WO2006015306A2

WO2006015306A2 - Methods, compositions and devices, including microfluidic devices, comprising coated hydrophobic surfaces

Info

Publication number: WO2006015306A2
Application number: PCT/US2005/027225
Authority: WO
Inventors: Robert G. Chapman; Mingqi Zhao; Jing Ni; Luc J. Boosse; John T. Stults; Say Yang; Roger Rudolf
Original assignee: Predicant Biosciences, Inc.
Priority date: 2004-07-29
Filing date: 2005-07-29
Publication date: 2006-02-09
Also published as: WO2006015306A3

Abstract

Methods are disclosed for coating at least a portion of a hydrophobic surface, including the surfaces of plastics or other polymers. Such methods include the use of a first coating layer and/or region that interacts with the hydrophobic surface, although the formation of a chemical bond between the first coating layer and the hydrophobic surface is not required. Subsequent layers may then interact chemically or non-chemically with at least a portion of the first coating layer and/or region. Such coated surfaces may be part of a device or apparatus, including microfluidic devices. Microfluidic devices provide substances to a mass spectrometer. The microfluidic devices include a substrate having at least one microchannel, a cover arranged on a surface of the microchannel, and at least one electrical potential source. Some embodiments include a microchannel widened at an outlet. Other embodiments position the electrical potential source along a surface of the cover. Still other embodiments include a well in which an electrode and a membrane are disposed. The various embodiments provide stable electrospray ionization of substances from a microfluidic device to a mass spectrometer.

Description

METHODS, COMPOSITIONS AND DEVICES, INCLUDING MICROFLUIDIC DEVICES, COMPRISING COATED HYDROPHOBIC SURFACES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part patent application of U.S. patent application Serial No.10/903,248, filed July 29, 2004, and U.S. patent application Serial No. 10/942,612, filed Sept 16, 2004, both of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to interfaces between microfluidic devices and mass spectrometers. More specifically, the invention relates to improved microfluidic devices for providing electrospray ionization of substances to a mass spectrometer. The invention also relates to methods of adding materials onto or coating of hydrophobic surfaces thereto. The present invention further relates to improved microfluidic devices and methods for making and using such devices to provide one or more substances to a mass spectrometer (MS) for analysis.

BACKGROUND OF THE INVENTION Many materials have at least one hydrophobic surface. Examples include the surfaces of plastics and other polymeric materials. These hydrophobic surfaces can be present on components of a device or apparatus. However, the requirements of the device or apparatus may dictate modification of at least one property of at least a portion of such hydrophobic surfaces. Many types of modifications can be envisioned; by way of example only, it might be desirable to decrease the hydrophobicity of the surface or to enhance the ionic content of the surface. One way to accomplish this modification would be to add at least one additional material in or onto (i.e., coat) at least a portion of the hydrophobic surface. Multiple materials may be added to create more complex surfaces or surfaces with properties tuned to a user's needs, for example, different channels in multi¬ channel microfluidic devices maybe comprised of differently coated surfaces based on a user's needs. Coating microfluidic devices may aid the separation and analysis of biological samples. Generally, such coatings should be stable and/or the stability controllable by the fabricator or user of the device or apparatus.

The use of microfluidic devices such as microfluidic chips is becoming increasingly common for such applications as analytical chemistry research, medical diagnostics and the like. Microfluidic devices may be used for separation and analysis of sample sizes as small as a few nanoliters or less and are thus generally quite promising for applications such as proteomics and genomics. One way to analyze substances using a microfluidic device is to mix and/or separate substances on the microfluidic device and then transfers the substances from the device to a mass spectrometer (MS) via electrospray ionization (ESI).

Some microfluidic devices simply act as a platform for delivering substances to a MS. In other words, one or more substances (typically fluids) are placed on such a microfluidic device and are made to move across the device and interface with the MS, typically via an ESI tip on the microfluidic device. Such microfluidic devices work well for the simple purpose of providing one or more substances to a MS. In other applications, however, it is advantageous to use microfluidic devices to separate, mix and/or otherwise manipulate one or more substances on the device and then provide one or more of the substances to the MS via an ESI tip. Such microfluidic devices typically include multiple fluid reservoirs connected to microchannels, with fluids being deposited in one or more reservoirs and driven along one or more microchannels using electrokinetic forces, pumps and/or other driving mechanisms. After passing through one or more microchannels and being separated, a fluid (or fluids) is then passed from an ESI tip of the microfluidic device to a MS for analysis. Electrospray ionization generates ions for mass spectrometric analysis. Some of the advantages of ESI include its ability to produce ions from a wide variety of samples such as proteins, peptides, small molecules, drugs and the like, and its ability to transfer a sample from the liquid phase to the gas phase, which may be used for coupling other chemical separation methods, such as capillary electrophoresis (CE), liquid chromatography (LC), or capillary electrochromatography (CEC) with mass spectrometry. One of the challenges in developing microfluidic devices has been to combine the ability of a device to separate, mix or otherwise manipulate sample substances with its ability to provide those substances to a MS device via ESI. One problem sometimes encountered in currently available microfluidic ESI devices is the challenge of applying a potential to substances in the device with a stable ionization current while minimizing dead volume and minimizing or preventing the production of bubbles in the channels or in the droplet at the microchannel outlet. A potential may be applied to substances, for example, to move them through microchannel(s) in a microfluidic device, to separate substances, to provide electrospray ionization, or typically a combination of all three of these functions. Some microfluidic devices use a conductive coating όfi the outer surface of the chip or capillary to achieve this purpose. The conductive coating, however, often erodes or is otherwise not reproducible. Furthermore, bubbles are often generated in currently available devices during water electrolysis and/or redox reactions of analytes. Such bubbles adversely affect the ability of an ESI device to provide substances to a mass spectrometer in the form of a spray having a desired shape. In particular, the presence of one or more bubbles in the microfluidic channel of a microfluidic device can interrupt both the flow and the electrical current needed to sustain electrospray ionization, thus destabilizing the electrospray and disabling the device. It has also been difficult to minimize dead volume at the tip of the microfluidic device which results in loss of sensitivity and separation performance of a microfluidic device. Another difficulty in developing microfluidic devices with ESI tips is to minimize or eliminate electrical breakdown between the ESI tip and the MS counter electrode.Therefore, it would be desirable to have improved microfluidic devices that provides robust transfer of substances to MSvia ESI and that are easily manufactured. Ideally, such microfluidic devices would include means for ESI to provide desired spray patterns to MS while minimizing electrical breakdown between the ESI tip and the MS counter electrode. Such microfluidic devices would also include means for providing a charge to substances with minimum generation of bubbles and dead volume. At least some of these objectives will be met by the present invention.

SUMMARY OF THE INVENTION

Presented herein are methods for adding another material in or onto, that is, coat, at least a portion of a hydrophobic surface. Also presented herein, are surfaces on or in which another material has been coated so that the properties of the original surface has been modified. Further presented are devices comprising at least one surface that has been coated, at least in part, with another material so that the properties of the original surface has been modified. Also presented are methods for making and using devices that comprise at least one surface on or in which another material has been coated. Further presented are multi-channel microfluidic devices in which at least two channels comprise differently coated surfaces. Also presented is the application of the coated microfluidic devices for the separation and analysis of biological samples. In one aspect is a surface comprising the structure S/A/Z, wherein S is selected from the group consisting of a hydrophobic surface, a covalently modified hydrophobic surface and a functionalized hydrophobic surface, A is an amphophilic region comprising a monolayer of an amphophilic polymer or a modified amphiphilic polymer, and Z is a charged region comprising a monolayer of a non-amphiphilic charged polymer or a modified non-amphiphilic charged polymer; wherein the interaction between S and A comprises hydrophobic interactions and/or covalent bonds, and the interaction between A and Z comprises electrostatic and/or covalent bonds. In one embodiment, the amphiphilic polymer or modified amphiphilic polymer is no more than a monolayer. In a further embodiment, the charged polymer or modified charged polymer is no more than a monolayer.

In a further embodiment of the aforementioned aspect, S is a hydrophobic surface comprising a hydrophobic polymer. In further embodiments, the amphiphilic polymer or modified amphiphilic polymer is no more than a monolayer. In yet further embodiment, the charged polymer or modified charged polymer is no more than a monolayer. In a further embodiment, the hydrophobic polymer is selected from the group consisting of a polyolefϊn, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a methacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefϊn polymer, a polysiloxane, a polycarbonate, and copolymers thereof. In still a further embodiment, the hydrophobic surface comprises a mixture or blend of at least two hydrophobic polymers. In further embodiments, the hydrophobic polymer is a methacrylate or cyclo-olefin polymer or the hydrophobic polymer is polycarbonate.

In a further embodiment of the aforementioned aspect, S is a modified hydrophobic surface comprising a modified hydrophobic polymer. In a further embodiment, the modified hydrophobic polymer is selected from the group consisting of a modified polyolefϊn, a modified styrene polymer, a modified halogenated hydrocarbon polymer, a modified vinyl polymer, a modified acrylic polymer, a modified acrylate polymer, a modified methacrylic polymer, a modified methacrylate polymer, a modified polyester, a modified anhydride polymer, a modified polyacrylamide, a modified cyclo-olefin polymer, a modified polysiloxane, a modified polycarbonate, and modified copolymers thereof. In further embodiments, the hydrophobic polymer is a modified methacrylate or cyclo-olefin polymer or the hydrophobic polymer is modified polycarbonate. In any of these embodiments, the modification can be a covalent modification and/or a partial modification.

Such modified hydrophobic polymers may be made by a method comprising exposing a hydrophobic polymer surface with a nucleophile and/or exposing a hydrophobic polymer surface with an electrophile. Further, in such methods, the exposing step may be sufficient to partially modify the hydrophobic polymer surface. Further, in such methods, the hydrophobic polymer surface may be either a methacrylate or cyclo- olefin polymer surface or a polycarbonate surface.

In any of the surfaces comprising the structure S/A/Z, A may comprise an amphiphilic polymer or a modified amphiphilic polymer. In further embodiments, the amphiphilic polymer comprises a moiety selected from the group consisting of an aryl, an alkyl, and a halogenated alkyl. In still further embodiments, the modified amphiphilic polymer comprises a moiety selected from the group consisting of an aryl, an alkyl, and a halogenated alkyl. In still further embodiments, the amphiphilic polymer comprises polystyrene units. In yet still further embodiments, the modified amphiphilic polymer comprises polystyrene units. In still further embodiments, the amphiphilic polymer comprises positively charged moieties or the amphiphilic polymer comprises negatively charged moieties. In yet still further embodiments, the amphiphilic polymer comprises maleic anhydride units or the amphiphilic polymer is derived from maleic anhydride units. The amphiphilic region described above may be made by a method comprising reacting a non- amphiphilic polymer with at least one nucleophile to form an amphiphilic polymer. In further embodiments, the nucleophile is a charged nucleophile or the nucleophile is a neutral nucleophile. In still further embodiments, the method further comprises of reacting the non-amphiphilic polymer with an additional nucleophile. In still further embodiments of such methods, at least a portion of the non-amphiphilic polymer is in contact with S prior to the reacting step. In still further embodiments, such methods further comprise exposing the amphiphilic polymer to S. In yet further embodiments, the exposing step is prior to the reacting step or the exposing step is after the reacting step or the exposing step is simultaneous with the reacting step. In still further embodiments, the method further comprises of reacting the amphiphilic polymer with an additional reagent thereby forming a modified amphiphilic surface. In still further embodiments of any of these methods, the non-amphiphilic polymer comprises maleic anhydride units.

In still further embodiments of any of these methods, S is a hydrophobic polymer selected from the group consisting of a polyolefm, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a methacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefϊn polymer, a polysiloxane, a polycarbonate, and copolymers thereof. In further embodiments, the hydrophobic polymer is a methacrylic polymer or the hydrophobic polymer is a polycarbonate polymer. In alternative further embodiments of any of these methods, S is a modified hydrophobic polymer selected from the group consisting of a modified polyolefm, a modified styrene polymer, a modified halogenated hydrocarbon polymer, a modified vinyl polymeria modified acrylic polymer, a modified acrylate polymer, a modified methacrylic polymer, a modified methacrylate polymer, a modified polyester, a modified anhydride polymer, a modified polyacrylamide, a modified cyclo-olefin polymer, a modified polysiloxane, a modified polycarbonate, and modified copolymers thereof.

In any of the surfaces comprising the structure SIkIZ, Z may be a non-amphiphilic charged polymer or Z may be a modified non-amphiphilic charged polymer. In further embodiments, Z comprises negatively- charged moieties or Z comprises positively-charged moieties. In further embodiments, the positively-charged moieties are quarternary amines. In further embodiments, the molecular weight of Z is greater than 20,000 atomic mass units.

In any of the aforementioned surfaces, Z may be made by a method comprising exposing a surface comprising the structure S/A to non-amphiphilic charged polymer. In still further embodiments, the method further comprises of reacting the non-amphiphilic charged polymer with a reagent thereby forming a modified non-amphiphilic charged polymer. In further embodiments, the exposing step is prior to the reacting step. In another embodiment described herein is a surface comprising the structure SIPIR, wherein S is selected from the group consisting of a hydrophobic surface, a covalently modified hydrophobic surface, and a functionalized hydrophobic surface, P is a functionalized region comprising a monolayer of a linkable hydrophobic polymer or a modified linkable hydrophobic polymer, and R is a charged region comprising a monolayer of a linkable charged hydrophilic polymer or a modified linkable charged hydrophilic polymer; wherein the interaction between S and P comprises hydrophobic interactions and/or covalent bonds, and the interaction between P and R comprises covalent bonds, and/or electrostatic bonds, and/or hydrophobic interactions.

In further embodiments of such surfaces, the linkable hydrophobic polymer or the modified linkable hydrophobic polymer is no more than a monolayer or the linkable charged hydrophilic polymer or modified linkable charged hydrophilic polymer is no more than a monolayer. In further embodiments, S is a hydrophobic surface comprising a hydrophobic polymer. In further embodiments, the linkable hydrophobic polymer or the modified linkable hydrophobic polymer is no more than a monolayer. In still further embodiments, the linkable charged hydrophilic polymer or modified linkable charged hydrophilic polymer is no more than a monolayer. In still further embodiments of such surfaces, the hydrophobic polymer is selected from the group consisting of a polyolefin, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a mefhacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefin polymer, a polysiloxane, a polycarbonate, and copolymers thereof. In still further embodiments, the hydrophobic surface comprises a mixture or blend of at least two hydrophobic polymers. In further embodiments, the hydrophobic polymer is a methacrylate or cyclo-olefin polymer or the hydrophobic polymer is polycarbonate.

In other embodiments of such surfaces, S is a modified hydrophobic surface comprising of a modified hydrophobic polymer. In further embodiments, the modified hydrophobic polymer is selected from the group consisting of a modified polyolefin, a modified styrene polymer, a modified halogenated hydrocarbon polymer, a modified vinyl polymer, a modified acrylic polymer, a modified acrylate polymer, a modified methacrylic polymer, a modified mefhacrylate polymer, a modified polyester, a modified anhydride polymer, a modified polyacrylamide, a modified cyclo-olefin polymer, a modified polysiloxane, a modified polycarbonate, and modified copolymers thereof. In further embodiments, the hydrophobic polymer is a modified methacrylate polymer or modified cyclo-olefin polymer or the hydrophobic polymer is modified polycarbonate. In further embodiments, the modification is a covalent modification and/or the modification is a partial modification.

Also described are methods for forming the modified hydrophobic polymer in a surface comprising the structure S/P/R, comprising exposing a hydrophobic polymer surface with a nucleophile or exposing a hydrophobic polymer surface with an electrophile. In further embodiments, the exposing step is sufficient to partially modify the hydrophobic polymer surface. In further embodiments, the hydrophobic polymer surface is a methacrylate or cyclo-olefin polymer surface or the hydrophobic polymer surface is a polycarbonate surface.

In further embodiments of a surface having the structure S/P/R, P comprises a linkable hydrophobic polymer or P comprises a modified linkable hydrophobic polymer. In further embodiments, the linkable hydrophobic polymer comprises a moiety selected from the group consisting of an aryl, an alkyl, and a halogenated alkyl or the linkable hydrophobic polymer comprises a moiety selected from the group consisting of a vinyl and a substituted vinyl. In still further embodiments, the modified linkable hydrophobic polymer comprises a moiety selected from the group consisting of an aryl, an alkyl, and a halogenated alkyl or the modified linkable hydrophobic polymer comprises a moiety selected from the group consisting of a vinyl, and a substituted vinyl. In still further embodiments, the linkable hydrophobic polymer comprises of poly(l,14- tetradecanediol dimethacrylate) units or the modified linkable hydrophobic polymer comprises of poly(l,14- tetradecanediol dimethacrylate) units. Also described herein are methods of making the functionalized region of surfaces having the structure S/P/R, comprising reacting a non-linkable hydrophobic polymer with at least one nucleophile to form the linkable hydrophobic polymer. In further embodiments, the nucleophile comprises a moiety selected from the group consisting of a vinyl and a substituted vinyl. In other embodiments, the method further comprises of reacting the non-linkable hydrophobic polymer with an additional nucleophile. In further embodiments, at least a portion of the non-linkable hydrophobic polymer is in contact with S prior to the reacting step. In other embodiments, the method further comprises, exposing the non-linkable hydrophobic polymer to S prior to the reacting step or exposing the non-linkable hydrophobic polymer to S simultaneous with the reacting step. In a further embodiment, the method comprises of exposing reactive monomeric units of the linkable hydrophobic polymer to S; further embodiments comprise polymerizing the reactive units thereby forming the linkable hydrophobic polymer on S. In any of such embodiments, the method may further comprise of reacting the linkable hydrophobic polymer with an additional reagent thereby forming a modified linkable hydrophobic surface.

In any of such methods embodiments, S may be a hydrophobic polymer is selected from the group consisting of a polyolefin, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a methacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefin polymer, a polysiloxane, a polycarbonate, and copolymers thereof or S may be a modified hydrophobic polymer is selected from the group consisting of a modified polyolefin, a modified styrene polymer, a modified halogenated hydrocarbon polymer, a modified vinyl polymer, a modified acrylic polymer, a modified acrylate polymer, a modified methacrylic polymer, a modified methacrylate polymer, a modified polyester, a modified anhydride polymer, a modified polyacrylamide, a modified cyclo- olefin polymer, a modified polysiloxane, a modified polycarbonate, and modified copolymers thereof. In further embodiments, the hydrophobic polymer is a methacrylate or a cyclo-olefin polymer or the hydrophobic polymer is a polycarbonate polymer. In further embodiments of a surface comprising the structure S/P/R, R is a linkable charged hydrophilic polymer or R is a modified linkable charged hydrophilic polymer. In further embodiments, R comprises negatively-charged moieties or R comprises positively-charged moieties or R comprises moieties with charge equal to zero. In further embodiments, the positively-charged moieties are quarternary amines. In still further embodiments, the molecular weight of R is greater than 20,000 atomic mass units. In further embodiments of a surface comprising the structure S/P/R, the charged region may be made by a method comprising exposing the linkable charged hydrophilic polymer to the linkable hydrophobic polymer on S, and reacting the linkable charged hydrophilic polymer with at least a portion of the linkable hydrophobic polymer on S. In further embodiments of a surface comprising the structure S/P/R, the charged region may be made by a method comprising exposing monomeric units of the linkable charged hydrophilic polymer to the linkable hydrophobic polymer on S, and reacting the monomeric units of the linkable charged hydrophilic polymer with at least a portion of the linkable hydrophobic polymer on S. In further embodiments of a surface comprising the structure S/P/R, the charged region may be made by a method comprising exposing the modified reactive charged hydrophilic polymer to the reactive hydrophobic polymer on S, and reacting the modified linkable charged hydrophilic polymer with at least a portion of the linkable hydrophobic polymer on S. In further embodiments of a surface comprising the structure S/P/R, the charged region may be made by a method comprising exposing monomelic units of the modified linkable charged hydrophilic polymer to the linkable hydrophobic polymer on S, and polymerizing the monomeric units of the modified linkable charged hydrophilic polymer with at least a portion of the linkable hydrophobic polymer on S.

In further embodiments is a surface comprising the structure S/N, wherein S is selected from the group consisting of a hydrophobic surface, a covalently modified hydrophobic surface, and a functionalized hydrophobic surface. N is a hydrophilic region comprising a monolayer of neutral hydrophilic polymer or a modified neutral hydrophilic polymer; wherein the interaction between S and N comprises physical entrapment of at least a portion of N in S.

In further embodiments, the neutral hydrophilic polymer or a modified neutral hydrophilic polymer is no more than a monolayer. In further embodiments, S is a hydrophobic surface comprising a hydrophobic polymer. In further embodiments, the hydrophobic polymer is selected from the group consisting of a polyolefin, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a methacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefϊn polymer, a polysiloxane, a polycarbonate, and copolymers thereof. In still further embodiments, the hydrophobic surface comprises a mixture or blend of at least two hydrophobic polymers. In further embodiments, the hydrophobic polymer is a methacrylate or cyclo-olefϊn polymer or the hydrophobic polymer is polycarbonate. In alternative embodiments, S is a modified hydrophobic surface comprising a modified hydrophobic polymer. In further embodiments, the modified hydrophobic polymer is selected from the group consisting of a modified polyolefin, a modified styrene polymer, a modified halogenated hydrocarbon polymer, a modified vinyl polymer, a modified acrylic polymer, a modified acrylate polymer, a modified methacrylic polymer, a modified methacrylate polymer, a modified polyester, a modified anhydride polymer, a modified polyacrylamide, a modified cyclo-olefϊn polymer, a modified polysiloxane, a modified polycarbonate, and modified copolymers thereof. In further embodiments, the hydrophobic polymer is a modified methacrylate or cyclo-olefin polymer or the hydrophobic polymer is modified polycarbonate. In further embodiments, the modification is a covalent modification and/or the modification is a partial modification.

Also described are methods for making such a modified hydrophobic polymer comprising exposing a hydrophobic polymer surface with a nucleophile or exposing a hydrophobic polymer surface with an electrophile. In further embodiments, the exposing step is sufficient to partially modify the hydrophobic polymer surface. In further embodiments, the hydrophobic polymer surface is a methacrylate or cyclo-olefϊn polymer surface or the hydrophobic polymer surface is a polycarbonate surface.

In further embodiments of surfaces comprising the structure S/N, N comprises a neutral hydrophilic polymer or N comprises a modified neutral hydrophilic polymer. In further embodiments, the neutral hydrophilic polymer is selected from the group consisting of a poly(ethylene glycol) derivative, a poly(ethylene oxide) derivative, a cellulose derivatives, and combinations thereof. In further embodiments, the modified hydrophilic polymer is selected from the group consisting of a modified poly(ethylene glycol) derivative, a modified poly(ethylene oxide) derivative, a modified cellulose derivatives, and combinations thereof. In further embodiments, the neutral hydrophilic polymer comprises poly(ethylene glycol) units. In further embodiments, the neutral hydrophilic polymer comprises poly(ethylene oxide) units or the neutral hydrophilic polymer comprises hydroxypropylmethyl cellulose units. In further embodiments, the modified neutral hydrophilic polymer comprises modified poly(ethylene glycol) units or the modified neutral hydrophilic polymer comprises modified poly(ethylene oxide) units or the modified neutral hydrophilic polymer comprises modified hydroxypropylmethyl cellulose units.

Also described are methods for making the neutral regions of surfaces comprising the structure S/N comprising swelling the hydrophobic surface with a solvent, and exposing the swollen hydrophobic surface to the neutral hydrophilic polymer. In further embodiments, such methods further comprise drying the swollen hydrophobic surface sufficient to entrap at least a portion of the neutral hydrophilic polymer within at least a portion of the hydrophobic surface. In further embodiments, such methods further comprise of reacting the neutral hydrophilic polymer with a reagent to form a modified neutral hydrophilic polymer. Also described herein are surfaces having the structure S/C, wherein S is selected from the group consisting of a hydrophobic surface, a covalently modified hydrophobic surface, and a functionalized hydrophobic surface, C is a hydrophilic region comprising a monolayer of a linkable hydrophilic polymer or a linkable modified hydrophilic polymer; wherein the interaction between S and C comprises covalent attachment of at least a portion of C onto S. In further embodiments, the linkable hydrophilic polymer or a linkable modified hydrophilic polymer is no more than a monolayer. In further embodiments, S is a hydrophobic surface comprising a hydrophobic polymer. In further embodiments, the hydrophobic polymer is selected from the group consisting of a polyolefin, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a methacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefm polymer, a polysiloxane, a polycarbonate, and copolymers thereof. In further embodiments, the hydrophobic surface comprises a mixture or blend of at least two hydrophobic polymers. In a further embodiment, the hydrophobic polymer is a methacrylate polymer or a cyclo-olefin polymer or the hydrophobic polymer is polycarbonate or the hydrophobic polymer is poly(styrene-co-maleic anhydride). In an alternative embodiment, S is a modified hydrophobic surface comprising a modified hydrophobic polymer. In a further embodiment, the modified hydrophobic polymer is selected from the group consisting of a modified polyolefin, a modified styrene polymer, a modified halogenated hydrocarbon polymer, a modified vinyl polymer, a modified acrylic polymer, a modified acrylate polymer, a modified methacrylic polymer, a modified methacrylate polymer, a modified polyester, a modified anhydride polymer, a modified polyacrylamide, a modified cyclo-olefin polymer, a modified polysiloxane, a modified polycarbonate, and modified copolymers thereof. In further embodiments, the hydrophobic polymer is a modified methacrylate or cyclo-olefin polymer or the hydrophobic polymer is a modified polycarbonate or the hydrophobic polymer is a modified poly(styrene-co-maleic anhydride). In further embodiments, the modification is a covalent modification and/or the modification is a partial modification.

Also described are methods for forming the modified hydrophobic polymer in surfaces having the structure S/C comprising exposing a hydrophobic polymer surface with a nucleophile or exposing a hydrophobic polymer surface with an electrophile. In further embodiments, the exposing step is sufficient to partially modify the hydrophobic polymer surface. In further embodiments, the hydrophobic polymer surface is a methacrylate or a cyclo-olefin polymer surface or the hydrophobic polymer surface is a polycarbonate surface.

In further embodiments of surfaces having the structure S/C, C comprises a linkable hydrophilic polymer or C comprises a linkable modified hydrophilic polymer. In further embodiments, the linkable hydrophilic polymer comprises positively charged moieties or the linkable hydrophilic polymer comprises negatively charged moieties or the linkable hydrophilic polymer is neutral. In further embodiments, linkable modified hydrophilic polymer comprises positively charged moieties or the linkable modified hydrophilic polymer comprises negatively charged moieties or the linkable modified hydrophilic polymer is neutral. In further embodiments, the linkable hydrophilic polymer is selected from the group consisting of polysaccharides, such as hydroxypropylmethyl cellulose, hydroxyethylmethyl cellulose, methyl cellulose and dextran; polyethers, such as polyethylene glycol and polyethylene oxide; polyalcohols, such as polyvinyl alcohol, polyglycerols, polyglycydols; polyamides; polyacrylamides; polyacylamide; polydimethylacrylamide; poly-N- hydroxyethylacrylamide; polyduramide; polyacryloxymorpholine; poly-N-methyloxazoline; poly-N- ethyloxazoline; polyvinylpyrrolidone; zwitterionic polymers, such as ρoly([3- (memacryloylamino)propyl]dimethyl(3-sulfopropyl)arnmonium hydroxide), and proteins such as albumin, gelatin and collagen. In still further embodiments, the linkable modified hydrophilic polymer is a modified version of any of the aforementioned linkable hydrophilic polymers.

In further embodiments are methods of making such hydrophilic region comprising exposing the hydrophobic surface or the modified hydrophobic surface with a hydrophilic polymer or a modified hydrophilic polymer comprised of linkable moieties; and reacting the linkable moieties with at least a portion of the hydrophobic surface or the modified hydrophobic surface. In further embodiments, the linkable unit is a nucleophile or the linkable unit is an electrophile or the linkable unit is chlorohydrin or epoxide.

Also described herein are microfluidic chips for mass spectrometric analysis comprising a microfluidic body layer formed with a plurality of fluid reservoirs; at least one separation channel and/or at least one side channel that are formed along a length of the microfluidic body layer in fluid communication with at least one fluid reservoir; wherein at least one of the separation channels and/or side channels comprises a charged polymer monolayer coated on a hydrophobic surface; and a cover plate for enclosing the separation channel and the side channel to provide a stable electrospray from the microfluidic chip. In further embodiments, the side channel provides electrical contact to the separation channel or the side channel provides sheath flow. In further embodiments, the charged coating of the side channel is a negatively charged coating, and the separation channel includes a positively charged coating. Such a charged coating may be made using any of the methods described herein. In further embodiments, the charged coating of the side channel is a negatively charged coating, and the separation channel is without a coating. In such methods, the negatively charged coating is produced using any of the methods described herein. In further embodiments, the charged coating of the side channel is a negatively charged coating, and the separation channel includes a neutral uncharged coating. In further embodiments, such a negatively charged coating is produced using any of the methods described herein, and the neutral uncharged coating is further produced using any of the methods described herein. In a further embodiment, the charged coating of the side channel is a positively charged coating, and the separation channel includes a negatively charged coating. In such embodiments, each of the charged coatings may also be produced using any of the methods described herein. In further embodiments, the charged coating of the side channel is a positively charged coating, and the separation channel is without a coating. In such embodiments, the positively charged coating may be further produced using any of the methods described herein. In further embodiments, the charged coating of the side channel is a positively charged coating, and the separation channel includes a neutral uncharged coating. In such embodiments, the positively charged coating may be further produced using any of the methods described herein and the neutral uncharged coating may be further produced using any of the methods described herein.

In further embodiments, side channel is without a coating, and the separation channel includes a positively charged coating. In such embodiments, the positively charged coating may be further produced using any of the methods described herein. In further embodiments, the side channel is without a coating, and the separation channel includes a negatively charged coating. In such embodiments, the negatively charged coating may be further produced using any of the methods described herein. In further embodiments, the side channel includes a neutral coating, and the separation channel includes a positively charged coating. In such embodiments, the neutral uncharged coating may be further produced using any of the methods described herein and the positively charged coating may be further produced using any of the methods described herein. In further embodiments, the side channel includes a neutral coating, and the separation channel includes a negatively charged coating. In such embodiments, the neutral uncharged coating may be further produced using any of the methods described herein, and the negatively charged coating may be further produced using any of the methods described herein. In further embodiments of such microfluidic chips, the microfluidic chips further comprise a plurality of electrodes positioned in each fluid reservoir to apply voltages to impart movement of materials within the separation channel and the side channel. In further embodiments, the^~ cover plate extends beyond the microfluidic body layer to form an open-ended distal tip portion at which the separation channel and the side channel terminate to provide an electrospray ionization tip that directs a stable electrospray from the microfluidic chip. In still further embodiments, at least a portion of the open-ended distal tip portion is covered with a hydrophilic material. In still further embodiments, the tapered end portion of the microfluidic body layer includes a tapered end formed along a substantially flat truncated portion of the tapered end portion.

Also described herein are microfluidic chips for electrospray ionization comprising a channel plate formed with a separation channel and at least two side channels that are each in fluid communication with at least one fluid reservoir included within the channel plate, and herein at least one side channel includes a charged coating; and a covering plate for substantially enclosing the non-intersecting fluid channels formed on the channel plate, wherein the covering plate includes an overhang that extends beyond the channel plate to provide an electrospray tip that includes an open-tip region at which each of the non-intersecting fluid channels terminate. In further embodiments, such a microfluidic chip further comprises a syringe in fluid communication with a side channel to provide sheath flow. In further embodiments, the charged coating of the side channel includes positively or negatively charged molecules. In further embodiments, the charged coating of the side channel includes negatively charged molecules, and wherein the separation channel has a charged coating that includes positively charged molecules. In further embodiments, the charged coating of the side channel is a positively charged coating, and the separation channel is without a coating. In further embodiments, the charged coating of the side channel is a positively charged coating, and the separation channel includes a neutral uncharged coating. In further embodiments, the charged coating of the side channel is a positively charged coating, and the separation channel includes a positively charged coating. In further embodiments, the charged coating of the side channel is a negatively charged coating, and the separation channel includes a negatively charged coating. In further embodiments, the coating of the side channel is a neutral uncharged coating, and the separation channel includes a neutral uncharged coating. In further embodiments, the side channel and the separation channel are uncoated. In further embodiments, the charged coating of the side channel is a negatively charged coating, and the separation channel includes a positively charged coating. In further embodiments, the charged coating of the side channel is a neutral uncharged coating, and the separation channel includes a negatively charged coating. In still further embodiments, the side channel is uncoated, and the separation channel includes a negatively charged coating.

Also described herein are any of the aforementioned microfluidic chips in which the is fabricated by pressure molding poly(styrene-co-maleic anhydride). In various embodiments, microfluidic devices include improved mechanisms for causing substances to pass from the microfluidic device to the MS via electrospray ionization (ESI). Generally, microfluidic devices include a substrate comprising at least one microchannel, a cover arranged on a surface of the substrate, at least one outlet in fluid communication with the microchannel for allowing egress of substances, and at least one tip surface extending the cover beyond the outlet. Devices also typically include one or more electrical potential sources, such as electrodes, to provide ESI. Improved design configurations and the like provide for enhanced ESI from a microfluidic device that may also provide for separation and/or other manipulation of substances. Another aspect of the invention relates to a microfluidic device for providing one or more substances to a mass spectrometer for analysis which includes: a substrate comprising at least one layer, the substrate including at least one microchannel, wherein the substances are movable within the at least one microchannel; a cover arranged on a surface of the substrate, the cover including at least one electrical potential source; at least one outlet in fluid communication with the microchannel for allowing egress of the substances from the microchannel; and at least one tip surface extending the cover beyond the outlet. In this device, the ^• microchannel in fluid communication with the outlet widens from a first cross sectional dimensions along the majority of its length to a second, wider cross sectional dimensions at the outlet.

In further embodiment of the aforementioned microfluidic device, the microchannel is enclosed between the substrate and the cover. In further embodiment, at least one microchannel comprises of at least two intersecting microchannels. In further embodiment, at least one microchannel may include a first microchannel in fluid communication with a first outlet and having first cross sectional dimensions and second, wider cross sectional dimensions, and at least a second microchannel in fluid communication with a second outlet disposed at the tip surface. In further embodiment of the microchannel, the second microchannel includes at least one substance for preventing substances exiting the first outlet from entering the second outlet. In further embodiment, this substance in the second microchannel may include at least one substance, such as but not limited to a cross-linked polyacrylamide, an agarose gel, a linear polyacrylamide, a cellulose polymer, polyethylene oxide, polyvinylpyrrolidone and other hydrophilic polymer solutions, for preventing substances exiting the first outlet from entering the second outlet. In still further embodiment of the microchannel, the second microchannel may have negatively charged walls for directing a buffer through the second microchannel to prevent substances exiting the first outlet from entering the second outlet. In another embodiment, the first microchannel may have positively charged walls, and the second microchannel may have walls with essentially no charge or a very low charge, for preventing substances from entering the second outlet.

In further embodiment of the aforementioned microfluidic device, the cover comprises at least one material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz and silica. In still further embodiment of the aforementioned cover, a polymer may be used, such as but not limited to, cyclic polyolefin, cyclo-olefin polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyetliylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ or other acrylic-based polymers. In further embodiment of the aforementioned microfluidic device, at least one electrical potential source of the cover comprises a strip of material disposed across the outlet. In further embodiments, the electrical potential source comprises a strip of metal film or a strip of conductive ink. In further embodiments, the electrical potential source may be embedded in the cover, In still further embodiment, the electrical potential source is coupled with the cover via adhesive or coupled with the cover via any other suitable means. In another aspect of the invention, a microfluidic device for providing one or more substances to a mass spectrometer for analysis includes: a substrate comprising at least one layer, the substrate including at least one microchannel, wherein the substances are movable within the at least one microchannel; a cover arranged on a surface of the substrate and having a first surface in contact with the substrate and a second surface opposite the first surface; at least one outlet in fluid communication with the microchannel for allowing egress of the substances from the microchannel; at least one tip surface extending the cover beyond the outlet; and at least one electrical potential source disposed on the second surface of the cover and ending near a distal end of the tip. In further embodiment of the aforementioned microfluidic device, microchannel is enclosed between the substrate and the cover. In still further embodiment, at least one microchannel comprises at least two intersecting microchannels. In further embodiment, at least one microchannel comprises at least two microchannels, each in fluid communication with a different outlet. In further embodiment of the aforementioned microfluidic device, the tip includes a V-shaped edge surface for providing electrospray ionization of the substances to the mass spectrometer. In further embodiments, one end of the electrical potential source may be disposed at the V-shaped edge surface. In further embodiment, one end of the electrical potential source or is recessed within the V-shaped edge surface. In any such embodiments, the electrical potential source may comprise a conductive wire.

In further embodiment of the aforementioned aspect, the tip includes at least one hole through the cover. In further embodiment, the electrical potential source may comprise a conductive wire shaped to extend into the hole. In further embodiment, the electrical potential source may comprise a conductive plate having a post extending into the hole. In further embodiment of the aforementioned aspect, the cover comprises at least one material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz and silica. In still further embodiment of the aforementioned cover, a polymer is selected from the group consisting of cyclic polyolefin, cyclo-olefin polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ or other acrylic-based polymers. In further embodiment, at least one electrical potential source is coupled with the cover via adhesive.The electrical potential source in any embodiment may be coupled with the cover via any suitable means, such as by adhesive or the like.

In another aspect of the invention, a microfluidic device for providing one or more substances to a mass spectrometer for analysis includes: a substrate comprising at least one layer; a cover arranged on a surface of the substrate; at least one outlet in fluid communication with the microchannel for allowing egress of the substances from the microchannel; and at least one tip surface extending the cover beyond the outlet. The substrate, in turn, includes at least one microchannel, wherein the substances are movable within the at least one microchannel; and at least one electrode reservoir in fluid communication with the microchannel, the electrode reservoir having a membrane, conductive fluid separated from the microchannel by the membrane, and an electrode. In further embodiment of the aforementioned microfluidic device, the microchannel is enclosed between the substrate and the cover. In further embodiment, at least one microchannel comprises of at least two intersecting microchannels. In further embodiment, at least one microchannel comprises at least two microchannels, each in fluid communication with a different outlet. This microfluidic device may be made of any suitable materials, such as those listed above, and may have any of the other device characteristics described above, such as multiple intersecting channels and the like.

In further embodiment of the aforementioned microfluidic device, the electrode reservoir comprises a reservoir portion containing the membrane, the conductive fluid and the electrode and a bridging channel between the reservoir portion and the microchannel, the bridging channel having smaller dimensions than the reservoir portion. In further embodiment, the membrane is disposed at the bottom of the reservoir portion, immediately adjacent the bridging channel, and the membrane comprises nanopores configured to allow only small ions to pass through the membrane from the reservoir portion to the bridging channel. In further embodiments, at least part of the electrode is disposed in the reservoir portion in contact with the conductive fluid. Further embodiments may optionally include a membrane fixture for holding the membrane in place at the bottom of the reservoir portion. In further embodiment, the membrane may be held in place at the bottom of the reservoir portion via adhesive. In further embodiment of the aforementioned aspect, the cover comprises at least one material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz and silica. In still further embodiment of the aforementioned cover, a polymer is selected from the group consisting of cyclic polyolefrn, cyclo-olefϊn polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ or other acrylic-based polymers.

In another aspect of the invention, a microfluidic device for providing one or more substances to a mass spectrometer for analysis includes: a substrate comprising at least one layer, a cover arranged on a surface of the substrate; a first outlet in fluid communication with a first microchannel for allowing egress of the substances from the first microchannel; at least a second outlet in fluid communication with the second microchannel for allowing electrical current from the second microchannel; and at least one tip surface extending the cover beyond the outlet. The substrate includes at least a first microchannel, wherein the substances are movable within the first microchannel, and at least a second microchannel coupled with an electrical contact and one of first and second microchannel include at least one substance for preventing the substances in the first microchannel from passing into the second microchannel. In further embodiment of the aforementioned microfluidic device, the microchannel is enclosed between the substrate and the cover. In further embodiment, at least a third microchannel is intersecting with the first microchannel.

In further embodiment of the aforementioned aspect, at least one substance in the second microchannel may comprise, for example at least one of a cross-linked polyacrylamide, an agarose gel, or a viscous polymeric solution such as a linear polyacrylamide, cellulose polymers, polyethylene oxide, polyvinylpyrrolidone, and other hydrophilic polymer solutions. In further embodiment, at least one substance in the second microchannel may comprise a buffer, and the second microchannel may have negatively charged walls for directing the buffer through the second microchannel to prevent the substances exiting the first outlet from entering the second outlet. In further embodiment, the first microchannel comprises positively charged walls, and the second microchannel comprises essentially neutral walls. In further embodiment of the aforementioned aspect, the cover comprises at least one material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz and silica. In still further embodiment of the aforementioned cover, a polymer is selected from the group consisting of cyclic polyolefm, cyclo-olefin polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ or other acrylic-based polymers.

In another aspect of the invention, a method of making a microfluidic device for providing one or more substances to a mass spectrometer for analysis involves: fabricating a substrate, fabricating a cover having at least one tip surface, and applying the cover to the substrate. Fabricating the substrate includes forming at least one microchannel having a microfabricated surface and forming an outlet in fluid communication with the microchannel and disposed along an edge surface of the substrate. The microchannel in fluid communication with the outlet is formed so as to widen ^"from a first ^"cross sectional dimensions along the maj ority of its length to a second, wider cross sectional dimensions at the outlet.

In a further embodiment of the aforementioned device, fabricating the substrate comprises forming at least two intersecting microchannels. In a further embodiment, the substrate and the cover are fabricated from a material such as but not limited to, glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz, silica and a combination thereof. In a still further embodiment, a polymer may contain, for example cyclic polyolefin, cyclo-olefin polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ or other acrylic-based polymers. In further embodiment, at least one microchannel comprises of forming a first microchannel having positively charged walls, and the second microchannel having essentially neutral walls.In further embodiment of the aforementioned aspect, it further involves coupling an electrical potential source with the device to move the substances through the microchannel by electrophoretic or electrokinetic mobility. In still further embodiment, the electrical potential source comprises an electrical potential microchannel, the electrical potential microchannel containing at least one electrically charged substance. In further embodiment, the electrical potential microchannel exits the microfluidic device immediately adjacent the microchannel. In further embodiment,the method involves disposing at least one substance in the electrical potential microchannel for preventing substances exiting the outlet from entering the electrical potential microchannel. In further embodiment, at least one substance in the electrical potential microchannel may comprise of at least one of a cross-linked polyacrylamide, an agarose gel, or a viscous polymeric solution such as a linear polyacrylamide, cellulose polymer, polyethylene oxide, polyvinylpyrrolidone, and other hydrophilic polymer solutions. In further embodiment, at least one substance in the electrical potential microchannel may comprise a buffer, and the electrical potential microchannel may have negatively charged walls for directing the buffer through the electrical potential microchannel. In another embodiment, the first microchannel may have positively charged walls, and the second microchannel may have walls with essentially no charge or very little charge, for preventing substances from entering the second outlet.

In further embodiment, the electrical potential source comprises at least one electrode on the microfluidic device. In further embodiment, at least one electrode may comprise of a strip of material, such as a metal film or conductive ink, coupled with the cover so as to be disposed across the outlet. In still further embodiments, the material is metal film or conductive ink. In further embodiments, at least one electrode may be embedded in the cover or coupled with the cover via adhesive, or coupled with the cover via any other suitable means. In further embodiments, at least one electrode provides potential for effecting at least one of electrophoretic separation of the substances and electrospray ionization. In further embodiments, at least one electrode provides potential for effecting at least one of electrokinetic movement of the substances in the microchannel and electrospray ionization. In further embodiment, at least one electrode may comprise any suitable material or materials, such as but not limited to at least one of copper, nickel, conductive ink, silver, silver/silver chloride, gold, platinum, palladium, iridium, aluminum, titanium, tantalum, niobium, carbon, doped silicon, indium tin oxide, other conductive oxides, polyanaline, sexithiophene, polypyrrole, polythiophene, polyethylene dioxythiophene, carbon black, carbon fibers, conductive fibers, and other conductive polymers and conjugated polymers. In further embodiment, at least one electrode provides the electrical potential without producing a significant quantity of bubbles in the substances.

In further embodiments of the aforementioned method of making a microfluidic device, the method further involves making at least two connected microfluidic devices from one or more common pieces of starting material and separating the at least two microfluidic devices by cutting the common pieces of starting material. In another embodiment of the method, at least one microchannel may be formed by at least one of photolithographically masked wet-etching, photolithographically masked plasma-etching, embossing, molding, compression molding, injection molding, photoablating, micromachining, laser cutting, laser ablation, milling, die cutting, reel-to-reel methods, photopolymerizing and casting. In another aspect of the invention, a method of making a microfluidic device for providing one or more substances to a mass spectrometer for analysis involves: fabricating a substrate; fabricating a cover having at least one tip surface, a substrate contacting surface, and an electrical potential surface opposite the substrate contacting surface; coupling at least one electrical potential source with the electrical potential surface; and applying the cover to the substrate. Fabricating the substrate comprises forming at least one microchannel having a micro fabricated surface and forming an outlet in fluid communication with the microchannel and disposed along an edge surface of the substrate. The substrate and cover may generally be made of any materials and have any characteristics described above in various embodiments. In further embodiment of the aforementioned device, fabricating the substrate comprises forming at least two intersecting microchannels. In a further embodiment, the substrate and the cover are fabricated from a material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz, silica and a combination thereof. In a still further embodiment, a polymer is selected from the group consisting of cyclic polyolefin, cyclo-olefin polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ or other acrylic-based polymers.In further embodiment of the aforementioned microfluidic device, the electrical potential source comprises at least one electrode. In a further embodiment, fabricating the cover may involve forming a V-shaped edge surface in the tip surface, and the electrode may comprise of a conductive wire with one end disposed in the V-shape. In further embodiment, fabricating the cover comprises forming a hole in the tip. In further embodiment, the electrode may optionally comprise a conductive wire shaped to extend into the hole. In further embodiment, the electrode may comprise a conductive plate having a post extending into the hole. The electrode may comprise any suitable substance and may be used for separation of the substances and/or electrospray ionization. In some emobodiments, the electrode provides the electrical potential without producing a significant quantity of bubbles in the substances. In further embodiments, at least one electrode provides potential for effecting at least one of electrophoretic separation of the substances and electrospray ionization. In further embodiment, at least one electrode provides potential for effecting at least one of electrokinetic movement of the substances in the microchannel and electrospray ionization. In further embodiment, at least one electrode comprises at least one of copper, nickel, conductive ink, silver, silver/silver chloride, gold, platinum, palladium, iridium, aluminum, titanium, tantalum, niobium, carbon, doped silicon, indium tin oxide, other conductive oxides, polyanaline, sexithiophene, polypyrrole, polythiophene, polyethylene dioxythiophene, carbon black, carbon fibers, conductive fibers, and other conductive polymers and conjugated polymers. In some embodiments, at least one electrode provides the electrical potential without producing a significant quantity of bubbles in the substances.In yet another aspect of the invention, a method of making a microfluidic device for providing one or more substances to a mass spectrometer for analysis involves: fabricating a substrate; fabricating a cover having at least one tip surface, a substrate contacting surface, and an electrical potential surface opposite the substrate contacting surface; and applying the cover to the substrate. Fabricating the substrate comprises: forming at least one microchannel having a microfabricated surface; forming an outlet in fluid communication with the microchannel and disposed along an edge surface of the substrate; and forming at least one electrode reservoir in fluid communication with the microchannel, the electrode reservoir having a membrane, conductive fluid separated from the microchannel by the membrane, and an electrode. In a further embodiment of the aforementioned device, fabricating the substrate comprises forming at least two intersecting microchannels. In a further embodiment, the substrate and the cover are fabricated from a material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz, silica and a combination thereof. In a still further embodiment, a polymer is selected from the group consisting of cyclic polyolefrn, cyclo-olefm polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ or other acrylic-based polymers.

In further embodiments of the aforementioned microfluidic device, at least one electrode provides potential for effecting at least one of electrophoretic separation of the substances and electrospray ionization. In further embodiment, at least one electrode provides potential for effecting at least one of electrokinetic movement of the substances in the microchannel and electrospray ionization. In further embodiment, at least one electrode comprises at least one of copper, nickel, conductive ink, silver, silver/silver chloride, gold, platinum, palladium, iridium, aluminum, titanium, tantalum, niobium, carbon, doped silicon, indium tin oxide, other conductive oxides, polyanaline, sexithiophene, polypyrrole, polythiophene, polyethylene dioxythiophene, carbon black, carbon fibers, conductive fibers, and other conductive polymers and conjugated polymers. In some embodiments, at least one electrode provides the electrical potential without producing a significant quantity of bubbles in the substances.

Another aspect of the invention is a method of making a microfluidic device for providing one or more substances to a mass spectrometer for analysis, the method comprising: fabricating a substrate; fabricating a cover having at least one tip surface; coupling an electrical potential source with the device to move the substances through the microchannel by electrophoretic or electrokinetic mobility; and applying the cover to the substrate. Fabricating the substrate comprises forming at least one microchannel having a microfabricated surface and forming an outlet in fluid communication with the microchannel and disposed along an edge surface of the substrate. In a further embodiment of the aforementioned device, fabricating the substrate comprises forming at least two intersecting microchannels. In a further embodiment, the substrate and the cover are fabricated from a material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz, silica and a combination thereof. In a still further embodiment, a polymer is selected from the group consisting of cyclic polyolefin, cyclo-olefm polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ or other acrylic-based polymers.

In further embodiment of the aforementioned microfluidic device, the electrical potential source comprises an electrical potential microchannel containing at least one electrically charged substance. In still further embodiment, the electrical potential microchannel exits the microfluidic device immediately adjacent the microchannel. Further embodiment involves disposing at least one substance in the electrical potential microchannel for preventing substances exiting the outlet from entering the electrical potential microchannel. In further embodiment, at least one substance in the electrical potential microchannel may comprise of a cross- linked polyacrylamide, an agarose gel, or a viscous polymeric solution such as a linear polyacrylamide, a cellulose polymer, polyethylene oxide, polyvinylpyrrolidone, and other hydrophilic polymer solutions. In still further embodiment, the substance in the electrical potential microchannel may comprise of a buffer, and the electrical potential microchannel may have negatively charged walls for directing the buffer through the electrical potential microchannel. In another embodiment, the first microchannel may have positively charged walls, and the second microchannel may have walls with essentially no charge or very little charge, for preventing substances from entering the second outlet. Also described herein are any of the aforementioned microfluidic chips which can be fabricated by pressure molding poly(styrene-co-nialeic anhydride).

These and other aspects and embodiments of the present invention are described in further detail below.

INCORPORATION BY REFERENCE AU publications, patents and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

A better understanding of the features and advantages of the present methods and compositions may be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of our methods, compositions, devices and apparatuses are utilized, and the accompanying drawings of which:

FIG. 1 is a flowchart presenting an illustrative synthesis and use of the coated surfaces.

FIG. 2 depicts various coating embodiments which utilize amphiphilic and charged polymers. FIG. 3 depicts various coating embodiments which utilize polymerization of hydrophobic and charged polymers.

FIG. 4A depicts various coating embodiments which utilize entrapment of neutral polymers.

FIG. 4B depicts various coating embodiments which utilize covalent attachment of charged or neutral polymers. FIG. 5 is an illustrative schematic displaying a hydrophobic surface (a) before coating, (b) after coating with an amphiphilic polymer (PSMA), and (c) after coating the PSMA region with a charged polymer (PDADMAC).

FIG. 6 is an illustrative schematic displaying a hydrophobic surface (a) before coating, (b) after coating with an amphiphilic polymer, precursor, or monomer and (c) after coating the amphiphilic region with a charged polymer, precursor, or monomer.

FIG. 7A is an illustrative schematic displaying a hydrophobic surface coated with (a) functionalized PSMA, and (b) functionalized positively charged polymer (PCPMEDMAC).

FIG.7B are illustrative reaction schemes for other methods to functionalize anhydride based copolymers. FIG. 8 is an illustrative schematic displaying a hydrophobic surface (a) before coating, (b) after coating with a polymerizable hydrophobic monomers (1,14-tetradecanediol dimethacrylate), n = 14, and (c) after co- polymerization of hydrophobic 1,14-tetradecanediol dimethacrylate monomers with charged reactive monomers (3-methylammonium propylmethacrylate (MAPTAC)).

FIG. 9 is an illustrative plot of fluorescence intensity vs. time for a mixture of bodipy labeled proteins/peptides separated using an electrophoresis microfluidic chip with the separation channel coated with a 1,14-tetradecanediol dimethacrylate /MAPTAC coating.

FIG. 1OA is an illustrative example of covalent attachment of a cationic polymer to a polycarbonate surface.

FIG. 1OB is an illustrative example of covalent attachment of a neutral polymer to a polycarbonate surface.

FIG. 11 is an illustrative plot of fluorescence intensity vs. time for a mixture of bodipy labeled proteins/peptides separated using an electrophoresis microfluidic chip with the separation channel coated via direct covalent attachment of a cationic polymer to polycarbonate.

FIG. 12 is an illustrative schematic of a neutral hydrophilic polymer coating on and/or in a hydrophobic surface.

FIG. 13 is an illustrative schematic of a neutral hydrophilic polymer coating on and/or in a hydrophobic surface.

FIG. 14 is an illustrative schematic of a hydrophilic polymer coating that is partially entrapped in a hydrophobic surface. FIG 15 is an enlarged perspective view of an illustrative microfluidic chip that is formed with a tip and a pair of fluid channels converging at a distal tip region.

FIG. 16A illustrates a configuration or set-up that may be incorporated with microfluidic devices including those provided elsewhere herein to provide more reliable separation and electrospray. FIG. 16B illustrates the distal end of a microfluidic chip wherein the separation channel is coated and the side channel is coated or uncoated.

FIG. 16C illustrates the distal end of a microfluidic chip wherein the separation channel is neutrally coated or uncoated and the side channel is coated with a charged polymer.

FIG. 17 illustrates the distal end of a microfluidic chip employing two side channels for sheath flow. FIG. 18 illustrates a multi-channel chip with sheath flow from one side and an integrated electrode positioned at the tip (3').

FIG. 19 is a fluorescence image of a separation channel coated with PSMA-Bodipy/PD ADMAC and an uncoated side channel.

FIG. 20 is a fluorescence image of separation channel coated with PSMA/MAPTAC-Bodipy and an uncoated side channel.

FIG. 21 is an illustrative plot of Mass Spectrometric detection vs. time for a mixture of native (unlabeled) proteins/peptides separated using an electrbphόresis/electro-spray microfluidic chip with the separation channel coated with PSMA/PD ADMAC and the side channel uncoated.

FIG. 22 presents illustrative stability data of the migration time for Bodipy-labeled ubiquitin and Angiotensin I plotted as a function of storage time.

FIG. 23 presents illustrative stability data of the theoretical plate number for Bodipy-labeled ubiquitin and Angiotensin I plotted as a function of storage time.

FIG. 24A depicts a side view of a microfluidic device according to an embodiment of the present invention. FIG. 24B depicts a top view of the microfluidic device shown in FIG. 24A.

FIGS. 25A-25E depict top views demonstrating methods of making a microfluidic device according to two embodiments of the present invention.

FIGS. 26A-26I depict top views demonstrating alternative methods of making a microfluidic device according to two embodiments of the present invention. FIGS. 27A-27D depict top views demonstrating alternative method of making a microfluidic device according to two embodiments of the present invention.

FIGS. 28A-28C depict top views of portions of three embodiments of a microfluidic device having an electrode well, according to three embodiments of the present invention.

FIG. 28D depicts a side view of an electrode well as in FIGS. 28A-28C. FIGS. 29A and 29B depict top views of a microfluidic device and a tip of the device, respectively, having multiple microchannels and multiple wells according to one embodiment of the present invention.

FIG. 30 depicts the through hole version of the bottom view of the plated contacts on top layer of the chip.

FIG. 31 depicts the bottom view of the contacts of the top layer with shorter bottom layer for access. DETAILED DESCRIPTION OF THE INVENTION

Methods for stably modifying a surface are needed in many different applications, including applications in the medical, biotechnology, pharmaceutical and other life sciences industries. Typically, applications in these industries utilize apparatuses/devices manufactured/fabricated from a polymer, glass, silicon, metal, or other inorganic or organic material. However, the initial surfaces of these apparatuses/devices may not have properties that are desired for a particular end user. For example, if the initial surface is hydrophobic and the end user needs a hydrophilic, positively-charged surface or region, then the original surface must be modified. Preferably, such modifications should be stable for the desired use, and even more preferably, such modifications should be stable for multiple uses. Furthermore, if such modifications are to be incorporated into a device or apparatus, then such modifications are preferably amenable to efficient, cost- effective and reproducible production. As used herein, coating refers to any means of modifying at least part of an exposed surface with another material in the form of a new region and/or layer. As described herein, the interactions between the original surface and the new region and/or layer can include hydrophobic interactions, covalent interactions, electrostatic interactions, hydrogen-bond interactions, non-covalent interactions as well as any combination of these interactions. As a result of such a coating, the properties of the new surface differ from the properties of the original surface.

One particular end use for a modified surface or region is in the field of micro-applications, including, by way of example only, miniaturized biosensors, microfluidic devices, microarrays, lab-on-a-chip devices, and other devices created on a "chip" or other miniature surface. These microfluidic devices incorporating modified surfaces or regions may be used in a variety of applications, including, e.g., the performance of high throughput screening assays in drug discovery, immunoassays, diagnostics, genetic analysis, and the like. Furthermore, these microfluidic devices incorporating modified surfaces or regions may also be used for the analysis of biological samples; wherein the biological samples may comprise, by way of example only, proteins, peptides, amino acids, steroids, fatty acids, lipids, saccharides, polysaccharides, nucleosides, nucleotides, oligonucleotides, DNA, RNA, hormones, drugs, pro-drugs, or drug metabolites.

One common surface or region that is created during the fabrication of such devices is a hydrophobic surface, whereas the final end product may have need for a hydrophilic and/or ionic surface or region. As a result, such hydrophilic and/or ionic surfaces or regions need to be created on or adjacent to the hydrophobic surface. Furthermore, for certain applications it may be desirable to control and/or tailor the surface charge density of an ionic surface. One illustrative application in which such control and/or tailoring is expected to find use is in miniaturized electrophoresis devices, i.e., allowing the fabricator to control the magnitude and direction of electroosmotic flow to suit the needs of the end user; in one example, the magnitude (regardless of sign) of the electroosmotic flow is at least 3 x 10^'4 (cm²/vs) in a solution of 20% isopropanol and 0.05% formic acid in water.

However, because the initial hydrophobic surface and the desired hydrophilic and/or ionic surface or regions have a transition in properties, the interface is potentially unstable; thus methods for stabilizing the interface between a hydrophobic surface or region and an adjacent hydrophilic and/or ionic surface or region are in demand. Covalent modification of a hydrophobic surface to create a hydrophilic surface is often impracticable. For certain types of hydrophobic surfaces, such as PMMA, covalent modification is limited by the functionality present on the surface, available chemistries used for attachment, and solvent systems used to enable covalent attachment to the hydrophobic surface. Often conditions must be utilized that are detrimental to the polymer, for example, the use of severe solvents and reagents, which becomes impractical for large scale manufacturing (see, e.g., S.A. Soper et al, Analytica Chimica Acta, 470. (2002), 87-99). The methodology described herein allows for modification of any hydrophobic surface, including hydrophobic surfaces that would otherwise require severe conditions in order to effect covalent modification, using solution chemistry (including, but not limited to aqueous-based methods), in a simple approach with a small number of manipulations. An "alkoxy" group refers to a (alkyl)O- group, where alkyl is as defined herein.

An "alkyl" group refers to an aliphatic hydrocarbon group. The alkyl moiety may be a "saturated alkyl" group, which means that it does not contain any alkene or alkyne moieties. The alkyl moiety may also be an "unsaturated alkyl" moiety, which means that it contains at least one alkene or alkyne moiety. An "alkene" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond, and an "alkyne" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be branched, straight chain, or cyclic.

The "alkyl" moiety may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as "1 to 10" refers to each integer in the given range; e.g., "1 to 20 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term "alkyl" where no numerical range is designated). The alkyl group could also be a "lower alkyl" having 1 to 8 carbon atoms. The alkyl group of the compounds described herein also may be designated as "Ci-C₄ alkyl" or similar designations. By way of example only, "C₁-C₄ alkyl" indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

The term "alkylamine" refers to the -N(alkyl)_xH_y group, where x and y are selected from the group x=l, y=l and x=2, y=0. When x=2, the alkyl groups, taken together, can optionally form a cyclic ring system. The term "alkenyl" refers to a type of alkyl group in which the first two atoms of the alkyl group form a double bond that is not part of an aromatic group. That is, an alkenyl group begins with the atoms -C(R)=C- R, wherein R refers to the remaining portions of the alkenyl group, which may be the same or different. Non- limiting examples of an alkenyl group include -CH=CH, -C(CH₃)=CH, -CH=CCH₃ and -C(CH₃)=CCH₃. The alkenyl moiety may be branched, straight chain, or cyclic (in which case, it would also be known as a "cycloalkenyl" group).

An "amide" is a chemical moiety with formula -C(O)NHR or -NHC(O)R, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). The procedures and specific groups to make such amides are known to those of skill in the art and can readily be found in reference sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3^rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.

The term "amphiphilic" refers to a molecule, polymer, composition or structure that has a attraction towards both polar solvents (like a hydrophile) and non-polar solvents (like a hydrophobe). The hydrophilic portion may be neutral, positively charged or negatively charged. By way of example only, an amphiphilic polymer has hydrophobic subunits and hydrophilic subunits. Such different subunits may result from the copolymerization of more than one polymerizable molecule, at least one of which has a hydrophobic portion and one of which has a hydrophilic portion. Alternatively, an amphiphilic polymer may result from the polymerization of an amphiphilic polymerizable molecule, the co-polymerization of an amphiphilic polymerizable molecule and a non-amphiphilic polymerizable molecule, or the co-polymerization of two different amphiphilic polymerizable molecules. In yet still another variation, a hydrophobic polymer may be converted into an amphiphilic polymer by reaction with a hydrophilic reagent; the reverse situation is also envisioned, that is, a hydrophilic polymer may be converted into an amphiphilic polymer by reaction with a hydrophobic reagent. Preferably, an amphiphilic polymer should be able to coat at least a portion of a hydrophobic surface so that the predominant interactions with such a surface are through the hydrophobic portions of the amphiphilic polymer. Further, the resulting exposed surface of the amphiphilic polymer should preferably be predominantly hydrophilic. By way of example only, Figure 5(b) presents an idealized coating of an amphiphilic polymer on a hydrophobic surface. In this figure, the amphiphilic polymer interacts with the hydrophobic surface via the hydrophobic units of the amphiphilic polymer, whereas the hydrophilic portion (here, the negatively charged units) of the amphiphilic polymer are exposed for subsequent interaction with other reagents, such as a positively-charged polymer (see Figure 5(c)).

Many types of amphiphilic polymers and co-polymers can be designed so as to satisfy the aforementioned requirements, i.e., being able to coat a surface predominantly with one type of group while exposing to the environment a different type of group. A preferred type of co-polymer is an alternating or alt co-polymer; however, deviations from this structure are also expected to be satisfactory.

The term "aromatic" or "aryl" refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes both carbocyclic aryl (e.g., phenyl) and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups. The term "carbocyclic" refers to a compound which contains one or more covalently closed ring structures, and that the atoms forming the backbone of the ring are all carbon atoms. The term thus distinguishes carbocyclic from heterocyclic rings in which the ring backbone contains at least one atom which is different from carbon.

The term "attached" refers to interactions including, but not limited to, covalent bonding, ionic bonding, electrostatic, physisorption (also referred to as physical adsorption), intercalation, entanglement, and combinations thereof.

The term "bilayer" refers to two single thin film monolayers, each of which has an average thickness less than about 500 nm. That is, each monolayer may be of a different thickness and each monolayer may also be less than 100 nm in thickness, less than 50 nm in thickness, less than 20 nm in thickness, or less than 10 nm in thickness. The term "bond" or "single bond" refers to a chemical bond between two atoms, or two moieties when the atoms joined by the bond are considered to be part of larger substructure.

The term "coverplate" refers to a substrate used in creating certain microfiuidic devices. Typically the channel network is fabricated into a separate substrate, and the separate substrate is mated or joined, at least in part, to a top substrate, forming the microfiuidic device of the invention, e.g., create the channels networks. In addition, the top substrate may include a plurality of holes or ports used for fluidic introduction and/or accessibility to the channels and/or for sample introduction.

The term "ester" refers to a chemical moiety with formula -COOR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). The procedures and specific groups to make such esters are known to those of skill in the art and can readily be found in reference sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3^rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.

The term "functionalized" refers to the covalent modification of chemical moieties on a polymer. The term "halo" or, alternatively, "halogen" means fluoro, chloro, bromo or iodo. Preferred halo groups are fluoro, chloro and bromo.

The terms "haloalkyl," "haloalkenyl," "haloalkynyl" and "haloalkoxy" include alkyl, alkenyl, alkynyl and alkoxy structures that are substituted with one or more halo groups or with combinations thereof. The terms "fluoroalkyl" and "fluoroalkoxy" include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine.

Broadly speaking, surfaces or regions interact with water in one of two ways. If the surface or region is resistant to wetting, or not readily wet by water, the interaction is termed hydrophobic. Such surfaces or regions have a lack of affinity for water. On the other hand, if the surface or region is readily wet by, or readily absorbs, water, the interaction is termed hydrophilic. Such surfaces or regions have an affinity for water. One common technique for determining whether, and to what degree, a surface is hydrophobic or hydrophilic is by contact angle measurements. In this technique, a drop of water is deposited on a test surface and the angle of the receding and advancing edges of the droplet with the surface are measured. The term "hydrophobic" is used to describe a surface or coating which forms a contact angle of greater than 60° when a droplet of water is deposited thereon. The term "hydrophilic" is used to describe a surface or coating which forms a contact angle of less than 60° when a droplet of water is deposited thereon.

The term "linkable" refers to the ability to form an attachment to a surface or region.

The term "modified hydrophobic" refers to a hydrophobic surface that has been physically and/or chemically modified; such a modified hydrophobic surface remains hydrophobic although the level of hydrophobicity may have been altered by the physical and/or chemical modification. In addition, a modified hydrophobic surface includes a hydrophilic surface that has been physically and/or chemically modified to become a hydrophobic surface.

The term "moiety" refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule. The term "monolayer" refers to a single thin film layer that has an average thickness less than about 500 nm. That is, the monolayer may also be less than 100 nm in thickness, less than 50 nm in thickness, less than 20 nm in thickness, or less than 10 nm in thickness.

The term "multilayer" refers to multiple single thin film monolayers, each of which has an average thickness less than about 500 nm. That is, each monolayer may be of different thicknesses, and further each monolayer may also be less than 100 nm in thickness, less than 50 nm in thickness, less than 20 nm in thickness, or less than 10 nm in thickness.

The terms "nucleophile" and "electrophile" as used herein have their usual meanings familiar to synthetic and/or physical organic chemistry. Selected examples of covalent linkages formed by reaction of a nucleophile and an electrophile are given in the following table.

Table 1: Examples of Covalent Linkages and Precursors Thereof

The term "optionally substituted" means that the referenced group may be substituted with one or more additional group(s) individually and independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl, perfluoroalkyl, silyl, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof. The protecting groups that may form the protective derivatives of the above substituents are known to those of skill in the art and may be found in references such as Greene and Wuts, above.

The term "polymer" refers to a molecule composed of smaller monomeric subunits covalently linked together. The term polymer encompasses the term homopolymer, which refers to a polymer made of only one type of monomer, as well as the term copolymer, which refers to a polymer made up of two or more types of monomer.

Examples of copolymers encompassed within the term "polymer," as well as the shorthand terminology used within, are presented in the following table:

The term "sealing" refers to the method of applying a cover plate on top of a substrate in which channels have been formed in, thus enclosing, at least in part, the channels.

The term "swell" refers to a material exhibiting expansion when in contact with liquid in at least one direction i.e. in the x transverse direction, the y longitudinal direction or the z vertical direction or a material which swells in any combination of these directions.

The term "swelling" refers to the act of causing a material to swell. The term "trilayer" refers to three single thin film monolayers, each of which has an average thickness less than about 500 nm. That is, each monolayer may have a different thickness and each monolayer may also be less than 100 nm in thickness, less than 50 nm in thickness, less than 20 nm in thickness, or less than 10 nm in thickness. The compounds and polymers presented herein may possess one or more chiral centers and each center may exist in the R or S configuration. The compounds and polymers presented herein include all diastereomeric, enantiomeric, and epimeric forms as well as the appropriate mixtures thereof. Stereoisomers may be obtained, if desired, by methods known in the art as, for example, the separation of stereoisomers by chiral chromatographic columns. EXAMPLES OF POLYMERIC MATERIALS

Examples of hydrophobic polymers that may be used with the surfaces, regions, coatings, methods, devices and apparatuses described herein, include, by way of example only (note that the categories presented below are provided for organizational purposes only and not to imply that a particular polymer may not fall within more than one sub-category) (a) polyolefins, including by way of example only, as polyethylene, poly(isobutene), poly(isoprene), poly(4-methyl-l-pentene), polypropylene, ethylene-propylene copolymers, ethylene-propylene- hexadiene copolymers, and ethylene-vinyl acetate copolymers;

(b) styrene polymers, including by way of example only, poly(styrene), poly(2- methylstyrene), styrene-acrylonitrile copolymers having less than about 20 mole-percent acrylonitrile, and styrene-2,2,3,3,-tetrafluoropropyl methacrylate copolymers,

(c) halogenated hydrocarbon polymers, including by way of example only, poly(chlorotrifluoroethylene), chlorotrifluoroethylene-tetrafluoiOethylene copolymers, poly(hexafluoropropylene), poly(tetrafluoroethylene), tetrafluoroethylene-ethylene copolymers, poly(trifluoroethylene), polyvinyl fluoride), and poly(vinylidene fluoride); (d) vinyl polymers, including by way of example only, poly( vinyl butyrate), poly(vinyl decanoate), poly(vinyl dodecanoate), poly(vinyl hexadecanoate), poly(vinyl hexanoate), poly(vinyl propionate), poly( vinyl octanoate), poly(heptafluoroisopropoxyethylene), poly(heptafluoroisopropoxypropylene), and poly(methacrylonitrile) ;

(e) acrylic and acrylate polymers, including by way of example only, poly(n-butyl acetate), poly(ethyl acrylate), poly[(l-chlorodifluoromethyl)tetrafluoroethyl acrylate], poly[di(chlorofluoromethyl)fluoromethyl acrylate], poly(l,l-dihydroheptafluorobutyl acrylate), poly(l,l- dihydropentafluoroisopropyl acrylate), poly(l,l-dihydropentadecafluorooctyl acrylate), poly(heptafluoroisopropyl acrylate), poly[5-(heptafluoroisopropoxy)pentyl acrylate], poly[ll- (heptafluoroisopropoxy)undecyl acrylate], poly[2-(heptafluoropropoxy)ethyl acrylate], and poly(nonafluoroisobutyl acrylate);

(f) methacrylic and methacrylate polymers, including by way of example only, poly(benzyl methacrylate), poly(n-butyl methacrylate), poly(isobutyl methacrylate), poly(t-butyl methacrylate), poly(t- butylaminoethyl methacrylate), poly(dodecyl methacrylate), poly(ethyl methacrylate), poly(2-ethylhexyl methacrylate), poly(n-hexyl methacrylate), poly(methyl methacrylate), poly(ρhenyl methacrylate), poly(n- propyl methacrylate), poly(octadecyl methacrylate), poly( 1 , 1 -dihydropentadecafluorooctyl methacrylate), poly(heρtafluoroisopropyl methacrylate), poly(heptadecafluorooctyl methacrylate), poly(l-hydrotetrafluoroethyl methacrylate), poly(l,l-dihydrotetrafluoropropyl methacrylate), poly(l-hydrohexafluoroisopropyl methacrylate), and poly(t-nonafluorobutyl methacrylate);

(g) polyesters including by way of example only, ρoly(ethylene terephthalate), poly(butylene terephthalate), poly(ethylene terenaphthalate), and polycarbonate;

(h) anhydride based polymers, including by way of example only, poly(styrene-α/tanaleic anhydride) (PSMAA), poly(styrene~co-maleic anhydride);

(i) polyacrylamides, including by way of example only, poly(N,N-dimethylacrylamide), polymethacrylamide; (J) cyclo-olefin polymers including by way of example only, Zeonor™ and Topas™

(k) polysiloxanes, including by way of example only, polydimethyl siloxane (PDMS); and (1) copolymers comprising at least two different monomeric subunits of any of the aforementioned homopolymers.

Table 2 shows examples of amphiphilic polymers that may be used with the surfaces, regions, coatings, methods, devices and apparatuses described herein, include, by way of example only (note that the categories presented below are provided for organizational purposes only and not to imply that a particular polymer may not fall within more than one sub-category). Other examples of amphiphilic polymers include, by way of example only the hydrolysis products of anhydride based polymers, such as maleic anhydride or glutaric anhydride, or polymers resulting from the reaction of anhydride polymers with nucleophiles other than water, such as those shown in Figure 7B.

Positively charged non-amphiphilic polymers that may be used with the surfaces, regions, coatings, methods, devices and apparatuses described herein, include, by way of example only (note that the categories presented below are provided for organizational purposes only and not to imply that a particular polymer may not fall within more than one sub-category) are shown in Table 3. Alternatively, a negatively charged non- amphiphilic polymers include, by way of example only, poly(acrylic acid), poly(styrenesulfonic acid), poly(vinylphosphonic acid), poly(stryrenesulfonic acid-co-maleic acid), poly(glutamic acid), poly(aspartic acid), poly(anilinesulfonic acid), poly(3-Sulfopropyl methacrylate), polyanetholesulfonic acid sodium salt and heparin. In one embodiment, the charged non-amphiphilic polymers, used for creating the desired charge on the coated surface, possess the desired charge at or near pH 7. By way of example only, charged non-amphiphilic polymers containing amine moieties would be used to create a positively charged coating at or near pH 7; whereas, by way of example only, charged non-amphiphilic polymers containing carboxylic, sulfonic, or phosphonic acid groups would be used to create a negatively charged coating at or near pH 7.

DESCRIPTIONS OF SYNTHETIC STRATEGIES AND METHODOLOGIES The general method for modifying a hydrophobic surface and/or region by means of an amphiphilic or modified amphiphilic polymer, as described herein, is presented in Figure 1. The fabricator has available a hydrophobic surface and/or region which requires modification. The hydrophobic surface and/or region may be all or part of a device, apparatus, or a component of either a device or an apparatus, or the surface and/or region may become or be incorporated into a device or apparatus. Further, the hydrophobic surface may also be modified, at least in part, so that the surface region is chemically different from the non-exposed (or bulk) portion of the hydrophobic polymer. In any case, at least a part of the hydrophobic surface is coated with an amphophilic region and/or layer. Such a coating step may occur in a single step or result from multiple sub-steps (see below). The amphiphilic coating step may occur by exposing the hydrophobic region and/or surface to an amphiphilic material (such as an amphiphilic polymer), or to a series of materials that will make an amphiphilic coating (such as an amphiphilic polymer) on the hydrophobic surface and/or region. The resulting amphiphilic region and/or layer may be a partial monolayer, a single monolayer, a partial multilayer, or it may be a multilayer, such as a bilayer; further, part of the amphiphilic region and/or layer may be embedded in the hydrophobic surface or region, or the amphiphilic region and/or layer may be a distinct surface or region adjacent to the hydrophobic region and/or layer; still further, the interaction of the amphiphilic region and/or layer with the hydrophobic surface or region may be covalent, or through non-covalent interactions, or combinations thereof. In any case, a portion of the amphiphilic region and/or layer interacts with the hydrophobic surface or region by means of the hydrophobic portion of the amphiphilic region and/or layer; at least a portion of the hydrophilic portion of the amphiphilic region and/or layer is then exposed to the environment. Further, this exposed hydrophilic portion may be ionically charged to various extents, depending upon the needs of the end user. For example, a significant ionic charge may be produced on the hydrophilic region and/or layer by reacting the hydrophilic region and/or layer with a strong acid or base; alternatively such reactions may occur prior to contacting the amphiphilic polymer with the hydrophobic surface. Further, a lesser ionic charge may be produced by reacting the amphiphilic polymer with a mixture of nucleopbiles, of which only a portion comprise ionic groups. The stability of the amphiphilic coating on the hydrophobic surface and/or region is derived in part from the hydrophobic-hydrophobic interactions between the hydrophobic surface and/or region and the hydrophobic portion of the amphiphilic coating. The thickness or properties of the amphiphilic region and/or layer need not be uniform; such non-uniformities may be a result of random fluctuations in the coating process, variations in the surface hydrophobicity, variations in buffer composition, buffer pH, flow rate, temperature, time of exposure, polymer concentration, or may result from the designs of the fabricator.

Following formation, at least in part, of the amphiphilic region and/or layer on or in (at least in part) the hydrophobic surface and/or region, the next region and/or layer may be added on or in (at least in part) the amphiphilic region and/or layer. In one embodiment, the subsequent region and/or layer is an ionically charged region and/or layer, wherein the predominant charge in the ionically charged region and/or layer is the opposite charge to the predominant ionic charge in the exposed hydrophilic surface of the amphiphilic region and/or layer. By way of example only, if the predominant charge in the exposed portion of the amphiphilic region and/or layer is a positive charge, then the predominant charge in the charged region and/or layer is preferably a negative charge; that is not to say that the only charge in the charged region and/or layer would be a negative charge, but rather that the predominant or majority charge would be a negative charge. As before, the concentration of ionic charges in the charged region and/or layer may range from a low concentration to a high concentration; further, the local charge density may vary, depending on random fluctuations of the coating process; further, the charged region and/or layer may, and most likely will, comprise non-charged moieties. If possible, an annealing step may be used to formulate a more even charge distribution within the charged region and/or layer. The charged region and/or layer need not be a charged region and/or layer upon first exposure to the amphiphilic region and/or layer; encompassed within the methods described herein, the ionic charges may be formed in the charged region and/or layer subsequent to contact with the amphiphilic region and/or layer. One of the interactions between the amphiphlic region and/or layer and the charged region and/or layer will be an ionic interaction, because as stated above, the two regions and/or layers preferably bear opposite ionic charges. However, there may also be additional interactions between the two regions and/or layers, including covalent bonds, hydrogen bonds, polar interactions, and even simple non-covalent interactions.

Although additional ionic regions and/or layers may be added on to or into the first ionically charged region and/or layer, one of the benefits of the methods, compositions and devices described herein is that this simple approach is sufficient to provide stability to the overall coating: that is, where the overall coating is comprised of a first amphiphilic region and/or layer and a second ionically charged region and/or layer. Such an approach is sufficient to provide stability even when the coating is placed on or in (at least in part) a hydrophobic surface, layer or region. For sake of simplicity, the combination of an amphiphilic region and/or layer and an ionically-charged region and/or layer will be referred to as the "two-layer coating," although such regions and/or layers may be simple or complex and composed of a single or a multiple chemical moieties or entities, and although additional regions and/or layers may be added onto or in (at least partially) the two-layer coating.

Although not required for stability, further stability may be imparted to the coating by treating or otherwise fusing the two-layer coating. Such a treatment step may occur by means of heating, chemical reaction, ionic bombardment, γ-radiation, photochemical activation, or any other means or combination of means of treating or fusing a coating that is known in the art. In addition, such a treatment step may also occur by applying an additional region(s) and/or layer(s) onto or in (at least in part) the two-layer coating, followed (if necessary) by any of the activation methods just described. As with any of the other regions and/or layers, the treatment need not be uniform over the entire surface, nor does it have to cover the entire surface. Such non- uniformity of the treated region and/or layer may result from random fluctuations of the coating process or by conscious design of the fabricator or other person(s). The treatment step need not immediately follow the formation of the two-layer coating process; for example additional modification to the two-layer coating may occur, or additional modifications may occur on other portions of the device or apparatus of which the two-layer coating is a component, portion or feature. In addition, further modifications may occur to the two-layer coating even after the treatment step if the two-layer coating is otherwise accessible to chemical and/or biological agents, light, ions, heat, or other means of activation or modifying a two-layer coaling. Examples of chemical and/or biological agents include, by way of example only, flurorophores, antibodies, peptides, ligands, catalysts, reactive groups, oligonucleotides and oligonucleosides, oligosaccharides, electron donors and electron acceptors, or a combination of such chemical agents. In addition, the treated region and/or layer may undergo further processing or modification, or the device or apparatus of which the two-layer surface is a component, portion or feature may undergo further processing, manipulation or modification until the final device or apparatus is made.

As an additional option, the unfinished or finished device or apparatus of which the two-layer coating is a component, portion or feature may be appropriately stored until further needed. Preferably, such a storage step (or even storage steps) will not result in degradation of the two-layer coating: proper storage conditions may involve control of temperature, humidity, atmosphere, or other components that may impact degradation of the two-layer coating. Further, the unfinished or finished device or apparatus of which the two-layer coating is a component, portion or feature may be stored wet, or dry.

Finally, when needed, the device or apparatus of which the two-layer coating is a component, portion or feature may be used by the end user. Examples of components, portions or features of a device or apparatus that may be coated as described herein include the separation channel of a microfluidic device, the side channel of a microfluidic device, the wells of a plate or device, sections of an array, reaction channels in a microfluidic device, storage areas on a chip or device, and the inner or outer portions of a tube. Preferably, the stability of the two-layer coating is sufficient to allow multiple uses of the device or apparatus. Furthermore, different components, features, or portions of a device or apparatus can have similar or different types of coatings, depending upon the needs of the user. The methods and coatings described herein are flexible enough to allow both the customization and the mass-production of a desired device or apparatus.

Figures 2-4 show various schematic embodiments of the methods and compositions described herein. Figure 2 presents various possible configurations for at least a portion of a hydrophobic surface (any part of which may be modified, functionalized, and/or unmodified) coated with an amphiphilic polymer, precursor or monomer (any of which may be in part modified, functionalized, and/or unmodified) and with a charged polymer, precursor or monomer (any of which may be in part modified, functionalized, and/or unmodified). Various methods for achieving such coatings, as well as the characteristics of such coatings are described herein. Figure 3 presents various possible configuration for at least a portion of a hydrophobic surface (any part of which may be modified, functionalized, and/or unmodified) coated with a reactive hydrophobic polymer, precursor or monomer (any of which may be in part modified, functionalized, and/or unmodified) and a reactive charged polymer, precursor or monomer (any of which may be in part modified, functionalized, and/or unmodified). Various methods for achieving such coatings, as well as the characteristics of such coatings are described herein. Figure 4A presents various possible configurations for at least a portion of a hydrophobic surface (any part of which may be modified, functionalized, and/or unmodified) coated with a neutral polymer, precursor or monomer (any of which may be in part modified, functionalized, and/or unmodified). Various methods for achieving such coatings, as well as the characteristics of such coatings are described herein. Figure 4B presents various configurations for at least a portion of a hydrophobic surface (any part of which may be modified, functionalized, and/or unmodified) coated with a covalently attached polymer, precursor or monomer (any of which may be in part modified, functionalized, and/or unmodified). Various methods for achieving such coatings, as well as the characteristics of such coatings are described herein.

Figure 5, presents a schematic representation in which an entire flat hydrophobic surface is coated; however, an analogous procedure may be used for any smaller portion of the surface or for any form of surface, including porous surfaces, as well as recessed, curved, twisted or other possible configurations, including the inner surface or outer surface of a tube, channel or chamber. All that is required is that chemical agents can access by some means (including pressure, percolation and diffusion) the desired surface or region. Various methods exist in the art for coating portions of a surface, including the use of masks.

The initial surface, shown at the top of Figure 5 is a hydrophobic surface. A goal of the first step is to create an amphiphilic region and/or layer or coating on or in (at least in part) the hydrophobic surface. This coating process may (but need not) comprise multiple steps. At its most simplest embodiment, an amphiphilic polymer is applied to the hydrophobic surface. Such an amphiphilic polymer is comprised of a hydrophobic portion that forms an interaction (covalent, non-covalent, or otherwise) with the hydrophobic surface. Polar and even ionic groups that may be components of the amphiphilic polymer may also interact with the hydrophobic surface; however, the predominant (at the least, the plurality of interactions) is an attractive interaction between the hydrophobic components of the amphiphilic polymer and the hydrophobic surface. Many methods are available for contacting the amphiphilic polymer with the hydrophobic surface, including simply exposing the hydrophobic surface to a solution containing the amphiphilic polymer, or spin coating the amphiphilic polymer onto the hydrophobic surface, chemical vapor deposition, techniques involving aerosols, and application of the pure polymer onto the surface, either as a neat solution or in vapor phase. The method of simply exposing the amphiphilic region and/or layer to a solution of the charged polymer further allows for molecular organization of the charged polymer as in interacts with the underlying amphiphilic region and/or layer. Furthermore, these aforementioned deposition methods can be undertaken at room temperature, or elevated temperature. An additional rinsing step may be utilized to remove excess amphiphilic polymer or other materials. A drying step (effected by heat, vacuum or use of drying agents) may also be included to remove excess solvent or other materials from the amphiphilic coating. The amphiphilic coating may be obtained using a) amphiphilic polymers, b) precursors to ampliiphilic polymers, followed by formation of the amphiphilic polymer, or c) monomers for (a) or (b) above, followed by further reaction if needed to make the amphiphilic polymer.

A goal of the second step in Figure 5 is to create a charged region and/or layer or coating on or in (at least in part) the amphiphilic region and/or layer, whereby creating a stable charged "two-layer coating" on the hydrophobic surface. This coating process may (but need not) comprise multiple steps. In one embodiment, a polymer of opposite charge to that of the amphiphilic region and/or layer is applied to the amphiphilic region and/or layer on the hydrophobic surface. In the example shown in Figure 5 the amphiphilic region and/or layer contains negatively charged moieties, while the charged polymer contains positively charged moieties, thus creating a positively charged bilayer. However, as shown in figure 6, a negatively charged bilayer could be formed using an amphiphilic region and/or layer containing positively charged moieties, with the charged polymer containing negatively charged moieties. Such charged polymers are comprised of charged moieties that ionically interact with the amphiphilic region and/or layer. Hydrophobic components of the charged polymer may also interact with the amphiphilic region and/or layer; however, the predominant (at the least, the plurality of interactions) is an attractive interaction between the oppositely charged moieties of the amphiphilic polymer and the charged polymer. Many methods are available for contacting the amphiphilic region and/or layer with the charged polymer, including by way of example only, exposing the amphiphilic region and/or layer to a solution of the charged polymer, or spin coating the charged polymer onto the amphiphilic region and/or layer, chemical vapor deposition, techniques involving aerosols, and application of the pure charged polymer onto the amphiphilic region and/or layer. The method of simply exposing the amphiphilic region and/or layer to a solution of the charged polymer further allows for molecular organization of the charged polymer as it interacts with the underlying amphiphilic region and/or layer. Furthermore, these aforementioned deposition methods can be undertaken at room temperature, or elevated temperature. The charged coating may be obtained using a) charged polymers, b) precursors to charged polymers, followed by formation of the charged polymer, or c) monomers for (a) or (b) above, followed by further reaction if needed to make the charged polymer. An additional rinsing step may be utilized to remove excess charged polymer or other materials. A drying step (via heat, vacuum or use of drying agents) may also be included to remove excess solvent or other materials from the coating.

In Figure 5 is shown one embodiment of the method described herein in which the amphiphilic polymer is poly(styrene-alt-maleic acid) (PSMA) generated by base hydrolysis of poly(styrene-alt-maleic anhydride) (PSMAA) and purified prior to application onto the hydrophobic surface. The hydrophobic surface is exposed to a solution containing the amphiphilic polymer, PSMA, which adsorbs to the hydrophobic surface creating the initial amphiphilic region and/or layer. Subsequently, the PSMA region and/or layer is exposed to a solution containing the charged polymer poly(diallyldimethylammonium chloride) (PDADMAC), which ionically interacts with the amphiphilic region and/or layer creating the charged second region and/or layer on the hydrophobic surface. In this case the use of the methodology described above has modified the hydrophobic surface into a positively charged surface.

A further methodology, which incorporates the adsorption of modified amphiphilic polymers onto a hydrophobic surface, can also be used to create a positively charged, negatively charged, or neutral coating on the hydrophobic surface. Modification of amphiphilic polymers incorporates functionality into the amphiphilic polymer which can be used for subsequent attachment of a second polymer region and/or layer, thereby generating a neutral or charged region and/or layer on the modified amphilic region and/or layer. Attachment of the second polymer layer can be via electrostatic interaction or covalent linkage.

Figure 7A shows one possible approach to the method just described. In this example the amphiphilic polymer, poly(styrene-alt-maleic acid) (PSMA), is modified by reaction with 2-aminoethanol, and a hydrophobic surface is exposed to the modified amphiphilic polymer. With aqueous chemistry a coating containing amine functionality may be created on a hydrophobic surface. This modified amphiphilic layer may then be exposed to a cationic polymer, such as poly(3-chloro-2-hydroxyproρyl-2-methacryloxyethyl- dimethylammonium chloride, (PCHPMEDMAC), which has been activated by base treatment to functionalize the cationic polymer with epoxide moieties. The modified PCHPMEDMAC can electrostatically interact with the modified amphiphilic layer, and/or form covalent linkages.

Other functional groups may be incorporated into the PSMA polymer by reacting PSMAA with other nucleophiles. The use of a nucleophile, such as an alcohol, in the PSMA layer allows covalent crosslinking with cationic polymers that contain an electrophilic group, such as chlorohydrin. Additional covalent linkages may also be formed by methods known in the art; by way of example only, see the table of nucleophiles and electrophiles and the resulting covalent linkage presented above. Thus, the presence of electrophilic groups such as epoxides or chlorohydrins in the PSMA layer allows for covalent crosslinking of cationic polymers that contain nucleophiles such as alcohols or primary amino groups. Also, activation of the carboxylic acid groups of PSMA with a reagent like N-(3-dimethylaminopropyl)-N'-ethyl-carbodimide (EDC) allows the activated PSMA to be covalently crosslinked with nucleophiles such as amines or alcohols. Figure 7B presents examples of nucleophiles that have been incorporated into maleic anhydride polymers that may be used with such covalent attachment strategies.

Another method for producing a very stable positively charged, negatively charged, or neutral, coating on/into a hydrophobic surface, or at least part of a hydrophobic surface, uses a radical polymerization procedure. This procedure is similar to that described in Figure 1, however, rather than initially exposing the hydrophobic surface to an amphiphilic polymer, the hydrophobic surface is initially exposed to a polymerizable material which adsorbs on/into the hydrophobic surface. This polymerizable material contains hydrophobic regions, for interaction with the hydrophobic surface, and reactive moieties to accomplish covalent linkage (including co- polymerization) with neutral or charged reactive monomers, thus producing in effect an amphiphilic polymer. A possible embodiment of the method and compositions described herein is presented in Figure 8 in which the polymerizable material is initially adsorbed on/in the hydrophobic surface, and a charged monomer species that subsequently reacts with the absorbed polymerizable material. In this particular example n is equal to 14, however the value for n may from 2 to 30. In Figure 8 an entire flat surface is covered by the resulting amphiphilic polymer; however, an analogous procedure may be used for any smaller portion of the surface or for any form of surface, including porous surfaces, as well as recessed, curved, twisted or other possible configurations, including the inner surface or outer surface of a tube, channel or chamber. Chemical agents should be able to access by some means (including pressure, percolation and diffusion) the desired surface or region. Various methods exist in the art for coating portions of a surface, including the use of masks.

The initial surface, shown at the top of Figure 8 is a hydrophobic surface. A goal of the first step is to create a reactive layer or coating on or in (at least in part) the hydrophobic surface. This coating process may (but need not) comprise multiple steps. In one embodiment, a hydrophobic polymer with reactive moieties is applied to the hydrophobic surface. Such a hydrophobic polymer is comprised of a hydrophobic portion that forms an interaction (covalent, ήon-covalent, or otherwise) with the hydrophobic surface. Polar, and even ionic groups that may be components of the hydrophobic polymer may also interact with the hydrophobic surface; however, the predominant (at the least, the plurality of interactions) is an attractive interaction between the hydrophobic components of the hydrophobic polymer and the hydrophobic surface. Many methods are available for contacting the hydrophobic polymer with the hydrophobic surface, including simply exposing the hydrophobic surface to a solution of the hydrophobic polymer, or spin coating the hydrophobic polymer onto the hydrophobic surface, chemical vapor deposition, techniques involving aerosols, and application of the pure polymer onto the surface. An additional rinsing step may be utilized to remove excess hydrophobic polymer or other materials. A drying step (effected by heat, vacuum or use of drying agents) may also be included to remove excess solvent or other materials from the hydrophobic coating. This results in a polymeric coating on/in the hydrophobic surface which has pendent reactive moieties, such reactive vinyl groups, used for subsequent radical polymerization with a charged species.

A goal of the second step in Figure 8 is to create a charged layer or coating on or in (at least in part) the polymeric layer, whereby creating a stable charged bilayer on the hydrophobic surface. This coating process may (but need not) comprise multiple steps. In one embodiment, a charged monomer, or a charged polymer with reactive moieties is applied to the absorbed polymeric layer on the hydrophobic surface followed by subsequent free-radical polymerization. Initiation of the free-radical polymerization process may be accomplished using heat, exposure to UV, and any other method known in the art. In the example shown in Figure 8, 3- methylammonium propylmethacrylate (MAPTAC) is co-polymerized via free-radical polymerization to create a positively charged layer covalently attached to the hydrophobic layer adsorbed on/in the hydrophobic surface. Although, the example demonstrates formation of a positively charged bilayer, the same methodology can be used to create a negatively charged bilayer. An additional rinsing step may be utilized to remove excess materials not bound to the adsorbed polymeric layer on/in the hydrophobic surface. A drying step (effected by heat, vacuum or use of drying agents) may also be included to remove excess solvent or other materials from the bilayer coating.

The methods described above create a bilayer to modify the surface characteristics of a hydrophobic surface. However, the hydrophobic surface can also be modified by covalent attachment of positively charged, negatively charged, or neutral polymers to generate positively charged, negatively charged, or neutral layers, respectively, on the hydrophobic surface. In the case of polycarbonate, the phenolic functionality of the surface can be used for reaction with chlorohydrin modified polymers, thus creating any desired surface characteristic from a wide range of chlorhydrin modifiable polymers; either positively charged, negatively charged or neutral. Figure 10A- 1OB depict examples of this approach, in particular Figure 1OA shows the covalent attachment of poly(3-chloro-2-hydroxyproρyl-2-methacryloxyethyldimethylammonium chloride) onto polycarbonate, while Figure 1OB shows covalent attachment of polyethylene oxide derivatives to polycarbonate. Alternatively, chemistry can be performed on the residual chlorohydrin groups.

Yet another embodiment utilizing covalent attachment of neutral hydrophilic polymers to hydrophobic surfaces is, by way of example only, reacting poly(ethylene glycol-co-maleic anhydride) (PEG-AO-MaI) with a surface with available nucleophiles. Also, any amino reactive polyethylene glycol molecule could be used in a similar manner. This modification imparts a neutral hydrophilic coating on the hydrophobic surface, which yields minimal or no EOF. This modified surface is also useful for resisting adsorption of protein from solution.

Another example of direct covalent attachment to the hydrophobic surface is to react polycarbonate with copolymers containing oligo ethylene glycol groups and chlorohydrins. Another embodiment involves exposing hydrophobic surface to PSMA which has been functionalized with electrophilic groups. This modified surface is then reacted with polyethylene glycol bearing nucleophilic moieties, such as, by way of example only, amino-terminated polyethylene glycol, thus forming a bilayer with exposed hydrophilic moieties on the original hydrophobic surface. This embodiment is presented schematically in Figure 12. This approach may be extended to any hydrophobic surface that can be functionalized with electrophilic groups, including, by way of example only, chlorohydrides, carboxylates, aldehydes, and or ketones.

Figure 13 shows an embodiment for the generation of a trilayer. The example shown is for a neutral coating; however this approach may also be extended to creating positively charged or negatively charged coatings. In this embodiment, PSMA is used to coat a hydrophobic surface via hydrophobic interaction, the resulting surface is then exposed to a functionalized polyionic polymer which electrostatically interacts with the PSMA surface. To complete the trilayer, the functionalized polyionic polymer is reacted with functionalized polyethylene glycol. The functional group on the polyethylene glycol polymer can be nucleophilic or electrophilic, depending on the functional groups on the polyionic polymer.

- Alternatively, a simple surface modification method that can be used to modify the surface characteristics of hydrophobic surfaces involves the following procedure. For example, assuming material A has the desired characteristics and the surface of material B is to be modified to possess the property of material A. Material A is dissolved in a solvent which swells/attacks/penetrates material B and material B is then exposed to this solution. During the time of exposure, material A physically interpenetrates the surface networks of material B, becomes embedded in the surface of material B. After exposure to the material solution, material B is dried, leaving the surface blended with material A. By way of example only, the method can be used to modify the hydrophobic surfaces of poly(methyl methacrylate) (PMMA) or polycarbonate (PC) with hydrophilic polymers; poly (ethylene oxide) (PEO) or hydroxypropyl methyl cellulose (HPMC). These hydrophilic polymers are dissolved in either a solution of at least 50% isopropanol for the PMMA surface or at least 50% acetonitrile for the PC surface. The PMMA or PC surfaces are then exposed to the respective solutions and then dried. The contact angle of water on the subsequently modified surfaces is smaller than the un-treated surfaces, suggesting that the surfaces have become more hydrophilic after blending in the hydrophilic polymer. Figure 14 shows a schematic of the entrapment of HPMC in PMMA.

The surface of anhydride based copolymers, such as, by way of example only, poly(styrene-co-maleic anhydride) (PSMAA), are reactive towards nucleophiles, such as amino groups. Additional examples of other anhydride base copolymers and nucleophiles used to modify them can be found in Table 2 and Figure 7B, respectively. Furthermore; these copolymers can be pressure molded into any desired configuration and used as the bulk material for a component or apparatus of interest. For example, PSMAA can be pressure molded to form microfluidic channels in a microfluidic apparatus. Treatment of the PSMAA surface with a polyamine under basic conditions covalently attaches the polyamine and generates a stable, hydrophilic surface in a one step procedure. This procedure can be applied prior to/or after sealing of the molded parts to create the microfluidic channel. Sealing of the molded parts with a cover plate can be achieved using lamination, ultra¬ sonic welding, and thermal bonding,^" or any other technique known to one skilled in the art. Reaction with a polyamine generates a positive charged surface; however, reaction of the PSMAA with an amino functionalized PEG derivative can generate neutral surfaces. MICROFLUIDIC DEVICES

Microfluidic chips are often constructed using conventional semiconductor processing methods including photolithographically masked wet-etching and photolithographically masked plasma-etching, or other processing techniques including embossing, molding, injection molding, photoablating, micro-machining, laser cutting, milling, and die cutting. These devices conveniently support the separation and analysis of sample sizes that are as small as a few nanoliters or less. In general, these chips are formed with a number of microchannels that are connected to a variety of reservoirs containing fluid materials. The fluid materials are driven or displaced within these microchannels throughout the chip using electrokinetic forces, pumps and/or other driving mechanisms. The microfluidic devices available today can conveniently provide mixing, separation, and analysis of fluid samples within an integrated system that is formed on a single chip. There are numerous design alternatives to choose from when constructing an interface for microfluidic chips and electrospray ionization mass spectrometers. Some electrospray ionization interfaces include microfluidic chips that attempt to spray charged fluid droplets directly from the edge of the chip. But the accompanying solvent is known to wet much of the edge surface of the chip so as not to offer a high-stability spray for many applications. Other attempts to spray ionized particles directly from the edge of a microfluidic chip edge therefore rely on the formation of a hydrophobic surface that can yield improved spray results; however, even that often proves to be insufficiently stable. At the same time, adequate results can be also achieved with other chip devices that incorporate fused silica capillary needles or micro-machined or molded tips. In particular, some recent electrospray ionization designs incorporate small silicon etched emitters positioned on the edge of a microfluidic chip. While it is possible to generate a relatively stable ionization spray for mass spectrometric analysis with some of these microfiuidic devices today, they generally require apparatus that is relatively impractical and economically unfeasible for mass production.

In one aspect described herein, are methods for providing coatings for multi-channel microfiuidic chips and devices; examples of such chips and devices are described in U.S. Patent Application Serial Nos. 10/649,350 and 10/871,498, which are herein incorporated by reference in their entirety. One embodiment provides microfiuidic chips that are formed with individual fluid channels. Such fluid channels extend through the body of the microfiuidic chip and converge at a common distal tip region. The distal tip region includes an open-ended distal tip formed along a defined surface of a microfiuidic chip body. The microfiuidic chip may be constructed from a pair of polymer plates in which the converging channels run through and lead up to the distal tip region. The microfiuidic chip can be also formed with multiple but separate channels that supply fluids such as samples and sheath flow solutions to a single common electrospray tip. One method for achieving the interface between the microfiuidic device and a mass spectrometer is illustrated by the three-dimensional representation in Figure 15. In Figure 15, a microfiuidic chip 10 for electrospray ionization (ESI) applications is formed with multiple fluid channels 12 converging at a distal tip region 14. The fluid channels 12 may be formed on a substrate layer 16 of the chip 10 that is composed of glass, quartz, ceramic, silicon, silica, silicon dioxide or other suitable material such as a polymer, copolymer, elastomer or a variety of commonly used plastics. The channels 12 can be created using a variety of methods, such as conventional semiconductor processing methods including photolithographically masked wet-etching and photolithographically masked plasma-etching, or other processing techniques including embossing, molding, injection molding, photoablating, micro-machining, laser cutting, milling, and die cutting. A variety of channel patterns and configurations may be also selected for the channels, including channels having a substantially rectangular, trapezoidal, triangular, or D-shaped cross-section. For example, these channels may be produced with an anisotropically etched silicon master having a trapezoidal or triangular cross-section. A channel having a D-shaped cross-section may be formed alternatively following isotropic etching processes. The pair of channels 12 formed on the substrate layer 16 can run relatively non-parallel as shown with respect to each other which substantially converge at the distal tip region 14. A cover plate 5 can be bonded to the substrate layer 16, whereby sealing the cover plate 5 onto the substrate 16 and enclosing the channels 12. The cover plate 5 is formed so as to terminate at the end of the channels 12 at the distal tip region 14. The distal tip region 14 of the ESI tip 15 may be formed with an open-ended construction where different fluids can emerge or emit therefrom for analysis by a mass spectrometer or other analytical apparatus or detection method. In addition, the open distal tip region 14 can be created in the embossed substrate layer 16 or in the cover plate 5.In another aspect described herein, are coating methods that may be used with multi-channel microfiuidic chips and devices that additionally have features to provide improved fluid flow control, with or without using sheath flow for electrospray stability. As an additional aspect described herein are the microfiuidic chips and devices that include the feature that provide improved fluid flow control, with or without using sheath flow for electrospray stability. Reliable methods and apparatus are provided for achieving stable electrospray with or without sheath flow on microfiuidic chips. The microfiuidic chips include (1) separation or main channels with charged coatings and side channels with charged coatings or without coatings that maintain stable separation and electrospraying; (2) separation or main channels with neutral coatings and modified side channels with charged coatings that maintain stable separation and electrospraying during application of a sheath flow as provided herein. The side channels can be used for sheath flow assisted electrospray, or sheathless electrospray. For the application of sheathless electrospray, the function of the side channel is to establish electrical contact and whereby allow for generation of an electrospray. These techniques and microfluidic devices can assist in system automation, and reduce system complexity. At the same time, the electrospray devices provided with such an embodiment can increase system reliability and allow for relatively longer separation times. The sheath flow provided by the microfluidic side channels can be driven by pressure and/or electroosmotic flow. The microfluidic chips and devices used for electrophoresis, for example, those described in U.S. Patent Appl. No. 10/649,350, can be coupled with a mass spectrometer to deliver an electrospray by either sheath flow assisted techniques or sheathless flow.

For sheathless applications, an electrospray may be achieved by conventional methods such as pressure or electroosmotic flow (EOF) in a separation channel. Meanwhile, when a sheath flow is applied as with certain applications of the invention herein, a more stable electrospray can be observed that can facilitate system optimization and calibration. In the past, sheath flow was initially used in capillary CE/MS systems and was later adopted for microchip-based CE/MS platforms such as those herein. By inserting a capillary tube to the chip to serve as an extension of the microchannel, a sheath flow interface with the capillary can be provided to assist and stabilize electrospraying from a microfluidic chip. Usually a syringe is connected to a sheath flow channel through Upchurch fitting or other acceptable fixtures, and a metal connector is placed in a fluid line positioned between a well or reservoir in a microfluidic chip and the syringe. However the following problems and other issues arise with this conventional setup which is addressed by this aspect of the invention: (1) bubbles will be often generated in the line during the electrophoresis and electrospray, and these bubbles could terminate the experiment under certain conditions such as when the applied current is > 5 μA; and (2) the reliable sealing of the sheath flow loop could pose a problem and leak.

Figure 16A illustrates a sheath flow configuration or set-up that may be incorporated with microfluidic devices including those provided elsewhere herein to provide more reliable separation and electrospray. In this configuration, four electrodes may be selected to provide fluid control within the device including a sheath flow emanating from a side channel via EOF to achieve bulk movement of aqueous solutions therein past stationary channel wall surfaces upon application of an electric field, that is upon application of current or voltage. To provide sheath flow via EOF action, an electrode is dipped in Well #3 that is in fluid communication with a side channel. Figure 16A also illustrates a configuration or set-up for separation and sheathless electrospray from microfluidic chips. In this case, the side channel is only used for electric contact. A coating selected for the side channel can be positive, negative, neutral, or no coating based on the surface charge states in the main separation channel, or channels. As illustrated in Figure 16B, the side channel may be coated negatively, neutrally, or no coating when a main separation channel has a positive coating (positive ion mode), or the side channel may be coated positively, neutrally, or no coating when a main separation channel has a negative coating (negative ion mode). In another preferable embodiment, as shown in Figure 16C, the side channel may be coated positively (positive ion mode) or negatively (negative ion mode) when the main separation channel includes a neutral coating or no coating at all (non-coating). The positive, negative or neutral charge coatings herein can be formed by lining channel walls as already described above. The desired electrical parameters, such as current, voltage, or power, selected for the separation of a sample in the main channel and electrospraying at the device tip are achieved by selectively applying a combination of voltages or currents in Wells #1, #2, #3 and #4. The presence of bubbles often generated on the electrodes during the separation and electrospray will therefore not readily enter into the channels of the microfluidic chip, if at all, and will thus not affect significantly or terminate a separation process. It shall be understood that these channel configurations may be formed in the body or channel layer portions of microfluidic chips herein and combined with other systems and aspects of the invention described throughout this disclosure. Figure 17 shows another variation of the invention that includes a four electrode approach but with two side channels for both sheath flow and electrical contact. As shown in other portions of this specification, multi¬ channel microfluidic chips herein can include channel layers formed with a plurality of separation and/or side channels to support various electrospray related functions. In this illustrated embodiment, a first side channel connected to a Well #5 is used for providing the sheath flow through a syringe, and a second side channel is mainly for electrical contact by dipping an electrode in corresponding Well #3. This configuration allows the sheath flow to change flexibly and allows for system optimization more easily and more reliable electrospray. To prevent the separated charged species of the separation channel from entering into the side channel leading from Well #3 in this case as illustrated, the side channel can be coated in the same way as previously described with Figures 16A-C. Moreover, this separation/side channel configuration can provide a sheath flow using a syringe that is connected to Well # 5 and its respective side channel and/or via EOF in another side channel connected to Well #3 that includes the electrode dipped into therein. Alternatively, the side channel connected to Well #5 can be also coated to prevent the separated charged species from the separation channel from entering therein. These coating can be positive, negative, neutral, or no coating at all based on the surface charge states in the main separation channel as explained previously. For certain applications, the separation channel may remain uncoated or contain a neutral uncharged coating. The desired electrical parameters, such as current, voltage, or power, required for the separation in the main channel and electrospray at the tip can be also achieved by applying voltages or currents in Wells #1, #2, #3 and #4 as described previously.

Figure 18 describes another variation of the invention to provide a multi-channel chip with sheath flow similar to those previously described except that an integrated electrode is positioned at the tip (3'). This alternative design and method of electrospraying employs five electrodes in total and can provide direct control in the separation and electrospray electrical parameters. The task of electrospray optimization can be thus accomplished much easier with this configuration. Sheath flow can be provided by EOF in a side channel connected to Well #3 where an electrode is dipped therein. A positive or negative charged coating can be applied to the side channel walls leading from Well #3 in order to prevent charged species from entering therein. The electrical parameters, such as current, voltage, or power, required to effect separation in the main channel and electrospray at the tip can be achieved by applying voltages or currents in Wells #1, #2, #3, #4 and at electrode 3'. Methods are thus provided herein for improved fluid control in a microfluidic chip with sheath flow for enabling separation and more stable electrospray. A microfluidic chip or device may be selected as an initial step having a separation channel and at least one side channel for providing sheath flow. The side channel may include a positively or negatively charged coating with molecules having groups of suitable charges exposed to sheath flow solutions therein. A sample may be introduced into a fluid well on the chip and directed to the separation channel whereupon electrical parameters can be applied to a network of wells and channels through a series of electrodes so that selected components therein can be electrophoretically separated and emitted from the microfluidic chip as an electrospray into a mass spectrometer for analysis. The separation process and stable electrospray can be therefore achieved substantially without any of the charged species from the separation channel from entering the side channels having positively or negatively charged coatings. It shall be understood that the application of voltages or currents to create electric fields can be carried out using known microfluidic control systems.

Figure 19 is photograph illustrating the selective coating of the separation channel, relative to the side channel, in which the separation channel has been coated with PSMA labeled with bodipy and then this fluorescent coating was electrostatically coated with PDADMAC. Similarly, Figure 20 also illustrates the selective coating of the separation channel, relative to the side channel, however, in this example the separation channel has been coated with unlabeled PSMA and tins coating was electrostatically coated with bodipy labeled MAPTAC. Both images show that the separation channel is selectively coated, while the side channel remains uncoated. Although theses photographs illustrate the ability to obtain selective coatings, it may be desirable to manufacture and utilize the microfluidic devices described above with both the separation channels and side channels having a positive coating. Additionally, it may be desirable to manufacture and utilize the microfluidic devices described above with both the separation channels and side channels having a negative coating. Further, it may be desirable to manufacture and utilize the microfluidic devices described above with both the separation channels and side channels having a neutral coating. Still further, it may be desirable to manufacture and utilize the microfluidic devices described above with both the separation channels and side channels uncoated. Additionally, it may be desirable to manufacture and utilize the fnicrofluidic devices described above with the separation channels having a negative coating and the side channels uncoated. Further, it may be desirable to manufacture and utilize the microfluidic devices described above with the separation channels having a negative coating and side channels having a neutral coating. Still further, it may be desirable to manufacture and utilize the microfluidic devices described above with the separation channels having a positive coating and the side channels having a negative coating.

Figure 21 shows an electropherogram of a mixture of proteins using mass spectrometric detection. The microfluidic device used for this exemplary separation utilized a separation channel selectively coated with PSMA/PD ADMAC, and an uncoated side channel. In this example the side channel was used as a means to provide electrical contact to the electrospray tip.

The stability of the PSMA/PD ADMAC coatings is shown in Figures 22 and 23. In Figure 22 the migration time of bodipy-labeled ubiquitin and bodipy labeled Angiotensin I as function of days stored is shown, while, in Figure 23, the number of theoretical plates for bodipy-labeled ubiquitin and bodipy labeled Angiotensin I as a function of days stored is shown. See example 11 for details. The data suggests that the bilayer as produced is stable for at least 60 days.

Another aspect described herein is the improved microfluidic devices and methods for making and using such devices to provide one or more substances to a mass spectrometer (MS) for analysis. The microfluidic devices generally include a substrate and a cover (or a substrate having first and second surfaces or the like), at least one microchannel formed by the surfaces, an outlet at an edge of the surfaces, and at least one electrical potential source. In various embodiments of the present invention, different features of the substrate, cover, outlet and/or electrical potential source are configured to enhance electrospray ionization (ESI) of substances from a microfluidic device to a MS device for analysis.

Figures 24A and 24B are schematic illustrations of a side view and a top view, respectively, of a microfluidic device 100 comprising a substrate 102 and a cover 104. (The device 100 is not drawn to scale.) The substrate 102 includes a one or more wells 110, into which substance(s) may be deposited, and a one or more microchannels 108 through which substance(s) may be directed and in which substance(s) may be separated into constituent parts. At least one of the microchannel 108 is typically in fluid communication with an outlet 113 to allow egress of substance(s) from the microchannel 108. The cover 104, arranged on a surface of the substrate 102, may extend beyond an edge of the substrate 102 to form an ESI tip 106. As shown in

Figure 24B, an electrospray 112 of one or more substances may be provided from the ESI tip 106, to deliver the substance(s) to a MS device.

The term "substrate" as used herein refers to any material that can be microfabricated (e.g., dry etched, wet etched, laser etched, molded or embossed) to have desired miniaturized surface features, which may be referred to as "microstructures." Microfabricated surfaces can define these microstractures and other, optionally larger structures. Microfabricated surfaces and surface portions can benefit from a dimensional tolerance of 100 μms or less, often being 10 μms or less, the tolerances of the microfabricated surfaces and surface portions more generally being significantly tighter than provided by dicing (substrate cutting or separating) techniques that may define adjacent portions and surfaces. Examples of microstructures include microchannels, which are described in further detail below. Microstructures can be formed on the surface of a substrate by adding material, subtracting material, a combination of both, pressing, or the like. For example, polymer channels can be formed on the surface of a glass substrate using photo-imageable polyimide.

The substrates herein, may comprise any suitable material or combination of materials, such as but not limited to a polymer, a ceramic, a glass, quartz, fused silica, a metal, a composite thereof, a laminate thereof, or the like. Examples of polymers include, but are not limited to, polyimide, polycarbonate, polyester, polyamide, polyether, polyolefin, polymethyl methacrylates, cyclo-olefϊn polymer, other acrylic polymers, polyurethanes, polyacrylonitrile-butadiene-styrene copolymers, polystyrene, polyfluorocarbons, and combinations thereof. Furthermore, substrates may suitably comprise one layer or multiple layers, as desired. When multiple substrate layers are provided, the layers will often be bonded together. Suitable bonding methods may include application of a combination of pressure and heat, thermal lamination, pressure sensitive adhesive, ultrasonic welding, laser welding, and the like. Generally, the substratescomprise any suitable material(s) and may be microfabricated by any suitable technique(s) to form any desired microstructure(s), shape, configuration and the like.

The term "cover" as used herein refers to one or more layers of any suitable material disposed on a surface of a substrate. In various embodiments, the cover 104 may be disposed on an upper surface, a lower surface (as in Figure 24A and 24B), or any other suitable surface of the substrate 102. In some embodiments, the cover 104 encloses the microchannels 108. The Cover generally comprises any suitable material, such as the materials described above in reference to the substrates. Thus, cover may comprise a polymer, a ceramic, a glass, a metal, a composite thereof, a laminate thereof, or any other suitable material or combination. As is described further below, in various embodiments the cover may comprise a simple, planar component without notable surface features, or may alternatively have one or more surface features, outlets or the like. Typically, the cover is bonded to the substrate, and such bonding can be achieved by any suitable method.

As mentioned above, in some embodiments the substrate 102 includes one or more of the microchannels 108, at least one of which is in fluid communication with the outlet 113. The microchannel (as with all microfluidic channels described herein) will often have at least one cross-sectional dimension (such as width, height, effective dimensions or dimensions) of less than 500 μm, typically in a range from 0.1 μm to 500 μm. As shown in Figure 24B, the substrate 102 may include a plurality of the microchannels 108 defining one, two, or more than two intersections. Typically, substances are moved through the microchannels 108 by electric charge, where they also may be separated, and the substances then exit the device 100 via the outlet 113 in the form of the electrospray 112 directed towards a mass spectrometer or other device. In some embodiments, the outlet 113 may be located in a recessed area, which is recessed from an edge 103 of the device 100. The recessed area generally serves the purpose of protecting the ESI tip 106, which extends beyond the outlet 113, from being damaged or broken during manufacture or use. The ESI tip 106, in some embodiments, may include a hydrophilic surface 110, such as a metalized surface, which may help form a desirable configuration of an electrospray, such as a Taylor cone.

In some embodiments, the microfiuidic device 100 includes at least one hydrophilic surface and at least one hydrophobic surface. Either type of surface may be used in portions of the substrate 102, the cover 104 or both. Generally, such hydrophilic and hydrophobic surfaces allow substances to be sprayed from the device in a desired manner, for example to direct fluidic substance(s) toward the MS device while preventing the substance(s) from exiting the outlet from spreading along the edge or the surface of device. At the same time, the hydrophilic surface on the microchannel 108 and/or tip 106 may help keep fluidic substance(s) generally moving along a desired path defined by the microchannel 108. Thus, a combination of the hydrophilic and the hydrophobic surfaces may be used to enhance ESI of substances to the device such as a mass spectrometer. For further description of such hydrophilic and hydrophobic surfaces, reference may be made to U.S. Patent Application Serial No. 10/794,572, entitled "Microfiuidic Devices and Methods," filed March 4, 2004, the full disclosure of which is hereby incorporated by reference.

Figures 25A-25E depict portions of two embodiments of a microfiuidic device 210, 220 which are shown from a top view. These Figures demonstrate a simplified method for making the microfiuidic devices 210, 220. Figure 25A illustrates one embodiment of a substrate 212, having a microchannel 213 with a widened outlet 214. The substrate 212 tapers as it approaches the outlet 214, as is the case in many embodiments. Figure 25B shows another embodiment of a substrate 222, this embodiment including a microchannel 223 with a widened outlet 224, as well as an additional microchannel 226 with an outlet 227.

Referring to Figure 25C, and as designated by the arrows with + signs, either substrate 212, 222 may be coupled with a cover 215 having an electrode 216 and a tip 217. The electrode 216 may comprise, for example, a conductive wire, a laminated metal trace, or the like. Figure 25D illustrates the cover 215 coupled with the first substrate 212, and Figure 25E shows the cover 215 coupled with the second substrate 222. In either embodiment, the electrode 216 of the cover 215 extends over the widened outlet 214, 224 of the substrate 212, 222. In some embodiments, the electrode 215 may also extend over the additional outlet 227. The widened outlet 214, 224 help to focus the electric field at the tip 217 for providing a desired electrospray while significantly reducing the possibility of an electric discharge between the electrode 216 and a counter electrode of a mass spectrometer orifice. Positioning the electrode 216 at the widened outlet 214, 224 also helps reduce the amount of bubbles generated in fluidic substances exiting the outlet 214, 224, since the electric field present in the fluid is reduced in proportion to the amount of widening. Embodiments like those shown may be used with or without electroosmotic flow. Figures 26A-26I illustrate another method of making various embodiments of a microfluidic device 240, 250. Figure 26A shows a tapered portion of a substrate 242 having one microchannel, while Figure 26B shows a tapered portion of another embodiment of a substrate 252 having three microchannels 253. In Figure 26, a cover 244 having a nib tip 245 is arranged on a surface of either substrate 242, 252, to form the substrate/cover combinations shown in Figures 26D or 26E. A conductive wire electrode 246 is then attached to the surface of the cover 244 that is opposite the substrate 252 to form the microfluidic device 240, 250. Figure 26F is a top view of the first embodiment, showing the electrode 246 tip disposed in the nib tip 245 of the cover 244. Figure 26G is a bottom view of the first embodiment, showing the electrode 246 attached to the bottom surface of the cover 244. Figures 26H and 261 are top and bottom views, respectively, of the second embodiment. Either embodiment may be used with or without electroosmotic flow.

Figures 27A-27D illustrate two alternative embodiments for making a microfluidic device 270, 280. Referring to Figures 27A, the tapered portion of one microfluidic device 270 includes a substrate 272 having a microchannel 273 and a cover having a tip 274. An electrode 275 may be attached to the bottom of the cover (not visible) such that a hooked portion of the electrode protrudes through the tip 274, as shown in Figure 27C. In another embodiment, illustrated in Figures 27B and 27D, a tapered portion of a microfluidic device

280 includes a substrate 282 having multiple microchannels 283 and a cover having a tip 284. An electrode 285 configured as a flat plate with a post member 286 may be attached to the bottom surface of the cover (not visible), such that the post member 286 protrudes through the tip 284, as in Figure 27D. In either of the two embodiments just described, either the linear, hooked electrode 275 or the plate with post electrode 285 may be used. In various alternative embodiments, the electrode may have any other suitable configuration, size, shape or the like and maybe made of any suitable material or combination of materials.

Figures 28A-28D illustrate another embodiment of a microfluidic deviceas follows. In Figures 28A- 28C, a tapered portion of a substrate 302, 312, 322 is shown, having various configurations and numbers of a microchannels 308, 318, 328 and coupled with a cover having a tip 304, 314, 324. In one microchannel 308, 318, 328 of each embodiment, a well 306, 316, 326 is disposed. In various embodiments, the well 306, 316, 326 may be placed in any suitable microchannel 308, 318, 328. In these embodiments, the well 306, 316, 326 provides the electrode function.

Figure 28D illustrates an electrode well 334 in further detail. The well is generally a hole formed in the substrate. Disposed in the well are a membrane 332 and a fixture 335 made of any suitable material and having any suitable configuration to hold the membrane 332 in place at the bottom of the well 334. A fluid 336, typically a buffer solution, is disposed in the well, and an electrode 330 is placed in contact with the fluid 336. The well 334 is in fluid communication with a smaller dimensions hole 337 in the substrate, which in turn is in fluid communication with a microchannel 338 of the substrate. However, the membrane 332 is configured to hold the fluid 336 within the well and prevent its passage into the hole 337. The membrane 332 includes nanopores to allow passage of ions but not other substances from the well 334 into the hole 337. In one embodiment, for example, the membrane 332 comprises a nanoporous polycarbonate material. Ions can pass through such a membrane 332 and continue along the path of the microchannel 338, thus providing the electrospray ionization function.

Figures 29A and 29B illustrate another embodiment of a microfluidic device 350 which includes multiple wells and multiple microchannels 355, 356, with no electrode immediately at a tip 358 of the device 350. Separation of substances in the separation microchannel 355 and electrospray at the tip 358 are achieved by applying a voltage to a well 1 351, which contains separation buffer, and a well 3 353. The second microchannel 356 coupled with the well 3 353 may be a sheath flow channel in some embodiments, while in other embodiments second microchannel may not have flow. Alternatively, voltage may be applied to well 1 351, well 2 352, well 4 354, and well 3 353. The applied voltages may be determined, for example, based on conductivity of the buffer solution, the dimensions of the separation microchannel 355 and/or the second microchannel 356, the electrospray needs at the tip 358, the electrospray mode (positive or negative), the separation performance, and the separation time window, and/or the like. In this embodiment, for charged coating in the sample loading and separation channels, it will often be desirable to limit or eliminate the amount of liquid flow in the second microchannel 356, or to influence its direction, in order to avoid analytes of interest moving into second microchannel 356, instead of being sprayed into a mass spectrometer. For neutral coating in the sample loading and separation channels, the second microchannel 356 is coated with charged molecules in a control way to provide the solution for the electrospray from the tip and minimize the dilution at the tip 358. This can be accomplished by a variety of methods, such as coating the walls of second microchannel 356 with a coating different from that used in the rest of the device. For instance, coating the channel 355 with a positive wall coating and the second microchannel 356 with a negative wall coating will result in an electroosmotic fluid flo^~w coming from both the channel 355 and the second microchannel 356 and flowing to the tip 358 when a positive voltage is applied to the well 3 relative to the voltage in the well 1. Alternatively, a neutral coating may be used in the second microchannel 356, with a positive coating elsewhere (or alternatively no coating, if the uncoated surface has sufficiently low electroosmotic flow). Another method to avoid loss of analyte in the channel 356 is to place in that channel a membrane, gel, viscous solution, or any other component that allows the passage of electrically charged ions, but that stops or reduces liquid flow. Examples of such a substance are a cross-linked polyacrylamide, an agarose gel, or a viscous polymeric solution such as linear polyacrylamides, cellulose polymers, polyethylene oxide, polyvinylpyrrolidone, or other hydrophilic polymer solutions. Another aspect of the invention provides dry electrodes that may offer certain advantages over microfluidic devices or chips configured with wet electrodes and plated through holes or vias. For example, Bousse et al., U.S. Patent Application Serial No. 11/031,963 filed on Jan 6, 2005 entitled "Electrospray Apparatus with an Integrated Electrode" which is incorporated by reference herein in its entirety, includes a description of microfluidic CE/ESI chips utilizing wet electrodes which are placed into already filled wells in the chip. Performance of the chip both from a CE and ESI standpoint can be influenced by the displacement of fluid and the meniscus change that occurs when the wet electrodes are inserted from above via a carrier assembly. The length of the electrode should be carefully controlled as they are easily damaged resulting in the possibility of experimental error. As the electrodes are often formed of platinum, they are generally far too expensive to replace for every test and as aforementioned, the height must be set carefully to minimize risk of damage. An added issue with wet electrodes is that the possibility of cross-contamination exists if they are not property cleaned between chips, each of which may have a different assay, serum or other sample sensitive to cross-contamination. Additionally, incorporating a separate electrode assembly can also complicate the sample automation eventually intended for a mass spectrometer diagnostic system. Adding motion control axes(s), along with an added wash station, and perhaps an added station for a preliminary/preparatory run before the actual tests, may add considerable cost and complication to an automation system. An embodiment of the invention herein provides externally contacted, dry electrodes which can be easily connected to a chip via spring contact pins such as "POGO" pins. These dry electrodes can be mounted in the base of a chip holder and connect automatically when the chip is inserted into its holder. A series of one or more surface electrodes may be provided on one or both of the layers of the chip. Accordingly, plated- through holes or vias are therefore not required in accordance with this aspect of the invention.

Electrode material may be deposited onto the surface of a layer by gold plating or any usual coating technique, including but not limited to sputtering, evaporation, or vapor deposition. A shadow mask can be typically used to define the coated area with relatively high degree of accuracy. A chrome, titanium, or layer made of similar material can be preferably applied for adhesion prior to the application of gold plating. It shall be understood that materials other than gold such as platinum, aluminum, etc. could also be used if desired. The thickness of the plating is preferably very thin, on the order of angstroms. Screen printing could also possibly be used by molding a series of recesses into the layer which can define the screen printing and control its features more accurately. Several exemplary embodiments of the invention are described further below.

A preferable embodiment of the invention includes a microfluidic device wherein a bottom side of a top layer is plated with an electrode and a contact lead pattern as shown in Figure 30. This bottom view of the top layer 400 depicts the contacts 402 in a back region of the chip, but it shall be understood that they could exit to any side as best desired for optimum mounting, ease of use, and automation. Fluid contact in this case is provided through the edge of the plating around well holes 404 and there is no plating in the region around the actual capillary channels. High voltage may be applied via spring pins that can make electrical contact with the electrodes through holes formed in the bottom layer.

In another embodiment of the invention, as shown in Figure 31 , a microfludic chip can be fabricated with a top layer 410 in a similar manner as described above in combination with a relatively shorter bottom layer. Instead of forming through holes therethrough, the bottom layer may be left shorter to allow relatively easy access to the electrode contacts. While the figure shows the contacts 412 exiting from the rear, again it shall be understood that contacts could also exit from the sides as desired. This embodiment of the invention may simplifiy the mold design for the layers by minimizing the forming of through holes, but at the same time an added level of complexity may be encountered when bonding or otherwise joining the layers together as in other embodiments of the invention herein.

For certain applications of the invention when relatively greater electrode contact area is preferred, the exposed edge of well hole plating may not be adequate. Accordingly, a full contact area under a capillary well can be provided whereby a coating of gold or other suitable materials can be applied to a bottom layer prior to lamination in the area under the well. An overlapping contact would then be made to top layer contacts that were shown in the earlier options. Although application of this coating involves another step in the manufacturing process and some added degree of complexity, a larger area of electrical conductivity can be thus provided as called for in certain applications of the invention.

Another alternative embodiment of the invention provides a relatively more simplified fabrication process involving the step of coating a bottom layer of the chip with a contact and electrode pattern. In this variation of the invention, selected spring pins may achieve electrical contact from above rather than an integral part of a chip mounting base. Accordingly, this series of through holes or vias formed in a top layer or a shortened top layer as described above would likely involve a carrier assembly as used presently with wet electrode configurations. Nevertheless this embodiment utilizing dry electrodes would still obviate some of the shortcomings of wet electrodes such as the aforementioned meniscus and cross-contamination issues. Additional adjustments and steps may be also involved with some aspects of the mounting and automation processes, but may ease fabrication requirements and well represent an intermediate step in dry electrode technology for CE/ESI spray chips.

One further embodiment of the invention with regards to replacing the sheath electrode, which is used to provide high voltage, but yields a significant voltage drop due to the sheath channel length. For example, the sheath electrode may be replaced as described in Bousse et al., U.S. Patent Application Serial No. 11/031,963 filed on Jan 6, 2005 entitled "Electrospray Apparatus with an Integrated Electrode" which is incorporated by reference herein in its entirety. Instead of using this electrode and channel, it may be possible to provide either a shorter sheath channel with an electrode near the tip, or possibly even eliminate the dual (sheath) channel chip design, by placing an electrode near the exit of the capillary near the spray tip. In this case, the electrode could be positioned on the bottom or top layer, and located in the channel to assure contact at the desired point. If the top layer is coated, the actual channel would have gold plating in it in a conformal manner. A selected number of leads and a contact pad would then come out as detailed in previously described embodiments above with a bottom or top contact as desired. Such a design would give closer and more accurate control of electrospray voltage. It may be therefore possible to eliminate a sheath channel and well altogether in this embodiment that allows lower voltages to be used due to the lack of voltage drop.

It shall be understood that the embodiments of the invention described herein referring to a top or a bottom layer should not be viewed in limiting sense. The terms "top" and "bottom" are used from a relative point of view and perspective that may vary so they should be considered interchangeable in certain applications herein. Several exemplary embodiments of microfluidic devices and methods for making and using those devices have been described. These descriptions have been provided for exemplary purposes only and should not be interpreted to limit the invention in any way. Many different variations, combinations, additional elements and the like may be used as part of the invention without departing from the scope of the invention as defined by the claims.The following examples are provided to further illustrate our devices, compositions and methods and are not provided to limit the scope of the current invention in any way.

EXAMPLES Example 1 : Preparation of PSMA-PD ADMAC Coating

Materials and solvents were analytical grade or better and were purchased from commercial vendors unless otherwise noted. 1,14-tetradecanediol dimethacrylate, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), glycidol, TEMED, [3-(methacryloylamino)proρyl]trimethylammonium chloride solution (MAPTAC), poly(diallyldimethylammonium chloride, poly(styrene-alt-maleic anhydride) (PSMAA), poly(styrene-co-maleic anhydride), 2,3-dihydrofuran, 2-aminoethyl methacrylate hydrochloride, poly( ethylene glycol) methyl ether methacrylate, 4-aminobenzophenone, octanohydrazide (fix in Figure 7B), (2-aminoethyl)trimethylammonium chloride hydrochloride, 3-amino-l-propanesulfonic acid, 8-aminooctanoic acid, 1-octanamine, N₅N- dimethylethylenediamine, N,N'-dimethylethylenediamine, 3-chloro-l,2-propanediol, (hydroxypropyl)methyl cellulose, branched polyethyleneimine (PEI), 4-acryloylmorpholine and ammonium persulfate as well as all peptides and proteins were purchased from Sigma/Aldrich/Fluka (also referred to herein as "Aldrich") (Milwaukee, WI); HPLC grade water (Burdick and Jackson), acetone (Burdick and Jackson), isopropanol (Burdick and Jackson), and sodium hydroxide (Baker) were purchased from VWR Scientific; 2-aminoethanol was purchased from TCI America. Poly(2-hydroxy-3-methacrylox3φropyl-trimethylammonium chloride), poly(3-chloro-2-hydroxypropyl-2-methacryloxyethyl-dimethylammonium chloride) (PCHPMEDMAC) and poly(ethylene glycol) methyl ether methacrylate were purchased from PolySciences Inc. Warrington, PA. N- (4,4-difluoro-5,7-dimethyl-4- bora-3a,4a-diaza-s-indacene-3- propionyl)cysteic acid, succinimidyl ester, triethylammonium salt

(BODIPY® FL, CASE, cat.#D6141) and 4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-propionyl ethylenediamine, hydrochloride (BODIPY® FL EDA cat.#D2390) were purchased from Molecular Probes, Eugene, OR. AO-MAL was purchased from Shearwater polymers, now Nektar Therapeutics.

Table 1. Name, structure and potential source for various reagents used in Example 1.

Example IA: Synthesis of polvCstyrene-alt-maleic acid) (PSMA^") from poMstyrene-alt-maleic anhydride) (PSMAA).

A 10% w/v solution of poly(styrene-α/f-maleic anhydride) (PSMAA, M_w 350,000) was prepared by dissolving 2.0 g of PSMAA in 20 mL of acetone. To this solution, 1 mL of water was added with vigorous mixing and the resulting solution stirred overnight. The partially hydrolyzed PSMAA acetone solution was added dropwise to a rapidly stirred aqueous solution of sodium hydroxide at 80-90 ⁰C (0.1 M, 180 mL). The solution was cooled and the pH was adjusted to ~ 6 using hydrochloric acid (6.0 M). Water was added to give a total volume of 200 mL resulting in a 1 % w/v solution of PSMA. A commercial PSMA polymer is also available.

PDADMAC is available from Aldrich as a 20% w/v solution in water in low, medium or high molecular weights (100,000-200,000; 200,000-350,000; and 400,000-500,000, respectively). Example IB: Preparation of a bilaver coating of PSMA-PD ADMAC:

electrostatic interaction

hydrophobic

interaction

Plastic Surface

A Harvard 22 syringe pump was used to serially flow fluids through the microfluidic chip while vacuum was used to simultaneously remove the excess fluid from tip of the chip thereby preventing cross contamination of the sheath flow channel, as shown below.

Main

Channel

UpChurch Scientific % - 20 flat bottom fittings were used in conjunction with a custom polycarbonate chip-mount that uses an o-ring pressure seal to connect to the microfluidic chip. Water was continuously flowed through the sheath flow channel (through well 4 at a rate of 20-30 μl /min) throughout all steps of the coating procedure. The main channel of the microfluidic chip was first washed with a 40% aqueous methanol solution followed by drying with vacuum at the tip. A 1% aqueous solution of PMSA was then pumped through the main channel (through wells 1, 2 and 3 at a rate of 15-75 μl/ min) for 3 minutes and then the fluidic top was removed and the microfluidic chip was allowed to equilibrate for 10-15 minutes. The PSMA solution was then removed from the wells and the wells and tip were thoroughly rinsed with water. Water was then pumped through the main channel (through wells 1, 2 and 3 at a rate of 15-25 μl/ min) for 2-3 minutes, followed by a 0.5% aqueous solution of PDADMAC pumped through the main channel (through wells 1, 2 and 3 at a rate of 15-75 μl/ min) for 3 minutes. The fluidic top was removed and the microfluidic chip was allowed to equilibrate for 10-15 minutes. The PDADMAC solution was removed from the wells and the wells and tip were thoroughly rinsed with water. Water was then pumped through the main channel (through wells 1, 2 and 3 at a rate of 15- 75 μl/ min) for 2-3 minutes. Finally, excess water was removed from all the wells using vacuum; vacuum at the tip removed water from the sheath flow and main channel of microfluidic chip. The microfluidic chip was stored dry until use. Example 2: Preparation of Additional Positively-Charged Coatings

Variations of the bilayer presented in Example 1 are made by substituting for PSMA one of the polymers (or the polymer products resulting from hydrolysis or reaction with other nucleophiles) shown in Table 2.

Table 2. Examples of Polymeric Reagents

Example 3: Preparation of Additional Positively-Charged Coatings

Variations of the bilayer presented in Example 1 are made by substituting for PDADMAC one of the polymers (or the polymer products resulting from hydrolysis or reaction with other nucleophiles) shown in Table 3.

Table 3. Examples of Polymeric Reagents

PDADMAC is available from Aldrich as a 20% w/v solution in water in low, medium or high molecular weights (100,000-200,000; 200,000-350,000; and 400,000-500,000, respectively).

Example 4: Preparation of Additional Positively-Charged Coatings

Positively-charged bilayers were prepared by functionalizing or incorporating other functional groups into the PSMA polymer. For example, reaction of PSMAA with ethanolamine produced the following polymer, which was coated onto the hydrophobic surface following the procedure described in Example 1.

The cationic polymer CHPMEDMAC was activated with a base, such as DBU, and then coated onto the HOCH₂CH₂NH₂-ftιnctionalized PSMA layer using the method described in Example 1. The presence of the nucleophile, i.e., the alcohol, in the PSMA layer allows covalent crosslinking with the activated cationic polymer.

Example 5: Preparation of Coatings Via Functionalized Amphiphilic Polymers

The presence of electrophilic groups such as epoxides or chlorohydrins in the PSMA layer (shown below) allows for covalent crosslinking of cationic polymers that contain nucleophiles, including by way of example only, alcohols or primary amino groups. The carboxylic acid groups of PSMA may also be covalently crosslinked with nucleophiles such as amines or alcohols following reaction with certain activating reagents, including by way of example only, N-(3-dimefhylaminopropyl)-N'-ethyl-carbodimide (EDC).

glycidol

Examples of cationic polymers that may be covalently attached and/or crosslinked to such reactive surfaces are shown below. For example, reaction of PHMAPTAC with glycidol functionalized PSMA produces a coating having the following proposed structure:

As an additional example, reaction of a co-polymer containing primary and quaternary amino groups with PSMA containing glycidol or chlorohydrin functional groups produces a coating having the following proposed structure:

Example 6: Preparation of Customized Cationic Polymers

Custom cationic polymers are made via co-polymerization of monomers containing amino groups and monomers containing functional groups that have no overall charge over a pH range of 1-14. Polyethylene glycol methyl ether methacrylate (or other oligoethylene glycol based acrylates), 3-chloro-2-hydroxy-propyl methacrylate, glycidyl methacrylate, [3-(methacryloylamino)propyl]-dimethyl (3-sulfopropyl)ammonium hydroxide, [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide, 4-acryloxymorpholine, dimethylacrylamide, methacrylamide, are examples of monomers containing functional groups that have no overall charge. 3-methacryloylamino)propyl]-trimethylammonium chloride (MAPTAC), - (methacryloyloxy_ethyl]-trimetylammonium chloride, 2-Aminoethyl methacrylate hydrochloride, 2- (Dimethylamino)ethyl methacrylate and N-[3-(dimethylamino)propyl]methacrylamide] are examples of monomers that contain positive charge at values of pH from 1-10. Examples of the synthesis of homopolymers and co-polymers are shown below. Example 6A. [3 -IUBtHaCrVlOvIaIiIiIiO)PrOpVn -trimethylammonium chloride (MAPTAC) Polymerization.

A 10% w/v solution of ammonium persulfate (APS, NH4S2O8) was prepared by adding 50 mg of ammonium persulfate to 0.5 mL of degassed water. A 5% v/v of MAPTAC (20 mL) was filtered through a 0.22 μm TEFLON syringe filter and degassed overnight in vacuo. To the degassed MAPTAC solution were added TEMED (44 uL) and 140 μL of the 10% solution of APS. The solution was mixed and polymerized in vacuo overnight. The resulting solution turned slightly yellow in color and has a much higher viscosity than the unpolymerized solution.

A 5% monomer concentration of 2-(methacryloyloxyethyl]-trimethylammonium chloride (TMAEMC 79% w/v of total monomer), 4-acryloylmorpholine (19% w/v of total monomer), and 2-aminoethyl methacrylate (2% w/v of total monomer), was prepared, filtered through a 0.22 μm TEFLON syringe filter and degassed in vacuo overnight. The degassed monomer solution was polymerized using APS and TEMED as described in Example 6A.

Various cationic polymers were prepared in this manner using a combination of the aforementioned monomers. The charge density of the resulting polymer may be selectively tuned by adjusting the relative concentration of charged and uncharged monomeric subunits.

Example 7: Preparation of Coating using Radical Polymerization

The channels of a microfluidic chip were first washed with an aqueous solution of methanol (40% v/v) for 1 minute and then dried using vacuum. Next, the channels were filled with neat 1,14-tetradecanediol dimethacrylate. After 1 hour the non-adsorbed 1,14-tetradecanediol dimethacrylate was removed using vacuum and the channels were rinsed with an aqueous solution of methanol (40% v/v) for 1 minute and dried using vacuum. Polymerization was performed by pumping an aqueous solution of 0.2% v/v N,N,N,N- tetramethylethylenediamine (TEMED), 0.07% w/v ammonium persulfate (APS) and 5% w/v MAPTAC through the channels for 3 hours. Finally, the chip was washed with water and stored dry until use. See Figure 8 for a schematic of this coating procedure.

An electrophoresis microfluidic chip, in which the separation channel was coated as described above, was used to separate a mixture of bodipy labeled proteins/peptides. The separation channel was 8 cm long and separation was performed at -450 V/cm in a buffer containing 20% v/v isopropanol and 0.05% v/v formic acid. Figure 9 is an illustrative plot of the resulting fluorescence intensity vs. time.

Example 8: Preparation of coating by covalent attachment.

In addition to physically adsorbing a polymer onto a hydrophobic surface, a hydrophilic or amph philic polymer may also be covalently attached to the hydrophobic surface; if needed, the hydrophobic surface or the hydrophilic or amphiphilic polymer may require initial activation with an appropriate reagent.

Example 8A. Covalent attachment of a chlorhydrin based polymer to the surface of polycarbonate fsee Figure

IQAV

Poly(3-chloro-2-hydroxypropyl-2-methacryloxyethyldimethylammonium chloride) was covalently attached to the surface of polycarbonate by application of an aqueous solution of poly(3-chloro-2- hydroxypropyl-2-methacryloxyethyldimethylammonium chloride) (1 % w/v) and l,8-diazabicyclo[5.4.0]undec-

7-ene (DBU 5% v/v) for 2 hours. The surface was washed with water and stored until used.

An electrophoresis microfluidic chip, in which the separation channel was coated as described above, was used to separate a mixture of bodipy labeled proteins/peptides. The separation channel was 8 cm long and separation was performed at -300 V/cm in a buffer containing 25% v/v ethanol and 0.1% v/v formic acid. Figure 11 is an illustrative plot of the resulting fluorescence intensity vs. time.

Example 8B. Covalent attachment of a chlorhydrin based polymer to the surface of polycarbonate fsee Figure

IQB).

A 5% monomer concentration of 3-chloro-2-hydroxy-propyl methacrylate (CHPMA 5% w/v of total monomer) and poly(ethylene glycol) methyl ether methacrylate (95% w/v of total monomer) was prepared, filtered through a 0.22 μτa TEFLON syringe filter and degassed in vacuo overnight. The degassed monomer solution was polymerized using APS and TEMED as described in Example 6A.

Poly£3-chloro-2-hydroxy-propyl methacrylate-co- poly(ethylene glycol) methyl ether methacrylate) was covalently attached to the surface of polycarbonate by application of an aqueous solution ofPolv(3-chloro- 2-hydroxy-propyl methacrylate-co- poly(ethylene glycol) methyl ether methacrylate) (1 % w/v) and 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU 5% v/v) for 8 hours. The surface was washed with water and stored until used. Example 9: Preparation of coating by covalent attachment.

The surface of molded PSMAA was exposed to a solution of 0.5% copolymer of 2- (memacryloyloxyemylj-trimethylammonium chloride (TMAEMC 79% w/v of total monomer), 4- acryloylmorpholine (19% w/v of total monomer), and 2-aminoethyl methacrylate (2% w/v of total monomer) in a pH 11 buffer for 1 hour. Contact angle measurements demonstrated that the resulting surface was hydrophilic.

Example 10: Stability Data.

Fifty 8 cm microfluidic chips were coated with a PSMA-PD ADMAC coating and stored dry in a clean room until use. The electrophoretic separation of a mixture of proteins/peptides that were tagged with a Bodipy fluorophor was measured at various time intervals. In each experiment, three separations were performed on each chip for each of three previously coated chips and for three control chips (coated that day). Graphs of the migration time and theoretical plate number for the Bodipy-labeled ubiquitin and Angiotensin I plotted as a function of time (See Figures 21 and 22).

Example 11: Preparation of coating by solution swelling (see Figure 14)

A 10 mL 50% isopropanol solution was prepared by mixing 5mL of isopropanol with 5 mL of deionized water. 15 mg of (Hydroxypropyl) methyl cellulose (Aldrich) was dissolved in the 50% IPA solution. The solution bottle was agitated on a shaker table overnight until the (Hydroxypropyl) methyl cellulose completely dissolved. The solution should not be vortexed. The coating solution may be stored with closed cap at room temperature.

5 μL of coating solution was added into each of the three reservoirs, sample inlet, sample outlet, and buffer inlet of a PMMA microfluidic chip. Vacuum was applied from the buffer outlet reservoir to draw the coating solution from the other three reservoirs into the channels until all were filled. It is important to watch for blocked channels. The vacuum was applied for an additional 10 min. The coating solution was emptied first from all the reservoirs and then the channels were dried using the vacuum. About 50 μL of deionized water was pushed from the buffer outlet reservoir using a syringe; it takes about 2 min to push through 50 μL of water. Again, it is important to watch for blocked channels. The chip was completely dried with vacuum, and stored dry in a clean box at room temperature.

Example 12: Preparation of a fluorescently-modified coating.

To a 10% w/v solution of PSMAA (10 ml in anhydrous acetone) was added 2.5 mg of 4,4-difluoro-5,7- dimethyl-4-bora-3a,4a- diaza-s-indacene-3-propionyl ethylenediamine, hydrochloride (Bodipy-amine, Molecular Probes, Eugene Oregon) and the solution was stirred for 3 hours. Water (1 ml) was added and the reaction was stirred overnight. The resulting solution was added drop wise to 100 ml of 0.1 N sodium hydroxide and then the pH was adjusted to ~6-7 with 6 N hydrochloric acid. The bodipy labeled PSMA (PSMA-Bodipy) was then dialyzed against 100 mM sodium chloride pH ~ 6-7 using a 10 ml Foat-A-Lyzer with a 25 K cutoff from Spectrum laboratories. PSMA-Bodipy was used for formation of the bilayer with PDADMAC as described in Example IB. Figure 20 presents a fluorescence image of a microfluidic chip in which the separation channel was coated with PSMA /PDADMAC-Bodipy while the side channel was not coated. Example 13: Preparation of a fluorescently-modified coating.

A 5% total monomer concentration of [3-methacryloylamino)propyl]-trimethylammonivxm chloride (MAPTAC 88% w/v of total monomer), N,N-dimethylmethacrylate (10% w/v of total monomer), and 2- aminoetliyl methacrylate (2% w/v of total monomer), was prepared, filtered through a 0.22 um TEFLON syringe filter and degassed in vacuo overnight. The degassed monomer solution was polymerized using APS and TEMED as described in Example 1. To 10 ml of this copolymer solution was added 50 mg of N- hydroxysuccinimide, 100 mg of EDAC and 2.0 mg of N-(4,4-difluoro-5,7-dimethyl-4- bora-3a,4a-diaza-s- indacene-3- propionyl)cysteic acid, succinimidyl ester, triethylammoniurii salt (Molecular Probes, Eugene, Oregon). The reaction was allowed to stir over night. The Bodipy labeled cationic polymer (MAPTAC-

Bodipy) was then dialyzed against water pH ~ 6 using a 10 ml Foat-A-Lyzer with a 25 K cutoff from Spectrum laboratories. MAPTAC-Bodipy was used for formation of the bilayer as a substitute for PDADMAC in the protocol described in Example IB. Figure 19 presents a fluorescence image of a microfluidic chip in which the separation channel was coated with P SMA-B odipy/M APTAC while the side channel was not coated.

While certain embodiments have been shown and described herein, such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the devices, compositions and methods described herein. It should be understood that various alternatives to the embodiments of the devices, compositions and methods described herein may be employed equivalently. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WE CLAIM:

1. A surface comprising the structure S/A/Z, wherein

S is selected from the group consisting of a hydrophobic surface, a covalently modified hydrophobic surface and a functionalized hydrophobic surface, A is an amphiphilic region comprising a monolayer of an amphiphilic polymer or a modified amphiphilic polymer, and

Z is a charged region comprising a monolayer of a non-amphiphilic charged polymer or a modified non-amphiphilic charged polymer; wherein the interaction between S and A comprises hydrophobic interactions and/or covalent bonds, and the interaction between A and Z comprises electrostatic and/or covalent bonds.

2. The surface of claim 1 wherein the amphiphilic polymer or modified amphiphilic polymer is no more than a monolayer.

3. The surface of any of claims 1 or 2, wherein the charged polymer or modified charged polymer is no more than a monolayer.

4. The surface of claim 1 wherein S is a hydrophobic surface comprising a hydrophobic polymer.

5. The surface of claim 4 wherein the amphiphilic polymer or modified amphiphilic polymer is no more than a monolayer.

6. The surface of any of claims 4 or 5, wherein the charged polymer or modified charged polymer is no more than a monolayer.

7. The surface of claim 4 wherein the hydrophobic polymer is selected from the group consisting of a polyolefin, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a methacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefin polymer, a polysiloxane, a polycarbonate, and copolymers thereof.

8. The surface of claim 4 wherein the hydrophobic surface comprises a mixture or blend of at least two hydrophobic polymers.

9. The surface of claim 1 wherein S is a modified hydrophobic surface comprising a modified hydrophobic polymer.

10. The surface of claim 9 wherein the modified hydrophobic polymer is selected from the group consisting of a modified polyolefin, a modified styrene polymer, a modified halogenated hydrocarbon polymer, a modified vinyl polymer, a modified acrylic polymer, a modified acrylate polymer, a modified methacrylic polymer, a modified methacrylate polymer, a modified polyester, a modified anhydride polymer, a modified polyacrylamide, a modified cyclo-olefin polymer, a modified polysiloxane, a modified polycarbonate, and modified copolymers thereof.

11. The surface of claim 7 wherein the hydrophobic polymer is a methacrylate or a cyclo-olefin polymer.

12. The surface of claim 7 wherein the hydrophobic polymer is polycarbonate.

13. The surface of claim 10 wherein the hydrophobic polymer is a modified methacrylate or a modified cyclo-olefin polymer.

14. The surface of claim 10 wherein the hydrophobic polymer is modified polycarbonate.

15. The surface of any of claims 13-14 wherein the modification is a covalent modification.

16. The surface of any of claims 13-14 wherein the modification is a partial modification.

17. A method for forming the modified hydrophobic polymer of claim 9 comprising exposing a hydrophobic polymer surface with a nucleophile.

18. A method for forming the modified hydrophobic polymer of claim 9 comprising exposing a hydrophobic polymer surface with an electrophile.

19. The method of any of claims 17 or 18 wherein the exposing step is sufficient to partially modify the hydrophobic polymer surface.

20. The method of any of claims 17 or 18 wherein the hydrophobic polymer surface is a methacrylate or a cyclo-olefin polymer surface.

21. The method of any of claims 17 or 18 wherein the hydrophobic polymer surface is a polycarbonate surface.

22. The surface of claim 1, wherein A comprises an amphiphilic polymer.

23. The surface of claims 1, wherein A comprises a modified amphiphilic polymer.

24. The surface of claim 22 wherein the amphiphilic polymer comprises a moiety selected from the group consisting of an aryl, an alkyl, and a halogenated alkyl.

25. The surface of claim 23 wherein the modified amphiphilic polymer comprises a moiety selected from the group consisting of an aryl, an alkyl, and a halogenated ^"alkyl.

26. The surface of claim 22 wherein the amphiphilic polymer comprises polystyrene units.

27. The surface of claim 23 wherein the modified amphiphilic polymer comprises polystyrene units.

28. The surface of claim 22 wherein the amphiphilic polymer comprises positively charged moieties.

29. The surface of claim 22 wherein the amphiphilic polymer comprises negatively charged moieties.

30. The surface of claim 22 wherein the amphiphilic polymer comprises maleic anhydride units.

31. The surface of claim 22 wherein the amphiphilic polymer is derived from maleic anhydride units.

32. A method of making the amphiphilic region of claim 1 comprising reacting a non-amphiphilic polymer with at least one nucleophile to form an amphiphilic polymer.

33. The method of claim 32 wherein the nucleophile is a charged nucleophile.

34. The method of claim 32 wherein the nucleophile is a neutral nucleophile.

35. The method of claim 32 further comprising reacting the non-amphiphilic polymer with an additional nucleophile.

36. The method of claim 32 wherein at least a portion of the non-amphiphilic polymer is in contact with S prior to the reacting step.

37. The method of claim 32 further comprising exposing the amphiphilic polymer to S.

38. The method of claim 37 wherein the exposing step is prior to the reacting step.

39. The method of claim 37 wherein the exposing step is after the reacting step.

40. The method of claim 37 wherein the exposing step is simultaneous with the reacting step.

41. The method of any of claims 38-40 further comprising reacting the amphiphilic polymer with an additional reagent thereby forming a modified amphiphilic surface.

42. The method of claim 32 wherein the non-amphiphilic polymer comprises maleic anhydride units.

43. The method of any of claims 38-40 wherein S is a hydrophobic polymer is selected from the group consisting of a polyolefin, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a methacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefin polymer, a polysiloxane, a polycarbonate, and copolymers thereof.

44. The method of any of claims 38-40 wherein S is a modified hydrophobic polymer is selected from the group consisting of a modified polyolefin, a modified styrene polymer, a modified halogenated hydrocarbon polymer, a modified vinyl polymer, a modified acrylic polymer, a modified acrylate polymer, a modified methacrylic polymer, a modified methacrylate polymer, a modified polyester, a modified anhydride polymer, a modified polyacrylamide, a modified cyclo-olefin polymer, a modified polysiloxane, a modified polycarbonate, and modified copolymers thereof.

45. The method of claim 43 wherein the hydrophobic polymer is a methacrylate or cyclo-olefin polymer.

46. The method of claim 43 wherein the hydrophobic polymer is a polycarbonate polymer.

47. The surface of any of claims 1, 22 or 23, wherein Z is a non-amphiphilic charged polymer.

48. The surface of any of claims 1, 22 or 23, wherein Z is a modified non-amphiphilic charged polymer.

49. The surface of claim 28 wherein Z comprises negatively-charged moieties.

50. The surface of claim 29 wherein Z comprises positively-charged moieties.

51. The surface of claim 50 wherein the positively-charged moieties are quarternary amines.

52. The surface of claim 47 wherein the molecular weight of Z is greater than 20,000 atomic mass units.

53. The surface of claim 48 wherein the molecular weight of Z is greater than 20,000 atomic mass units.

54. A method for making the charged region of claim 1 comprising exposing a surface comprising the structure S/A to non-amphiphilic charged polymer.

55. The method of claim 54 further comprising reacting the non-amphiphilic charged polymer with a reagent thereby forming a modified non-amphiphilic charged polymer.

56. The method of claim 55 wherein the exposing step is prior to the reacting step.

57. A surface comprising the structure S/P/R, wherein S is selected from the group consisting of a hydrophobic surface, a covalently modified hydrophobic surface, and a functionalized hydrophobic surface,

P is a functionalized region comprising a monolayer of a linkable hydrophobic polymer or a modified linkable hydrophobic polymer, and

R is a charged region comprising a monolayer of a linkable charged hydrophilic polymer or a modified linkable charged hydrophilic polymer; wherein the interaction between S and P comprises hydrophobic interactions and/or covalent bonds, and the interaction between P and R comprises covalent bonds, and/or electrostatic bonds, and/or hydrophobic interactions.

58. The surface of claim 57 wherein the linkable hydrophobic polymer or the modified linkable hydrophobic polymer is no more than a monolayer.

59. The surface of any of claim 57 or 58, wherein the linkable charged hydrophilic polymer or modified linkable charged hydrophilic polymer is no more than a monolayer.

60. The surface of claim 57 wherein S is a hydrophobic surface comprising of a hydrophobic polymer.

61. The surface of claim 60 wherein the linkable hydrophobic polymer or the modified linkable hydrophobic polymer is no more than a monolayer.

62. The surface of any of claims 60 or 61, wherein the linkable charged hydrophilic polymer or modified linkable charged hydrophilic polymer is no more than a monolayer.

63. The surface of claim 60 wherein the hydrophobic polymer is selected from the group consisting of a polyolefin, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a methacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefin polymer, a polysiloxane, a polycarbonate, and copolymers thereof.

64. The surface of claim 60 wherein the hydrophobic surface comprises a mixture or blend of at least two hydrophobic polymers.

65. The surface of claim 57 wherein S is a modified hydrophobic surface comprising of a modified hydrophobic polymer.

66. The surface of claim 65 wherein the modified hydrophobic polymer is selected from the group consisting of a modified polyolefin, a modified styrene polymer, a modified halogenated hydrocarbon polymer, a modified vinyl polymer, a modified acrylic polymer, a modified acrylate polymer, a modified methacrylic polymer, a modified methacrylate polymer, a modified polyester, a modified anhydride polymer, a modified polyacrylamide, a modified cyclo-olefin polymer, a modified polysiloxane, a modified polycarbonate, and modified copolymers thereof.

67. The surface of claim 63 wherein the hydrophobic polymer is a methacrylate or a cyclo-olefin polymer.

68. The surface of claim 63 wherein the hydrophobic polymer is polycarbonate.

69. The surface of claim 66 wherein the hydrophobic polymer is a modified methacrylate or a cyclo-olefin polymer.

70. The surface of claim 66 wherein the hydrophobic polymer is modified polycarbonate.

71. The surface of any of claims 69 or 70 wherein the modification is a covalent modification.

72. The surface of any of claims 69 or 70 wherein the modification is a partial modification.

73. A method for forming the modified hydrophobic polymer of claim 65 comprising exposing a hydrophobic polymer surface with a nucleophile.

74. A method for forming the modified hydrophobic polymer of claim 65 comprising exposing a hydrophobic polymer surface with an electrophile.

75. The method of any of claims 73 or 74 wherein the exposing step is sufficient to partially modify the hydrophobic polymer surface.

76. The method of any of claims 73 or 74 wherein the hydrophobic polymer surface is a methacrylate or a cyclo-olefin polymer surface.

77. The method of any of claims 73 or 74 wherein the hydrophobic polymer surface is a polycarbonate surface.

78. The surface of claim 57, wherein P comprises a linkable hydrophobic polymer.

79. The surface of claim 57, wherein P comprises a modified linkable hydrophobic polymer.

80. The surface of claim 78 wherein the linkable hydrophobic polymer comprises a moiety selected from the group consisting of an aryl, an alkyl, and a halogenated alkyl.

81. The surface of claim 78 wherein the linkable hydrophobic polymer comprises a moiety selected from the group consisting of a vinyl and a substituted vinyl.

82. The surface of claim 80 wherein the linkable hydrophobic polymer further comprises a moiety selected from the group consisting of a vinyl and a substituted vinyl.

83. The surface of claim 79 wherein the modified linkable hydrophobic polymer comprises a moiety selected from the group consisting of an aryl, an alkyl, and a halogenated alkyl.

84. The surface of claim 79 wherein the modified linkable hydrophobic polymer comprises a moiety selected from the group consisting of a vinyl, and a substituted vinyl.

85. The surface of claim 83 wherein the modified linkable hydrophobic polymer further comprises a moiety selected from the group consisting of a vinyl, and a substituted vinyl

86. The surface of claim 78 wherein the linkable hydrophobic polymer comprises poly( 1,14- tetradecanediol dirnethacrylate) units.

87. The surface of claim 79 wherein the modified linkable hydrophobic polymer comprises poly( 1,14- tetradecanediol dirnethacrylate) units.

88. A method of making the functionalized region of claim 57 comprising reacting a non-linkable hydrophobic polymer with at least one nucleophile to form the linkable hydrophobic polymer.

89. The method of claim 88 wherein the nucleophile comprises a moiety selected from the group consisting of a vinyl and a substituted vinyl.

90. The method of claim 88 further comprising reacting the non-linkable hydrophobic polymer with an additional nucleophile.

91. The method of claim 88 wherein at least a portion of the non-linkable hydrophobic polymer is in contact with S prior to the reacting step.

92. The method of claim 88 further comprising exposing the non-linkable hydrophobic polymer to S prior to the reacting step.

93. The method of claim 88 further comprising exposing the non-linkable hydrophobic polymer to S simultaneous with the reacting step.

94. A method of making the functionalized region of claim 57 comprising exposing reactive monomeric units of the linkable hydrophobic polymer to S.

95. The method of claim 94 further comprising polymerizing the reactive units thereby forming the linkable hydrophobic polymer on S.

96. The method of any of claims 92, 93 or 95 further comprising reacting the linkable hydrophobic polymer with an additional reagent thereby forming a modified linkable hydrophobic surface.

97. The method of any of claims 92, 93 or 95 wherein S is a hydrophobic polymer is selected from the group consisting of a polyolefϊn, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a methacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefin polymer, a polysiloxane, a polycarbonate, and copolymers thereof.

98. The method of any of claims 92-93 wherein S is a modified hydrophobic polymer is selected from the group consisting of a modified polyolefin, a modified styrene polymer, a modified halogenated hydrocarbon polymer, a modified vinyl polymer, a modified acrylic polymer, a modified acrylate polymer, a modified methacrylic polymer, a modified methacrylate polymer, a modified polyester, a modified anhydride polymer, a modified polyacrylamide, a modified cyclo-olefin polymer, a modified polysiloxane, a modified polycarbonate, and modified copolymers thereof.

99. The method of claim 97 wherein the hydrophobic polymer is a methacrylate or cyclo-olefin polymer.

100. The method of claim 97 wherein the hydrophobic polymer is a polycarbonate polymer.

101. The surface of any of claims 57, 78 or 79, wherein R is a linkable charged hydrophilic polymer.

102. The surface of any of claims 57, 78 or 79, wherein R is a modified linkable charged hydrophilic polymer.

103. A method of making the charged region of claim 57 comprising exposing the linkable charged hydrophilic polymer to the linkable hydrophobic polymer on S, and reacting the linkable charged hydrophilic polymer with at least a portion of the linkable hydrophobic polymer on S.

104. A method of making the charged region of claim 57, comprising exposing monomelic units of the linkable charged hydrophilic polymer to the linkable hydrophobic polymer on S reacting the monomeric units of the linkable charged hydrophilic polymer with at least a portion of the linkable hydrophobic polymer on S.

105. A method of making the charged region of claim 57 comprising exposing the modified reactive charged hydrophilic polymer to the reactive hydrophobic polymer on S reacting the modified linkable charged hydrophilic polymer with at least a portion of the linkable hydrophobic polymer on S.

106. A method of making the charged region of claim 57, comprising exposing monomeric units of the modified linkable charged hydrophilic polymer to the linkable hydrophobic polymer on S polymerizing the monomeric units of the modified linkable charged hydrophilic polymer with at least a portion of the linkable hydrophobic polymer on S.

107. The surface of claim 101 wherein R comprises negatively-charged moieties.

108. The surface of claim 101 wherein R comprises positively-charged moieties.

109. The surface of claim 101 wherein R comprises moieties with charge equal to zero.

110. The surface of claim 102 wherein R comprises negatively-charged moieties.

111. The surface of claim 102 wherein R comprises positively-charged moieties.

112. The surface of claim 102 wherein R comprises moieties with charge equal to zero.

113. The surface of claim 108 wherein the positively-charged moieties are quarternary amines.

114. The surface of claim 111 wherein the positively-charged moieties are quarternary amines.

115. The surface of claim 101 wherein the molecular weight of R is greater than 20,000 atomic mass units.

116. The surface of claim 102 wherein the molecular weight of R is greater than 20,000 atomic mass units.

117. A surface comprising the structure S/N, wherein

S is selected from the group consisting of a hydrophobic surface, a covalently modified hydrophobic surface, and a functionalized hydrophobic surface,

N is a hydrophilic region comprising a monolayer of neutral hydrophilic polymer or a modified neutral hydrophilic polymer; wherein the interaction between S and N comprises physical entrapment of at least a portion of N in S.

118. The surface of claim 117 wherein the neutral hydrophilic polymer or a modified neutral hydrophilic polymer is no more than a monolayer.

119. The surface of claim 117 wherein S is a hydrophobic surface comprising a hydrophobic polymer.

120. The surface of claim 119 wherein the hydrophobic polymer is selected from the group consisting of a polyolefin, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a methacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefin polymer, a polysiloxane, a polycarbonate, and copolymers thereof.

121. The surface of claim 119 wherein the hydrophobic surface comprises a mixture or blend of at least two hydrophobic polymers.

122. The surface of claim of claim 117 wherein S is a modified hydrophobic surface comprising a modified hydrophobic polymer.

123. The surface of claim 122 wherein the modified hydrophobic polymer is selected from the group consisting of a modified polyolefin, a modified styrene polymer, a modified halogenated hydrocarbon polymer, a modified vinyl polymer, a modified acrylic polymer, a modified acrylate polymer, a modified methacrylic polymer, a modified methacrylate polymer, a modified polyester, a modified anhydride polymer, a modified polyacrylamide, a modified cyclo-olefin polymer, a modified polysiloxane, a modified polycarbonate, and modified copolymers thereof.

124 The surface of claim 119 wherein the hydrophobic polymer is a methacrylate or a cyclo-olefin polymer.

125 The surface of claim 119 wherein the hydrophobic polymer is polycarbonate.

126 The surface of claim 122 wherein the hydrophobic polymer is a modified methacrylate or a modified cyclo-olefin polymer.

127 The surface of claim 122 wherein the hydrophobic polymer is modified polycarbonate.

128 The surface of claim 122 wherein the modification is a covalent modification.

129 The surface of claim 122 wherein the modification is a partial modification.

130 A method for forming the modified hydrophobic polymer of claim 122 comprising exposing a hydrophobic polymer surface with a nucleophile.

131 A method for forming the modified hydrophobic polymer of claim 122 comprising exposing a hydrophobic polymer surface with an electrophile.

132. The method of any of claims 130 or 131 wherein the exposing step is sufficient to partially modify the hydrophobic polymer surface.

133. The method of any of claims 130 or 131 wherein the hydrophobic polymer surface is a methacrylate or a cyclo-olefin polymer surface.

134. The method of any of claims 130 or 131 wherein the hydrophobic polymer surface is a polycarbonate surface.

135. The surface of any of claims 117, 118, 119 or 122, wherein N comprises a neutral hydrophilic polymer.

136. The surface of any of claims 117, 118, 119 or 122, wherein N comprises a modified neutral hydrophilic polymer.

137. The surface of claim 135 wherein the neutral hydrophilic polymer is selected from the group consisting of a ρoly(ethylene glycol) derivative, a poly( ethylene oxide) derivative, a cellulose derivatives, and combinations thereof.

138. The surface of claim 136 wherein the modified hydrophilic polymer is selected from the group consisting of a modified poly(ethylene glycol) derivative, a modified poly(ethylene oxide) derivative, a modified cellulose derivatives, and combinations thereof.

139. The surface of claim 137 wherein the neutral hydrophilic polymer comprises poly(ethylene glycol) units.

140. The surface of claim 137 wherein the neutral hydrophilic polymer comprises poly(ethylene oxide) units.

141. The surface of claim 137 wherein the neutral hydrophilic polymer comprises hydroxypropylmethyl cellulose units.

142. The surface of claim 138 wherein the modified neutral hydrophilic polymer comprises modified poly(ethylene glycol) units.

143. The surface of claim 138 wherein the modified neutral hydrophilic polymer comprises modified poly(ethylene oxide) units.

144. The surface of claim 138 wherein the modified neutral hydrophilic polymer comprises modified hydroxypropylmethyl cellulose units.

145. A method of making the neutral region of claim 117 comprising swelling the hydrophobic surface with a solvent, and exposing the swollen hydrophobic surface to the neutral hydrophilic polymer.

146. The method of claim 145 further comprising drying the swollen hydrophobic surface sufficient to entrap at least a portion of the neutral hydrophilic polymer within at least a portion of the hydrophobic surface.

147. The method of claim 145 further comprising reacting the neutral hydrophilic polymer with a reagent to form a modified neutral hydrophilic polymer.

148. A surface comprising the structure S/C, wherein

S is selected from the group consisting of a hydrophobic surface, a covalently modified hydrophobic surface, and a functionalized hydrophobic surface, C is a hydrophilic region comprising a monolayer of a linkable hydrophilic polymer or a linkable modified hydrophilic polymer; wherein the interaction between S and C comprises covalent attachment of at least a portion of C onto S.

149. The surface of claim 148 wherein the linkable hydrophilic polymer or a linkable modified hydrophilic polymer is no more than a monolayer.

150. The surface of claim 148 wherein S is a hydrophobic surface comprising a hydrophobic polymer.

151. The surface of claim 150 wherein the hydrophobic polymer is selected from the group consisting of a polyolefϊn, a styrene polymer, a halogenated hydrocarbon polymer, a vinyl polymer, an acrylic polymer, an acrylate polymer, a methacrylic polymer, a methacrylate polymer, a polyester, an anhydride polymer, a polyacrylamide, a cyclo-olefϊn polymer, a polysiloxane, a polycarbonate, and copolymers thereof.

152. The surface of claim 150 wherein the hydrophobic surface comprises a mixture or blend of at least two hydrophobic polymers.

153. The surface of claim of claim 148 wherein S is a modified hydrophobic surface comprising a modified hydrophobic polymer.

154. The surface of claim 153 wherein the modified hydrophobic polymer is selected from the group consisting of a modified polyolefϊn, a modified styrene polymer, a modified halogenated hydrocarbon polymer, a modified vinyl polymer, a modified acrylic polymer, a modified acrylate polymer, a modified methacrylic polymer, a modified methacrylate polymer, a modified polyester, a modified anhydride polymer, a modified polyacrylamide, a modified cyclo-olefin polymer, a modified polysiloxane, a modified polycarbonate, and modified copolymers thereof.

155 The surface of claim 151 wherein the hydrophobic polymer is a methacrylate or a cyclo-olefin polymer.

156 The surface of claim 151 wherein the hydrophobic polymer is polycarbonate.

157 The surface of claim 151 wherein the hydrophobic polymer is poly(styrene-co-maleic anhydride).

158 The surface of claim 154 wherein the hydrophobic polymer is a modified methacrylate or a modified cyclo-olefin polymer.

159 The surface of claim 154 wherein the hydrophobic polymer is a modified polycarbonate.

160 The surface of claim 151 wherein the hydrophobic polymer is a modified ρoly(styrene-co-maleic anhydride).

161 The surface of any of claims 158-160 wherein the modification is a covalent modification.

162 The surface of any of claims 158-160 wherein the modification is a partial modification.

163 A method for forming the modified hydrophobic polymer of claim 153 comprising exposing a hydrophobic polymer surface with a nucleophile.

164 A method for forming the modified hydrophobic polymer of claim 153 comprising exposing a hydrophobic polymer surface with an electrophile.

165. The method of any of claims 163 or 164 wherein the exposing step is sufficient to partially modify the hydrophobic polymer surface.

166. The method of any of claims 163 or 164 wherein the hydrophobic polymer surface is a methacrylate or a cyclo-olefin polymer surface.

167. The method of any of claims 163 or 164 wherein the hydrophobic polymer surface is a polycarbonate surface.

168. The surface of any of claims 148, 149, 150 or 153, wherein C comprises a linkable hydrophilic polymer.

169. The surface of any of claims 148, 149, 150 or 153, wherein C comprises a linkable modified hydrophilic polymer.

170. The surface of claim 168, wherein the linkable hydrophilic polymer comprises positively charged moieties.

171. The surface of claim 168, wherein the linkable hydrophilic polymer comprises negatively charged moieties.

172. The surface of claim 168, wherein the linkable hydrophilic polymer is neutral.

173. The surface of claim 169, wherein the linkable modified hydrophilic polymer comprises positively charged moieties.

174. The surface of claim 169, wherein the linkable modified hydrophilic polymer comprises negatively charged moieties.

175. The surface of claim 169, wherein the linkable modified hydrophilic polymer is neutral.

176. The surface of claim 168 wherein the linkable hydrophilic polymer is selected from the group consisting of a polysaccharide, a polyether, a poly(alcohol), a polyamide, a protein, a polyacrylonitrile, a zwitterionic polymer, a poly(acrylic acid), a polystyrenesulfonic acid, a polyvinylphosphonic acid, a poly(glutamic acid), a poly(aspartic acid), a poly(anilinesulfonic acid), a poly(3-sulfopropyl methacrylate), a polyethanolesulfonate, a heparin, a polyamine, a polyethyleneimine, a polyallylamine, a poly(N-methyl vinylamine, a poly(vinylamine), a poly(lysine), a poly(3-chloro-2-hydroxyproρyl-2- methacryloxyethyl-dimethylammonium chloride), and copolymers thereof.

177. The surface of claim 169 wherein the linkable modified hydrophilic polymer is selected from the group consisting of a modified polysaccharide, a modified polyether, a modified poly(alcohol), a modified polyamide, a modified protein, a modified polyacrylonitrile, a modified zwitterionic polymer, a modified poly(acrylic acid), a modified polystyrenesulfonic acid, a modified polyvinylphosphonic acid, a modified poly(glutamic acid), a modified poly(aspartic acid), a modified poly(anilinesulfonic acid), a modified poly(3-sulfopropyl methacrylate), a modified polyethanolesulfonate, a modified heparin, a modified polyamine, a modified polyethyleneimine, a modified polyallylamine, a modified poly(N- methyl vinylamine, a modified poly( vinylamine), a modified poly(lysine), a modified poly(3-chloro-2- hydroxypropyl-2-methacryloxyethyl-dimethylammonium chloride), and copolymers thereof.

178. The surface of claim 168 wherein the linkable hydrophilic polymer is a poly(ethyeneimine).

179. The surface of claim 169 wherein the linkable modified hydrophilic polymer is poly(N-methyl vinylamine).

180. A method of making the hydrophilic region of claim 148 comprising: exposing the hydrophobic surface or the modified hydrophobic surface with a hydrophilic polymer or a modified hydrophilic polymer comprised of linkable moieties; and reacting the linkable moieties with at least a portion of the hydrophobic surface or the modified hydrophobic surface.

181. The method of claim 180 wherein the linkable unit is a nucleophile.

182. The method of claim 180 wherein the linkable unit is an electrophile.

183. The method of claim 180 wherein the linkable unit is chlorohydrin or an epoxide.

184. A microfiuidic chip for mass spectrometric analysis comprising: a microfluidic body layer formed with a plurality of fluid reservoirs; at least one separation channel and/or at least one side channel that are formed along a length of the microfluidic body layer in fluid communication with at least one fluid reservoir; wherein at least one of the separation channels and/or side channels comprises a charged polymer monolayer coated on a hydrophobic surface; and a cover plate for enclosing the separation channel and the side channel to provide a stable electrospray from the microfluidic chip.

185. The microfluidic chip as recited in claim 184, wherein the side channel provides electrical contact to the separation channel.

186. The microfluidic chip as recited in claim 184, wherein the side channel provides sheath flow.

187. The microfluidic chip as recited in claim 184, wherein the charged coating of the side channel is a negatively charged coating, and the separation channel includes a positively charged coating.

188. The microfluidic chip of claim 187, wherein each of the charged coatings are produced using the method of claim 17.

189. The microfluidic chip of claim 187, wherein each of the charged coatings are produced using the method of claim 18.

190. The microfluidic chip of claim 187, wherein each of the charged coatings are produced using the method of claim 32.

191. The microfluidic chip of claim 187, wherein each of the charged coatings are produced using the method of claim 54.

192. The microfluidic chip as recited in claim 184, wherein the charged coating of the side channel is a negatively charged coating, and the separation channel is without a coating.

193. The microfluidic chip of claim 192, wherein the negatively charged coating is produced using the method of claim 17.

194. The microfluidic chip of claim 192, wherein the negatively charged coating is produced using the method of claim, 18.

195. The microfluidic chip of claim 192, wherein the negatively charged coating is produced using the method of claim 32.

196. The microfluidic chip of claim 192, wherein the negatively charged coating is produced using the method of claim 54.

197. The microfluidic chip as recited in claim 184, wherein the charged coating of the side channel is a negatively charged coating, and the separation channel includes a neutral uncharged coating.

198. The microfluidic chip of claim 197, wherein the negatively charged coating is produced using the method of claim 17.

199. The microfluidic chip of claim 197, wherein the negatively charged coating is produced using the method of claim 18.

200. The microfluidic chip of claim 197, wherein the negatively charged coating is produced using the method of claim 32.

201. The microfluidic chip of claim 197, wherein the negatively charged coating is produced using the method of claim 54.

202. The microfluidic chip of claim 197, wherein the neutral uncharged coating is produced using the method of claim 130.

203. The microfluidic chip of claim 197, wherein the neutral uncharged coating is produced using the method of claim 131.

204. The microfluidic chip of claim 197, wherein the neutral uncharged coating is produced using the method of claim 145.

205. The microfluidic chip as recited in claim 184 wherein the charged coating of the side channel is a positively charged coating, and the separation channel includes a negatively charged coating.

206. The microfluidic chip of claim 205, wherein each of the charged coatings are produced using the method of claim 17.

207. The microfluidic chip of claim 205, wherein each of the charged coatings are produced using the method of claim 18.

208. The microfluidic chip of claim 205, wherein each of the charged coatings are produced using the method of claim 32.

209. The microfluidic chip of claim 205, wherein each of the charged coatings are produced using the method of claim 54.

210. The microfluidic chip as recited in claim 184, wherein the charged coating of the side channel is a positively charged coating, and the separation channel is without a coating.

211. The microfluidic chip of claim 210, wherein the positively charged coating is produced using the method of claim 17.

212. The microfluidic chip of claim 210, wherein the positively charged coating is produced using the method of claim 18.

213. The microfluidic chip of claim 210, wherein the positively charged coating is produced using the method of claim 32.

214. The microfluidic chip of claim 210, wherein the positively charged coating is produced using the method of claim 54.

215. The microfluidic chip as recited in claim 184, wherein the charged coating of the side channel is a positively charged coating, and the separation channel includes a neutral uncharged coating.

216. The microfluidic chip of claim 215, wherein the positively charged coating is produced using the method of claim 17.

217. The microfluidic chip of claim 215, wherein the positively charged coating is produced using the method of claim 18.

218. The microfluidic chip of claim 215, wherein the positively charged coating is produced using the method of claim 32.

219. The microfluidic chip of claim 215, wherein the positively charged coating is produced using the method of claim 54.

220. The microfluidic chip of claim 215, wherein the neutral uncharged coating is produced using the method of claim 130.

221. The microfluidic chip of claim 215, wherein the neutral uncharged coating is produced using the method of claim 131.

222. The microfluidic chip of claim 215, wherein the neutral uncharged coating is produced using the method of claim 145.

223. The microfluidic chip as recited in claim 184, wherein the side channel is without a coating, and the separation channel includes a positively charged coating.

224. The microfluidic chip of claim 223, wherein the positively charged coating is produced using the method of claim 17.

225. The microfluidic chip of claim 223, wherein the positively charged coating is produced using the method of claim 18.

226. The microfluidic chip of claim 223, wherein the positively charged coating is produced using the method of claim 32.

227. The microfluidic chip of claim 223, wherein the positively charged coating is produced using the method of claim 54.

228. The microfluidic chip as recited in claim 184, wherein the side channel is without a coating, and the separation channel includes a negatively charged coating.

229. The microfluidic chip of claim 228, wherein the negatively charged coating is produced using the method of claim 17.

230. The microfluidic chip of claim 228, wherein the negatively charged coating is produced using the method of claim 18.

231. The microfluidic chip of claim 228, wherein the negatively charged coating is produced using the method of claim 32.

232. The microfluidic chip of claim 228, wherein the negatively charged coating is produced using the method of claim 54.

233. The microfluidic chip as recited in claim 184, wherein the side channel is includes a neutral coating, and the separation channel includes a positively charged coating.

234. The microfluidic chip of claim 233, wherein the neutral uncharged coating is produced using the method of claim 130.

235. The microfluidic chip of claim 233, wherein the neutral uncharged coating is produced using the method of claim 131.

236. The microfluidic chip of claim 233, wherein the neutral uncharged coating is produced using the method of claim 145.

237. The microfluidic chip of claim 233, wherein the positively charged coating is produced using the method of claim 17.

238. The microfluidic chip of claim 233, wherein the positively charged coating is produced using the method of claim 18.

239. The microfluidic chip of claim 233, wherein the positively charged coating is produced using the method of claim 32.

240. The microfluidic chip of claim 233, wherein the positively charged coating is produced using the method of claim 54.

241. The microfluidic chip as recited in claim 184, wherein the side channel is includes a neutral coating, and the separation channel includes a negatively charged coating.

242. The microfluidic chip of claim 241, wherein the neutral uncharged coating is produced using the method of claim 130.

243. The microfluidic chip of claim 241 , wherein the neutral uncharged coating is produced using the method of claim 131.

244. The microfluidic chip of claim 241, wherein the neutral uncharged coating is produced using the method of claim 145.

245. The microfluidic chip of claim 241, wherein the negatively charged coating is produced using the method of claim 17.

246. The microfluidic chip of claim 241 , wherein the negatively charged coating is produced using the method of claim 18.

247. The microfluidic chip of claim 241, wherein the negatively charged coating is produced using the method of claim 32.

248. The microfluidic chip of claim 241, wherein the negatively charged coating is produced using the method of claim 54.

249. The microfluidic chip as recited in claim 184, further comprising: a plurality of electrodes positioned in each fluid reservoir to apply voltages to impart movement of materials within the separation channel and the side channel.

250. The microfluidic chip as recited in claim 184, wherein the cover plate extends beyond the microfluidic body layer to form an open-ended distal tip portion at which the separation channel and the side channel terminate to provide an electrospray ionization tip that directs a stable electrospray from the microfluidic chip.

251. The microfluidic chip as recited in claim 184, wherein at least a portion of the open-ended distal tip portion is covered with a hydrophilic material.

252. The microfluidic chip as recited in claim 184, wherein the tapered end portion of the microfluidic body layer includes a tapered end formed along a substantially flat truncated portion of the tapered end portion.

253. A microfluidic chip for electrospray ionization comprising: a channel plate formed with a separation channel and at least two side channels that are each in fluid communication with at least one fluid reservoir included within the channel plate, and wherein at least one side channel includes a charged coating; and a covering plate for substantially enclosing the non-intersecting fluid channels formed on the channel plate, wherein the covering plate includes an overhang that extends beyond the channel plate to provide an electrospray tip that includes an open-tip region at which each of the non-intersecting fluid channels terminate.

254. The microfluidic chip as recited in claim 253, further comprising: a syringe in fluid communication with a side channel to provide sheath flow.

255. The microfluidic chip as recited in claim 253, wherein the charged coating of the side channel includes positively or negatively charged molecules.

256. The microfluidic chip as recited in claim 253, wherein the charged coating of the side channel includes negatively charged molecules, and wherein the separation channel has a charged coating that includes positively charged molecules.

257. The microfluidic chip as recited in claim 253, wherein the charged coating of the side channel is a positively charged coating, and the separation channel is without a coating.

258. The microfluidic chip as recited in claim 253, wherein the charged coating of the side channel is a positively charged coating, and the separation channel includes a neutral uncharged coating.

259. The microfluidic chip of any of claims 184 or 253 fabricated by pressure molding poly(styrene-co- maleic anhydride).

260. A microfluidic device for providing one or more substances to a mass spectrometer for analysis, the microfluidic device comprising: a substrate comprising at least one layer, the substrate including at least one microchannel, wherein the substances are movable within the at least one microchannel; a cover arranged on a surface of the substrate, the cover including at least one electrical potential source; at least one outlet in fluid communication with the microchannel for allowing egress of the substances from the microchannel; and at least one tip surface extending the cover beyond the outlet, wherein the microchannel in fluid communication with the outlet widens from a first cross sectional dimensions along the majority of its length to a second, wider cross sectional dimensions at the outlet.

261. A microfluidic device as in claim 260, wherein the at least one microchannel is enclosed between the substrate and the cover.

262. A microfluidic device as in claim 260, wherein the at least one microchannel comprises at least two intersecting microchannels.

263. A microfluidic device as in claim 260, wherein the at least one microchannel comprises: a first microchannel in fluid communication with a first outlet and having the first cross sectional dimensions and the second, wider cross sectional dimensions; and at least a second microchannel in fluid communication with a second outlet disposed at the tip surface.

264. A microfluidic device as in claim 263, wherein the second microchannel includes at least one substance for preventing substances exiting the first outlet from entering the second outlet.

265 . A microfluidic device as in claim 264, wherein the at least one substance in the second channel comprises at least one of a cross-linked polyacrylamide, an agarose gel, a linear polyacrylamide, a cellulose polymer, polyethylene oxide, polyvinylpyrrolidone and other hydrophilic polymer solutions.

266. A microfluidic device as in claim 263, wherein the second microchannel has negatively charged walls for directing a buffer through the second microchannel to prevent substances exiting the first outlet from entering the second outlet.

267. A microfluidic device as in claim 260, wherein the cover comprises at least one material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz and silica.

268. A microfluidic device as in claim 267, wherein the polymer comprises a material selected from the group consisting of cyclic polyolefm, cyclo-olefrn polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ and other acrylic-based polymers.

269. A microfluidic device as in claim 260, wherein the at least one electrical potential source of the cover comprises a strip of material disposed across the outlet.

270. A microfluidic device as in claim 269, wherein the at least one electrical potential source comprises a strip of metal film.

271. A microfluidic device as in claim 269, wherein the at least one electrical potential source comprises a strip of conductive ink.

272. A microfluidic device as in claim 269, wherein the at least one electrical potential source is embedded in the cover.

273. A microfluidic device as in claim 269, wherein the at least one electrical potential source is coupled with the cover via adhesive.

274. A microfluidic device for providing one or more substances to a mass spectrometer for analysis, the microfluidic device comprising: a substrate comprising at least one layer, the substrate including at least one microchannel, wherein the substances are movable within the at least one microchannel; a cover arranged on a surface of the substrate and having a first surface in contact with the substrate and a second surface opposite the first surface; at least one outlet in fluid communication with the microchannel for allowing egress of the substances from the microchannel; at least one tip surface extending the cover beyond the outlet; and at least one electrical potential source disposed on the second surface of the cover and ending near a distal end of the tip.

275. A microfluidic device as in claim 274, wherein the at least one microchannel is enclosed between the substrate and the cover.

276. A microfluidic device as in claim 274, wherein the at least one microchannel comprises at least two intersecting microchannels.

277. A microfluidic device as in claim 274, wherein the at least one microchannel comprises at least two microchannels, each in fluid communication with a different outlet.

278. A microfluidic device as in claim 274, wherein the tip includes a V-shaped edge surface for providing electrospray ionization of the substances to the mass spectrometer.

279. A microfluidic device as in claim 278, wherein one end of the electrical potential source is disposed at the V-shaped edge surface.

280. A microfluidic device as in claim 279, wherein the one end of the electrical potential source is recessed within the V-shaped edge surface.

281. A microfluidic device as in claim 278, 279 or 280, wherein the electrical potential source comprises a conductive wire.

282. A microfluidic device as in claim 274, wherein the tip includes at least one hole through the cover.

283. A microfluidic device as in claim 282, wherein the electrical potential source comprises a conductive wire shaped to extend into the hole.

284. A microfluidic device as in claim 282, wherein the electrical potential source comprises a conductive plate having a post extending into the hole.

285. A microfluidic device as in claim 274, wherein the cover comprises at least one material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz and silica.

286. A microfluidic device as in claim 285, wherein the polymer comprises a material selected from the group consisting of cyclic polyolefrn, cyclo-olefin polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ and other acrylic-based polymers.

287. A microfluidic device as in claim 274, wherein the at least one electrical potential source is coupled with the cover via adhesive.

288. A microfluidic device for providing one or more substances to a mass spectrometer for analysis, the microfluidic device comprising: a substrate comprising at least one layer, the substrate including: at least one microchannel, wherein the substances are movable within the at least one microchannel; and at least one electrode reservoir in fluid communication with the microchannel, the electrode reservoir having a membrane, conductive fluid separated from the microchannel by the membrane, and an electrode; a cover arranged on a surface of the substrate; at least one outlet in fluid communication with the microchannel for allowing egress of the substances from the microchannel; and at least one tip surface extending the cover beyond the outlet.

289. A microfiuidic device as in claim 288, wherein the at least one microchannel is enclosed between the substrate and the cover.

290. A microfiuidic device as in claim 288, wherein the at least one microchannel comprises at least two intersecting microchannels.

291. A microfiuidic device as in claim 288, wherein the at least one microchannel comprises at least two microchannels, each in fluid communication with a different outlet.

292. A microfiuidic device as in claim 288, wherein the electrode reservoir comprises: a reservoir portion containing the membrane, the conductive fluid and the electrode; and a bridging channel between the reservoir portion and the microchannel, the bridging channel having a smaller dimensions than the reservoir portion.

293 . A microfiuidic device as in claim 292, wherein the membrane is disposed at a bottom of the reservoir portion, immediately adjacent the bridging channel, and wherein the membrane comprises nanopores configured to allow only small ions to pass through the membrane from the reservoir portion to the bridging channel.

294. A microfiuidic device as in claim 293, wherein at least part of the electrode is disposed in the reservoir portion in contact with the conductive fluid.

295. A microfluidic device as in claim 293, further comprising a membrane fixture for holding the membrane in place at the bottom of the reservoir portion.

296. A microfluidic device as in claim 293, wherein the membrane is held in place at the bottom of the reservoir portion via adhesive.

297. A microfluidic device as in claim 288, wherein the cover comprises at least one material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz and silica.

298. A microfluidic device as in claim 297, wherein the polymer comprises a material selected from the group consisting of cyclic polyolefin, cyclo-olefin polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™,

Teflon™ and other acrylic-based polymers.

299. A microfluidic device for providing one or more substances to a mass spectrometer for analysis, the microfluidic device comprising: a substrate comprising at least one layer, the substrate including: at least a first microchannel, wherein the substances are movable within the first microchannel; and at least a second microchannel coupled with an electrical contact, wherein one of the first and second microchannels includes at least one substance for preventing the substances in the first microchannel from passing into the second microchannel; a cover arranged on a surface of the substrate; a first outlet in fluid communication with the first microchannel for allowing egress of the substances from the first microchannel; at least a second outlet in fluid communication with the second microchannel for allowing electrical current from the second microchannel; and at least one tip surface extending the cover beyond the outlet.

300. A microfluidic device as in claim 299, wherein the microchannels are enclosed between the substrate and the cover.

301. A microfluidic device as in claim 299, further comprising at least a third microchannel intersecting with the first microchannel.

302. A microfluidic device as in claim 299, wherein the at least one substance in the second microchannel comprises at least one of a cross-linked polyacrylamide, an agarose gel, a linear polyacrylamide, a cellulose polymer, polyethylene oxide, polyvinylpyrrolidone and other hydrophilic polymer solutions.

303. A microfluidic device as in claim 299, wherein the at least one substance in the second microchannel comprises a buffer, and wherein the second microchannel has negatively charged walls for directing the buffer through the second microchannel to prevent the substances exiting the first outlet from entering the second outlet.

304. A microfluidic device as in claim 299, wherein the first microchannel comprises positively charged walls, and the second microchannel comprises essentially neutral walls.

305. A microfluidic device as in claim 299, wherein the cover comprises at least one material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz and silica.

306. A microfluidic device as in claim 305, wherein the polymer comprises a material selected from the group consisting of cyclic polyolefin, cyclo-olefin polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ and other acrylic-based polymers.

307. A method of making a microfluidic device for providing one or more substances to a mass spectrometer for analysis, the method comprising: fabricating a substrate comprising: forming at least one microchannel having a microfabricated surface; and forming an outlet in fluid communication with the microchannel and disposed along an edge surface of the substrate, wherein the microchannel in fluid communication with the outlet widens from a first cross sectional dimensions along the majority of its length to a second, wider cross sectional dimensions at the outlet; fabricating a cover having at least one tip surface; and applying the cover to the substrate.

308. A method as in claim 307, wherein fabricating the substrate comprises forming at least two intersecting microchannels.

309. A method as in claim 307, wherein at least one of the substrate and the cover are fabricated from a material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz, silica and a combination thereof.

310. A method as in claim 309, wherein the polymer comprises a material selected from the group consisting of cyclic polyolefin, cyclo-olefϊn polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™,

Teflon™ and other acrylic-based polymers.

311. A method as in claim 307, wherein forming at least one microchannel comprises:

forming a first microchannel having positively charged walls; and forming a second microchannel having essentially neutral walls.

312. A method as in claim 307, further comprising coupling an electrical potential source with the device to move the substances through the microchannel by electrophoretic or electrokinetic mobility.

313. A method as in claim 312, wherein the electrical potential source comprises an electrical potential microchannel, the electrical potential microchannel containing at least one electrically charged substance.

314. A method as in claim 313, wherein the electrical potential microchannel exits the microfluidic device immediately adjacent the microchannel.

315. A method as in claim 314, further comprising disposing at least one substance in the electrical potential microchannel for preventing substances exiting the outlet from entering the electrical potential microchannel.

316. A method as in claim 315, wherein the at least one substance in the electrical potential microchannel comprises at least one of a cross-linked polyacrylamide, an agarose gel, a linear polyacrylamide, a cellulose polymer, polyethylene oxide, polyvinylpyrrolidone and other hydrophilic polymer solutions.

317. A method as in claim 315, wherein the at least one substance in the electrical potential microchannel comprises a buffer, and wherein the electrical potential microchannel has negatively charged walls for directing the buffer through the electrical potential microchannel.

318. A method as in claim 312, wherein the electrical potential source comprises at least one electrode on the microfluidic device.

319. A method as in claim 318, wherein the at least one electrode comprises a strip of material coupled with the cover so as to be disposed across the outlet.

320. A method as in claim 319, wherein the at least one electrode comprises a strip of metal film.

321. A method as in claim 319, wherein the at least one electrode comprises a strip of conductive ink.

322. A method as in claim 319, wherein the at least one electrode is embedded in the cover.

323. A method as in claim 319, wherein the at least one electrode is coupled with the cover via adhesive.

324. A method as in claim 318, wherein the at least one electrode provides potential for effecting at least one of electrophoretic separation of the substances and electrospray ionization.

325. A method as in claim 318, wherein the at least one electrode provides potential for effecting at least one of electrokinetic movement of the substances in the microchannel and electrospray ionization.

326. A method as in claim 318, wherein the at least one electrode comprises at least one of copper, nickel, conductive ink, silver, silver/silver chloride, gold, platinum, palladium, iridium, aluminum, titanium, tantalum, niobium, carbon, doped silicon, indium tin oxide, other conductive oxides, polyanaline, sexithiophene, polypyrrole, polythiophene, polyethylene dioxythiophene, carbon black, carbon fibers, conductive fibers, and other conductive polymers and conjugated polymers.

327. A method as in claim 318, wherein the at least one electrode provides the electrical potential without producing a significant quantity of bubbles in the substances.

328. A method as in claim 307, further comprising: making at least two connected microfluidic devices from one or more common pieces of starting material; and separating the at least two microfluidic devices by cutting the common pieces of starting material.

329. A method as in claim 307, wherein the at least one microchannel is formed by at least one of photolithographically masked wet-etching, photolithographically masked plasma-etching, embossing, molding, compression molding, injection molding, photoablating, micromachining, laser cutting, laser ablation, milling, die cutting, reel-to-reel methods, photopolymerizing and casting.

330. A method of making a microfiuidic device for providing one or more substances to a mass spectrometer for analysis, the method comprising: fabricating a substrate comprising: forming at least one microchannel having a microfabricated surface; and forming an outlet in fluid communication with the microchannel and disposed along an edge surface of the substrate; fabricating a cover having at least one tip surface, a substrate contacting surface, and an electrical potential surface opposite the substrate contacting surface; coupling at least one electrical potential source with the electrical potential surface; and applying the cover to the substrate.

331. A method as in claim 330, wherein fabricating the substrate comprises forming at least two intersecting microchannels.

332. A method as in claim 330, wherein at least one of the substrate and the cover are fabricated from a material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz, silica and a combination thereof.

333. A method as in claim 332, wherein the polymer comprises a material selected from the group consisting of cyclic polyolefin, cyclo-olefϊn polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ and other acrylic-based polymers.

334. A method as in claim 330, wherein the electrical potential source comprises at least one electrode.

335. A method as in claim 334, wherein fabricating the cover comprises forming a V-shaped edge surface in the tip surface, and wherein the electrode comprises a conductive wire with one end disposed in the V- shape.

336. A method as in claim 334, wherein fabricating the cover comprises forming a hole in the tip.

337. A method as in claim 336, wherein the electrode comprises a conductive wire shaped to extend into the hole.

338. A method as in claim 336, wherein the electrode comprises a conductive plate having a post extending into the hole.

339. A method as in claim 334, wherein the at least one electrode provides potential for effecting at least one of electrophoretic separation of the substances and electrospray ionization.

340. A method as in claim 334, wherein the at least one electrode provides potential for effecting at least one of electrokinetic movement of the substances in the microchannel and electrospray ionization.

341. A method as in claim 334, wherein the at least one electrode comprises at least one of copper, nickel, conductive ink, silver, silver/silver chloride, gold, platinum, palladium, iridium, aluminum, titanium, tantalum, niobium, carbon, doped silicon, indium tin oxide, other conductive oxides, polyanaline, sexithiophene, polypyrrole, polythiophene, polyethylene dioxythiophene, carbon black, carbon fibers, conductive fibers, and other conductive polymers and conjugated polymers.

342. A method as in claim 334, wherein the at least one electrode provides the electrical potential without producing a significant quantity of bubbles in the substances.

343. A method of making a microfluidic device for providing one or more substances to a mass spectrometer for analysis, the method comprising: fabricating a substrate comprising: forming at least one microchannel having a microfabricated surface; forming an outlet in fluid communication with the microchannel and disposed along an edge surface of the substrate; and forming at least one electrode reservoir in fluid communication with the microchannel, the electrode reservoir having a membrane, conductive fluid separated from the microchannel by the membrane, and an electrode; fabricating a cover having at least one tip surface, a substrate contacting surface, and an electrical potential surface opposite the substrate contacting surface; and applying the cover to the substrate.

344. A method as in claim 343, wherein fabricating the substrate comprises forming at least two intersecting microchannels.

345. A method as in claim 343, wherein at least one of the substrate and the cover are fabricated from a material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz, silica and a combination thereof.

346. A method as in claim 345, wherein the polymer comprises a material selected from the group consisting of cyclic polyolefin, cyclo-olefin polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™, Teflon™ and other acrylic-based polymers.

347. A method as in claim 343, wherein the electrode provides potential for effecting at least one of electrophoretic separation of the substances and electrospray ionization.

348. A method as in claim 343, wherein the electrode provides potential for effecting at least one of electrokinetic movement of the substances in the microchannel and electrospray ionization.

349. A method as in claim 343, wherein the electrode comprises at least one of copper, nickel, conductive ink, silver, silver/silver chloride, gold, platinum, palladium, iridium, aluminum, titanium, tantalum, niobium, carbon, doped silicon, indium tin oxide, other conductive oxides, polyanaline, sexithiophene, polypyrrole, polythiophene, polyethylene dioxythiophene, carbon black, carbon fibers, conductive fibers, and other conductive polymers and conjugated polymers.

350. A method as in claim 343, wherein the electrode provides the electrical potential without producing a significant quantity of bubbles in the substances.

351. A method of making a microfluidic device for providing one or more substances to a mass spectrometer for analysis, the method comprising: fabricating a substrate comprising: forming at least one microchannel having a microfabricated surface; and forming an outlet in fluid communication with the microchannel and disposed along an edge surface of the substrate; fabricating a cover having at least one tip surface; coupling an electrical potential source with the device to move the substances through the microchannel by electrophoretic or electrokinetic mobility; and applying the cover to the substrate.

352. A method as in claim 351, wherein fabricating the substrate comprises forming at least two intersecting microchannels.

353. A method as in claim 351, wherein at least one of the substrate and the cover are fabricated from a material selected from the group consisting of glass, silicon, ceramic, polymer, copolymer, silicon dioxide, quartz, silica and a combination thereof.

354. A method as in claim 353, wherein the polymer comprises a material selected from the group consisting of cyclic polyolefin, cyclo-olefin polymer, polycarbonate, polystyrene, PMMA, acrylate, polyimide, epoxy, polyethylene, polyether, polyethylene terephtalate, polyvinyl chloride, polydimethylsiloxane, polyurethane, polypropylene, phenol formaldehyde, polyacrylonitrile, Mylar™,

Teflon™ and other acrylic-based polymers.

355. A method as in claim 351, wherein the electrical potential source comprises an electrical potential microchannel, the electrical potential microchannel containing at least one electrically charged substance.

356. A method as in claim 355, wherein the electrical potential microchannel exits the microfluidic device immediately adjacent the microchannel.

357. A method as in claim 356, further comprising disposing at least one substance in the electrical potential microchannel for preventing substances exiting the outlet from entering the electrical potential microchannel.

358. A method as in claim 357, wherein the at least one substance in the electrical potential microchannel comprises at least one of a cross-linked polyacrylamide, an agarose gel, a linear polyacrylamide, a cellulose polymer, polyethylene oxide, polyvinylpyrrolidone and other hydrophilic polymer solutions.

359. A method as in claim 357, wherein the at least one substance in the electrical potential microchannel comprises a buffer, and wherein the electrical potential microchannel has negatively charged walls for directing the buffer through the electrical potential microchannel.

360. A method of making a microfluidic device for providing one or more substances to a mass spectrometer for analysis, the method comprising the following steps of:

fabricating a device substrate comprising:

forming a plurality of microchannels having microfabricated surfaces, including a first microchannel with positively charged walls and a second microchannel with substantially neutral walls; and

forming an outlet in fluid communication with the microchannel and disposed along an edge surface of the substrate, wherein the microchannel in fluid communication with the outlet widens from a first cross sectional dimensions along the majority of its length to a second, wider cross sectional dimensions at the outlet;

fabricating a cover having at least one tip surface; and

applying the cover to the device substrate.

361. A microfluidic device for mass spectrometric analysis comprising:

a first laminate layer formed with at least one electrode and contact lead pattern; and

a second laminate layer formed with plated-through holes for providing access to the at least one electrode contact with a spring contact pin.

362. A microfluidic device for mass spectrometric analysis comprising;

a first laminate layer formed with at least one electrode and contact lead pattern; and a second laminate layer formed with a predetermined length that is shorter than the first laminate layer, wherein the at least one electrode contact is exposed for electrical contact when the first laminate layer is combined with the second laminate layer.