis. Electrophoresis, Vol 22, pp 644-655;
Fouque, B., S. Porte, M. Balakirev, M. Berger, F. Perraut, and F. Chatelain. 2003. Light directed assembly of microarray in a glass capillary. Abstracts of the 7th International Conference on Miniaturized Chemical and Biochemical Analysis Systems. October 5-9. Squaw Valley, Calif., USA;
Towbin, H., T. Staehlin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76, 4350 (1979);
Jalal Zohuriaan-Mehr, M., Advances in chitin and chitosan modification through graft copolymerization: a comprehensive review, Iranian Polymer Journal, 14 (3), 235 (2005);
Fleming S. A., Tetrahedron, Vol 51, No 46, 12479, (1995); C. Thiele, F. Fahrenholz, Biochemistry, 32, 2741, (1993); and
R. N. Zuckermann, E. J. Martin, D.C. Spellmeyer, G. B. Stauber, K. R. Shoemaker, J. M. Kerr, G. M. Figliozzi, D. A. Goff, M. A. Siani, R. J. Simon, S. C. Banville, E. G. Brown, L. Wang, L. S. Richter, and W. H. Moos, J. Med. Chem. 37, 2678, (1994).
While the invention has been described in connection with specific embodiments it is evident that many variations, substitutions, alternatives and modifications will be apparent to those skilled in the art in light of the foregoing description and teaching. Accordingly, this description is intended to encompass all such variations, substitutions, alternatives and modifications as fall within the spirit of the appended claims.
This Patent Application claims the benefit of, and priority to, U.S. Provisional Patent application Ser. No. 60/833,060 filed on Jul. 24, 2006 titled “Adsorbed Coatings for Capillaries Capable of Capturing Analytes” and U.S. Provisional Patent application Ser. No. 60/838,766 filed on Aug. 17, 2006 titled “Absorbed and Covalent Coatings for Capillaries Capable of Capturing Analytes,” the disclosures of both of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60833060 | Jul 2006 | US | |
| 60838766 | Aug 2006 | US |
