WO2006110177A2

WO2006110177A2 - Microfluidic pumps and mixers driven by induced-charge electro-osmosis

Info

Publication number: WO2006110177A2
Application number: PCT/US2005/037387
Authority: WO
Inventors: Jeremy Levitan; Martin Z. Bazant; Martin Schmidt; Todd Thorsen
Original assignee: Massachusetts Institute Of Technology
Priority date: 2004-10-19
Filing date: 2005-10-19
Publication date: 2006-10-19
Also published as: WO2006110177A3

Abstract

This invention provides devices and apparatuses comprising the same, for the mixing and pumping of relatively small volumes of fluid. Such devices utilize nonlinear electrokinetics as a primary mechanism for driving fluid flow. Methods of cellular analysis and high-throughput, multi-step product formation using devices of this invention are described.

Description

MICROFLUIDIC PUMPS AND MIXERS DRIVEN BY INDUCED-CHARGE

ELECTRO-OSMOSIS

GOVERNMENT SUPPORT

[001] This invention was made with U.S. Government support under Giant Number DAAD- 19-02-002, awaided by US Army Research Office The government has certain rights in the invention

BACKGROUND OF THE INVENTION

[002] The invention relates to the fields of microfluidics, micro-total-analysis systems (μTAS) and miciO-electro-mechanical systems (MEMS), in particular microfluidic pumps and mixers driven by induced-charge electro-osmosis.

[003] The ability to transport fluids in micion-sized channels is essential for many emerging technologies, such as in vivo diug deliver)' devices, micro-electro-mechanica! systems (MEMS), and micro-total -analysis systems (μTAS). New methods foi the iapid mixing of non- homogeneous fluids in micron-scale devices are also required, since the absence of tuibulent mixing on these small length scales implies that mixing occurs by molecular diffusion alone This typically takes from seconds to minutes — far too slow foi envisioned applications. New technologies are thus lequired for the manipulation, transport and mixing of fluids on these small length scales.

[004] Although MEMS-based mechanical pumps with moving parts have recently been developed, including peristaltic pumps, a variety of non-mechanical pumping strategies without moving parts have been used, e.g based on electrical fields, thermal gradient, electrochemical reactions, surface tensions gradients, and patterned surfaces Non-mechanical strategies for fluid manipulation become more efficient at very small scales because they are driven by surface phenomena. Moreover, they can be much cheaper to implement than mechanical MEMS-based strategies because they take advantage of nano-scale chemical effects already exhibited by many fluids used in biomedical and chemical engineering applications. They can also possess fewer parts, and are better suited for flexible devices, such as microfluidic fibers [005] A popular non-mechanical fluid manipulation strategy is based on the phenomena of electro-osmosis, i.e. the fluid slip at a solid-electrolyte interface induced by a tangential electric field The fluid is set into motion by strong electrostatic body forces exerted by excess ionic charge in diffuse boundary layers of thickness λ=l-100 nm near a solid interface This effect, which has been studied extensively for more than a century in colloidal science and electrochemistry, is well suited for biomedical applications because the majority of bodily fluids, such as blood or lymph, are electrolytes with comparable ionic strengths. Moreover, the working electrode imposing spatially or temporally varying electric fields can be easily and cheaply built into microchannels with existing silicon-based micio-fabrication technology- Driving fluids with electric fields also facilitates integration with logic circuits foi sensing and integration microfluidic devices

[006] The simplest electro-osmotic pumping technique is based on applying a DC field tangential to a solid channel surface, presumed to have a uniform equilibrium zeta potential ζ or diffuse charge density q. In this case, the fluid-solid surface develops a 'slip velocity' given by the classical Helmholtz-Smoluchowsld formula defined as:

with a prescribed ζ ^η or q, J where S Lo is ⁿ the pεrmissivity of vacuum, and E and η dielectric constant and viscosity of the electrolytic fluid.

[007] In spite of its appealing simplicity, however, there are several drawbacks to the use of DC electric fields, related to the fact that a steady current (J = σE ) must exi^i in ojder to maintain a steady field because every electrolyte has a non-negligible bulk conductivity. A steady current in turn implies the creation of ions at one electrode and removal of ions at the other via electrochemical reactions. This can cause a variety of pioblems. For example, the dissolution of the anode eventually destroys the electric circuit, causing irreversible failure, Microfluidic devices employing DC electric fields thus typically have short lifetimes, which can be acceptable in some applications, such as one-time diug delivery, but not in others, such as μTAS. A shorter lifetime also translates into a higher cost per unit of time of operation. The dissolution of the anode also injects metallic ions into the fluid, which can present safety hazards in biomedical applications or can interfere with chemical reactions in μTAS. Also, the depositions of ions at the cathode can lead to unstable deposits, which can break off or otherwise interfere with the bulk fluid. Furthermore, electrochemical reactions at electrodes inevitably cause electrolyte concentration gradients, which create complicated and potentially unwanted secondary bulk electric fields, as well as secondary electrokinetic phenomena at surfaces [008] These problems can be solved using high-frequency AC fields, which can be safer, more reliable and more durable than using DC fields. Because AC fields are typically applied along closely spaced electrode arrays, much smaller voltages are required to achieve strong electric fields. Furthermore, the change in electrode polarity frustrates electrochemical reactions, helping avoid unwanted electrolysis reactions at the electrodes.

[009] Since the fluid slip velocity of standard electro-osmosis used in EQ. 1 is linear in the applied field E, it averages to zero in an AC field Therefore, different phenomena must be used to drive steady microfluidic flows using AC fields. For example, AC traveling waves on electrode arrays have been used to drive flows by coupling to thermal gradients, A pair¹ of electrodes adjacently located on a glass slide, to which an AC voltage is applied, has recently been shown to drive a steady swirling flow, and a stationary AC wave on a locally asymmetric electrode array has been shown to pump fluid. Both of these applications work in a limited range of frequencies and rely on a subtle form of electro-osmosis involving induced charges on the electrodes The electro-osmotic flow is driven by transient interactions between the high- frequency field and the self-induced changes in the diffuse-layer charge density along the electrode surfaces. The pumping effect is therefore a strictly non-equilibrium phenomenon which violates the ubiquitous assumption of a constant zeta potential underlying the classical theory of electro-osmosis.

[0010] Although the available pumping techniques based on AC electric fields offer various advantages over DC methods, there are still serious drawbacks. Foremost among these is the need to microfabricate complex patterned-surface electrodes with elaborate micro-circuitry, which can be more costly, difficult, and prone to failure than their very simple DC counterparts. Another potential drawback is that patteined-surface devices aie "hard-wired" into the electrical circuitry and the physical structure of the surface itself, rendering them less versatile.

SUMMARY OF THE EWENTION

[0011] In one embodiment, this invention provides a miciofluidic device comprising two or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or more micropumps, one or more micromixers, or a combination thereof, wherein: said micropumps comprise a passageway for transmitting an electrolyte fluid; a source providing an electric field in said microchannel; at least one conductor element in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and said at least one conductor element produce electro-osmotic flows so that said electrolyte fluid is driven across said mierofluidic channels; and said micromixeis comprise a passageway for transmitting an electrolyte fluid; a source providing an electric field in said mierofluidic channels; at least one conductor element in an orientation that is perpendicular to the axis of said electric field, at a location within oi proximal to said microcharrael, whereby interactions between said electric field and each conductoi element produce electro-osmotic flows with varied trajectories, and said electrolyte fluid is driven across said miciofluidϊc channels so that said electrolyte fluid is mixed in said miciofluidic channels.

[0012] hi one embodiment;, the electric field is comprised of a DC electric field, or in anothei embodiment, the electric field is comprised of an AC or pulsed AC electric field [0013] In one embodiment, the field source is comprised ol electrodes of different polarities In one embodiment, the conductor clement is comprised of a symmetric cylinder of a defined radius In one embodiment, the conductor clement is comprised of an asymmetric conductor element, with either non-uniform surface composition or non-circular cross section In one embodiment, the conductor element is comprised of a conducting strip In one embodiment, the wall of the mierofluidic channels comprises the conducting strip.

[0014] In one embodiment, the mierofluidic channels form a cross-junction, or in another embodiment, an elbow-junction, or in anothei embodiment, a T-junction, or in another embodiment, a Y-jimction.

[0015] In one embodiment, conductor element is comprised of a symmetric conductor clement, In another embodiment, the mierofluidic channels are comprised of a transparent material, or in another embodiment, a metal, which, in one embodiment, is a metal bi-Iayer, [0016] In another embodiment, this invention provides an apparatus comprising a device of this invention,

[0017} In another embodiment, this invention provides a method of cellular analysis, comprising the steps of: a, introducing a buffered suspension comprising cells to a first inlet port of a mierofluidic device; b. introducing a reagent for cellular analysis to said first inlet port or a second inlet port of said microfluidic device, said microfluidic device comprising; i. one or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or more micropumps, one or more micromixers, or a combination thereof, wherein: β said micropumps comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microchannel; at least one conductor element in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby intei actions between said electric field and said at least one conductoi element produce electio-osmotic flows so that said suspension and said reagent are driven acioss said miciofluidic channels; and o said miciomixers comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said miciofluidic channels; at least one conductor element in an orientation that is peipendicular to the axis of said electric field, at a location within oi proximal to said microchannel, whereby interactions between said electric field and said conductor element produce electro-osmotic flows with varied trajectories, and said suspension and said reagent aie driven across said microfluidic channels so that said suspension and said reagent are mixed in said microfluidic channels; and analyzing at least one parameter affected by contact between said suspension and said reagent [0018] Li one embodiment, the reagent is an antibody, a nucleic acid, an enzyme, a substrate, a ligand, oi a combination thereof. In another embodiment, the reagent is coupled to a detectable marker, which in another embodiment is a fluorescent compound In another embodiment, the device is coupled to a fluorimeter or fluorescent microscope. In another embodiment, the device is comprised of a tiansparent material

[0019] In another embodiment, the method further comprises the step of introducing a cellular¹ lysis agent in an inlet port of said device. According to this aspect of the invention, and in one embodiment, the reagent specifically interacts or detects an intracellular compound.

[0020] In anothei embodiment, this invention provides a method of Iiigh-throughput, multi-step pioduct formation, the method comprising the steps of: a. introducing a first liquid comprising a precuisor to a first inlet port of a miciofluidic device; b. introducing a second liquid comprising a reagent, catalyst, reaclant, cofactor, or combination thereof to said first inlet port or a second inlet port of said microfluidic device, said microfluidic device comprising:

L one or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or more micropumps, one or more micromixers, or a combination thereof, wherein: o said micropumps comprise a passageway for transmitting said suspension and said reagent; a source providing a electric field in said microchannel; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or pioximal to said miciochannel, whereby interactions between said electric field and said at least one conductor element produce electro-osmotic flows so that said first liquid and said second liquid are driven across said microfluidic channels; and o said micromixers comprise a passageway for transmitting said suspension and said reagent; a source providing a electric field in said microfluidic channels; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and each conductor element produce electro-osmotic flows with varied trajectories, and said first liquid and said second liquid are driven across said microfluidic channels so that said first liquid and said second liquid are mixed in said microΩuidic channels; and c collecting said mixed liquid formed from an outlet poit of said device [0021] In one embodiment, the method further comprises the step of carrying out iterative introductions of said second liquid, as in (b), to additional inlet poits In one embodiment, the ieagent is an antibody, a nucleic acid, an enzyme, a substrate, a ligand, a reactant or a combination thereof.

[0022] In another embodiment, this invention piovides a method of drug processing and delivery, the method comprising the steps of; a. introducing a first liquid comprising a drug to a first inlet port of a microfluidic device; b. introducing a second liquid comprising a buffer, a catalyst, or combination thereof to said first inlet port or to a second inlet port of said microfluidic device, said microfluidic device comprising: i. two or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or more micropurnps, one or more micromixers, or a combination thereof, wherein: © said micropumps comprise a passageway for liansmilting said first and said second liquids; a source providing a electric field in said microchannel; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and said at least one conductor element produce electro-osmotic flows so that said first liquid and said second liquid are driven across said micioffuidic channels; and o said micromixers comprise a passageway foi transmitting said first liquid and said second liquid; a source pioviding a electric field in said rnicrofluidic channels; one or more conductor elements placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said miciofluidic channels, whereby interactions between said electric field and each conductor element produce electio-osmotic flows with varied tiajectories, and said fϊist liquid and said second liquid are driven acioss said microfluidic channels so that said first liquid and said second liquid are mixed in said microfluidic channels; and c. delivering the pioduct of (b) to a subject, through an outlet port of said device [0023] In one embodiment, the method further comprises carrying out Relative introductions of said second liquid to said inlet ports Ln another embodiment, the second liquid serves to dilute the drug to a desired concentration.

[0024] In another embodiment, this invention provides a method of analyte detection or assay, comprising the steps of: a. introducing a fluid comprising an analyte to a first inlet port of a microfluidic device, said microfluidic device comprising:

L one or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or more micropumps, one or more micromixers, or a combination thereof, wherein: a, said micropumps comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microchannel; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and said at least one conductor element produce electro- osmotic flows so that said suspension and said reagent are driven across said microfluidic channels; and b. said rnicromixers comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microfluidic channels; an array of conductor elements placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microfluidic channels, whereby interactions between said electric field and each conductor element produce electro-osmotic flows with varied trajectories, and said suspension and said reagent are driven across said microfluidic channels so that said suspension and said reagent are mixed in said microfluidic channels; c said microchannels being coated with a reagent for the detection, assay, or combination thereof of said analyte; and ©detecting, analyzing, 01 a combination thereof, of said analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0026] Figure 1 schematically depicts one embodiment of a microfluidic two-stage mixer, comprising inlet ports 1-1O₅ 1-20, and 1-30 leading to channels 1-40, 1-50, and 1-80 respectively. Channels 1-40 and 1-50 conjoin to a Y-junction 1-60 and lead to channel 1-70, containing a nonlinear electroldnetic mixer Channels 1-70 and 1-80 conjoin to a Y-junction 1-90 and lead to channel 1-100 containing a nonlinear electrokinetic mixer. Channel 1-100 connects to channel 1~ 110 and outlet 1-40.

[0027] Figure 2 schematically depicts an embodiment of a fabrication process for the device described in Figure 1. labeled as section A-A.

[0028] Figure 3 schematically depicts induced-charge electro-osmotic micropump designs for sample cross, elbow, and T junctions.

[0029] Figuie 4 schematically depicts induced-charge electro-osmotic mixers [0030] Figure 5 schematically depicts sample pumps driven by induced-charge electro- osmotic flows generated at asymmetric conducting posts

[0031] Figure 6 schematically depicts linear-channel pump-mixers driven by electro-osmotic flows

[0032] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity Further, where consideied appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements,

DETAILED DESCRIPTION OF THE PRESENT INVENTION [0033] In the following detailed description, numerous specific details are set forth in oider to provide a thoiough understanding of the invention However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

[0034] This invention piovides, in some embodiments, devices and apparatuses comprising the same, for the mixing and pumping of relatively small volumes of fluid. Such devices utilize nonlinear electrokinetics as a primary mechanism for driving fluid flow. [0035] In one embodiment, this invention provides a miciofluidic device comprising two or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or more micropumps, one or more micromixers, or a combination thereof, wherein: said micropumps comprise a passageway foi transmitting an electrolyte fluid; a soiuce providing an electric field in said miciochannel; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby intei actions between said electric field and said at least one conductor element produce electro- osmotic flows so that said electrolyte fluid is driven across said microfluidic channels; and said micromixers comprise a passageway for transmitting an electrolyte fluid; a source providing an electric field in said microfluidic channels; an an ay of conducioi elements placed in an orientation that is perpendicular to the axis of said electric field, at a location within or pioximal to said microfluidic channels, whereby interactions between said electric field and each conductor element produce electio-osrnotic flows with varied trajectories, and said electiolyte fluid is driven across said microfluidic channels so that said electrolyte fluid is mixed in said microfluidic channels

[0036] In one embodiment, the subshate and/or other components of the device can be made from a wide variety of materials including, but not limited to, silicon, silicon dioxide, silicon nitride, glass and fused silica, gallium aisenide, indium phosphide, ID-V materials, PDMS. silicone lubber, aluminum, ceramics, polyimide, quartz, plastics, resins and polymers including π polymethylmethacrylate (PMMA). acrylics, polyethylene, polyethylene terepthalate, polycarbonate, polystyrene and other styrene copolymers, polypropylene, polytetxafluoioethylene, superalloys, zircaloy?. steel, gold, silver, copper, tungsten, molybdeumn, tantalum, KOVAR, KEVLAR, KAPTON, MYLAR, teflon, brass, sapphire, othei plastics, or other flexible plastics (polyimide), ceramics, etc., or a combination thereof. The substrate may be ground or processed flat. High quality glasses such as high melting borosilicate oi fused silicas may be used, in some embodiments, for their UV transmission properties when any of the sample manipulation and/oi detection steps require light based technologies. In addition, as outlined herein, portions of the internal and/or external surfaces of the device may be coated with a variety of coatings as needed, to facilitate the manipulation or detection technique performed.

[0037] In one embodiment, the substrate comprises a metal-bi layer, In some embodiments, the substrate may be further coated with a dielectiic and/or a self-assembled monolayer (SAM), to provide specific functionality to the surface of the device to which the mateiial is applied. [0038] In one embodiment, the microchannels comprise the same materials as the subsliate, or in another embodiment, are compiised of a suitable material which prevents adhesion to the channels..

[0039] In anothei embodiment, the substrate and/ot microchannels of the devices of this invention comprise a material which is functionalized to minimize, reduce or prevent adherence of materials introduced into the device. For example, in one embodiment, the functionalization comprises coating with extiacellular matrix protεin/s, amino acids, PEG, oi PEG functionalized

SAM's or is slightly charged to prevent adhesion of cells or cellular material to the surface In

< another embodiment, functionalization comprises treatment of a surface to minimize, icduce or pievent background fluoiescen.ee. Such functionalization may comprise, for example, inclusion of anti-quenching materials, as are known in the art. In another embodiment, the functionalization may comprise treatment with specific materials to alter flow properties of the material through the device In another embodiment, such functionalization may be in discrete regions, randomly, or may entirely functionalize an exposed surface of a device of this invention. [0040] In one embodiment, the invention provides for a microchip comprising the devices of this invention In one embodiment, the microchip may be made of a wide variety of materials and can be configured in a large nurnbei of ways, as described and exemplified herein, in some embodiments and other embodiments will be apparent to one of skill in the art The composition of the substrate will depend on a variety of factors, including the techniques used to create the device, the use of the device, the composition of the sample, the molecules to be assayed, the type of analysis conducted following assay, the size of internal structures, the placement of electronic components, etc. In some embodiments, the devices of the invention will be sterilizable as well, in some embodiments, this is not required. In some embodiments, the devices are disposable or, in anothei embodiment, re-usable.

[0041] Microfluidic chips used in the methods and devices of this invention may be fabricated using a variety of techniques, including, but not limited to, hot embossing, such as described in H Becker, et ah, Sensors and Materials, 11, 297, (1999), hereby incorpoiated by reference, molding of elastomers, such as described in D. C. Duffy, et. al., Anal. Chem., 70, 4974, (1998), heieby incorporated by reference, injection molding, LIGA, soft lithography, silicon fabrication and ielated thin film processing techniques, as known in the art, photolithography and reactive ion etching techniques, as exemplified herein In one embodiment, glass etching and diffusion bonding of fused silica substrates may be used to prepare microfluidic chips. [0042] In one embodiment, microfabrication technology, or microtechnology oi MEMS, applies the tools and processes of semiconductoi fabrication to the foimation of, foi example, physical structures. Microfabrication technology allows one, in one embodiment, to piecisely design features (e.g., reservoirs, wells, channels) with dimensions in the range of <1 μm to several centimeters on chips made, in other embodiments, of silicon, glass, oi plastics. Such technology may be used to construct the niicrochannels of the devices of this invention, in one embodiment. [0043] In one embodiment, fabrication of the device may be accomplished as follows: first, a glass substrate is metallized. The choice of metal can be made with respect to a variety of desired design specifications, including resistance to oxidation, compatibility with biological materials, compatibility with substrates, etc The metallization layer may be deposited in a specific pattern (i.e through adhesive or shadow-masked metal evaporation or sputtering), in one embodiment, or, in another embodiment, it may be etched subsequent to deposition. Metals can include, but arc not limited to gold, copper, silver, platinum, ihodium, chromium, etc. In some embodiments, the substrate may be coated with an initial layer of a thin metal, which promotes adhesion of another metal to the substiate. In some embodiments, metals may also be adhered to the substrate via adhesive. In some embodiments, the substrate is ground flat to promote adhesion. In some embodiments, the substrate is roughened to piomote metal adhesion, [0044] According to this aspect of the invention, and in one embodiment, the deposited metal may either be deposited in the final topology (i.e. through a mask) or, in another embodiment, patterned post-deposition. According to the latter embodiment, a variety of methods may be used to create the final pattern, as will be understood by one skilled in the art, including inier-alia, etching and laser ablation. Mechanical forms of lemoval (milling, etc ) may be used, in other embodiments

[0045] In one embodiment, gold is deposited on chromium and the gold is etched using a photoresist mask and a wet gold etchant. The chromium remains a uniform film, providing electi ical connection for subsequent electrodeposition (forming the anode connection), Ln another embodiment, gold is deposited via electron-beam evaporation onto an adhesion layer of titanium The gold is patterned using a wet etchant and photoresist mask. The titanium is left undisturbed for subsequent electrodeposition.

[0046] In another embodiment, the metal may be patterned prioi to deposition. A shadow mask can be utilized in one embodiment. The desired shape is etched or machined through a thin metal pattern or other substrate The etched substrate is then held parallel to the base substrate and the material is deposited via evaporation or sputtering through the mask onto the substrate

This method i educes the number of etch steps.

[0047] In anothei embodiment, the patterned surface is formed by transferring a pre-etched or stamped metal film with adhesive onto the subsuale In one embodiment, the various devices on the layer have a common electrical connection enabling subsequent eieclrodeposition, and aie deposited strategically so that release and dicing iesults in piopcr electrical isolation-

[0048] In another embodiment, a rigid stamp is used to puncture a thin metal film on a relatively pliable elastic (plastic) substrate The rigid stamp can have, in some embodiments sharp or blunt edges.

[0049] hi some embodiments, the thickness of deposited metals is tailored to specific applications. Ln one embodiment, thin metal is deposited onto the surface of the wafer and patterned, According to this aspect of the invention, and in one embodiment, the patterned surface forms a common anodic connection for electroplating into a mold

[0050] In one embodiment, molding may be used, In one embodiment, molding comprises a variety of plastics, ceramics, or other material which is dissimilar to the base substrate. In one embodiment, the molding material is removed following electroplating In some embodiments, the molding material is sacrificial.

[0051] In another embodiment, thick (greater than a few microns) metal is deposited and subsequently etched to form raised metal features.

[0052] In other embodiments, welding, assembly via SAMs, selective oxidation of thin metals

(conversion of, for instance, aluminum to aluminum oxide) comprise some of the methods used to form insulating areas and provide electrical isolation. [0053] In other embodiments, passivation of the metal surfaces with dielectric materials may be conducted, including, but not limited to, spin-on-glass, low temperature oxide deposition, plastics, photoresists, and other sputtered, evaporated, or vapor-deposited insulators. [0054] In some embodiments, the microfluidic channels used in the devices and/or methods of this invention, which convey fluid, may be constructed of a material which renders it transparent or semitiansparent, in order to image the materials being assayed, or in another embodiment, to ascertain the progress of the assay, etc. In some embodiments, the materials jxuther have low conductivity and high chemical resistance to buffer solutions and/or mild oiganics. In other embodiments, the material is of a machinable or moldable polymeric material, and may comprise iπsulatois, ceiamics, metals or insulator-coated metals. In other embodiments, the channel may be constructed from a polymei material mat is resistant to alkaline aqueous solutions and mild organics. In another embodiment, the channel comprises at least one suiface which is transparent oi semi-transparent, such that, in one embodiment, imaging of the device is possible. [0055] In one embodiment, the inlet, or in another embodiment, the outlet may comprise an area of the substiate in fluidic communication with one oi more microfluidic channels, in one embodiment, and/or a sample leservoir, in another embodiment. Inlets and outlets may be fabricated in a wide variety of ways, depending upon, in one embodiment, on the substrate material utilized and/or in another embodiment, the dimensions used. In one embodiment inlets and/or outlets are formed using conventional tubing, which prevents sample leakage, when fluid is applied to the device, under pressure In one embodiment inlets and/or outlets are formed of a material which withstands application of voltage, even high voltage, to the device In one embodiment, the inlet may further comprise a means of applying a constant pressure, to generate pressure-driven flow in the device.

[0056] hi one embodiment, a "device^" or "apparatus" of this invention will comprise at least the elements as described herein. In one embodiment, the devices of this invention comprise at least one microchannel, which may be formed as described heien, or via using other miciofabiication means known in the art. In one embodiment, the device may comprise a plurality of channels. In one embodiment, the phrase "a plurality of channels" iefers to more than two channels, or, in another embodiment, moie than 5, or. in other embodiments, more than 10, 96, 100. 384, 1 ,000, 1,536, 10,000, 100,000 or 1 ,000.000 channels.

[0057] In one embodiment, the devices of this invention comprise micropumps and/or micromixers as defined herein. In one embodiment, the miciopump comprises a passageway for transmitting an electrolyte fluid, which, in one embodiment, is a microchannel as described herein

[0058] In one embodiment, the micropump also comprises a source providing an electric field in the microchannel and at least one conductoi element that is placed in an orientation that is perpendicular to the axis of the electric field, at a location within oi pioximal to the microchannel. Interactions between the electric field and the conductor element produce electro- osmotic flows so that said electrolyte fluid is driven across the microfiuidic channels [0059] In one embodiment, the term "electrolyte fluid" refers to a solution, or in another embodiment, a suspension, or, in anothei embodiment, any liquid which will be conveyed upon the operation of a device of this invention In one embodiment, such a fluid may comprise a liquid comprising salts or ionic species. In one embodiment, the ionic species may be present, at any concentration, which facilitates conduction through the devices of this invention In one embodiment, the liquid is water, or in another embodiment, distilled ionized water, which has an ionic concentration ranging from about 1OnM to about 0,1 M. In one embodiment, a salt solution, ianging ranging in concentration from about 1OnM to about 0.1M is used. In one embodiment, a ImM KCl solution, when applied in a device of this invention may provide fluid velocities in excess of 2 miυ/s. In one embodiment, higher flows may be obtained when a 100V/cm applied field is used and 50V is applied to the conductor,

[0060] In another embodiment, the fluid comprises solutions oi buffered media for use suitable for the particular application of the device, for example, with regards to the method of cellular analysis, the buffer will be appropriate foi the cells being assayed. In one embodiment, the fluid may comprise a medium in which the sample material is solubilized or suspended. In one embodiment, such a fluid may comprise bodily fluids such as, in some embodiments, blood, urine, serum, lymph, saliva, anal and vaginal secretions, perspiration and semen, oi in another embodiment, homogenates of solid tissues, as described, such as, for example, liver, spleen, bone marrow, lung, muscle, neivous system tissue, etc., and may be obtained from virtually any organism, including, for example mammals, rodents, bacteria, etc. In some embodiments, the solutions or buffered media may comprise environmental samples such as, for example, materials obtained from air, agricultural, water or soil sources, which are present in a fluid which can be subjected to the methods of this invention. In another embodiment, such samples may be biological warfare agent samples; research samples and may comprise, for example, glycoproteins, biotoxins, purified proteins, etc. [0061] In one embodiment, the pH, ionic strength, temperature or combination thereof of the media/solution, etc., may be varied, to affect the assay conditions, as described herein, the rate of transit through the device, oi combination thereof.

[0062] As will be appreciated by those in the art, virtually any experimental manipulation may have been done on the sample prior to its use in embodiments of the present invention. For example, a variety of manipulations may be performed to generate a liquid sample of sufficient quantity fiom a raw sample. In some embodiments, gas samples and aerosol samples are so processed to generate a liquid sample containing molecules whose separation may be accomplished according to the methods of this invention.

[0063] Micropumps of this invention make use of non-linear, electroosmotic flow. In one embodiment, such flow is generated by the elements of the device, and their respective positioning in the device, as exemplified and described herein. In one embodiment, the conductor element is placed in an orientation that is perpendicular to the axis of the electric field, in a device of this invention In one embodiment, the term "perpendicular" oi "perpendicularly" refers to an orientation of a 90° angle with respect to the field axis, +/-5, or in another embodiment, at a 90° angle of +/- 10°, or in another embodiment, at a 90° angle +/- 20°.

[0064] Device operation relies upon the evolution of an electric field mound a solid conducting cylinder imrneised in a liquid electrolyte, in one embodiment Just aftei an electric field is applied, it must intersect a conducting surface at right angles Mobile ions in the liquid electrolyte are driven along electric field lines — positive ions in the direction of the field, and negative ions opposite the field direction. At the conductor/electrode surface, the field lines terminate, causing ions to accumulate in a small 'diffuse layer' and inducing an opposite

'image charge' in the conductor. Thus, according to this embodiment, positive ions accumulate around the side of the conductor nearest the field source, and negative ions around the side nearest the field sink. This induced-charge 'diffuse layer' grows, gradually expelling field lines, until all field lines are expelled. The steady state field configuration, is the same as that found around a perfect dielectric cylinder, and is attained after a time tc=λa/D, which is essentially the "RC" time of an equivalent resistor-capacitoi circuit, where D is the diffusivity constant of the electrolyte

[0065] This has important implications for the induced electro-osmotic fluid velocity The cylinder is surrounded by a dipolar diffuse charge cloud that is positive on one hemisphere and negative on the other. On the top of the cylinder, the positively-charged diffuse cloud is driven along the field lines towards the 'equator' of the cylinder; on the bottom, the negatively-charged diffuse cloud is driven against the field direction — also towards the 'equator' of the cylinder. The resulting 'induced-charge electro-osmotic' slip velocity is quadrupolar in nature. Generically, the induced fluid flow is driven from the 'poles' of the conducting body, towards its 'equator', [0066] The classical theory of electro-osmosis is based on the assumption that a solid object has a uniform charge density, or zeta potential, which is taken to be a constant material propeity. While tlαis can be appropriately applied to insulating materials, such as latex, it is certainly not for conductors with free charges, especially out of equilibrium. Although it is not commonly appreciated, the double layers in such conductors will generally develop non-unifoim polarizations in space and time in response to applied fields, In simple terms, the interfacial double layer acts as a nonlinear capacitor "skin^"' between the bulk liquid electrolyte and the conducting solid, and the local electro-osmotic slip, which varies in space and time, is simply given by the product of the tangential field and the potential difference acioss the capacitor "skin" For an arbitrary shaped conductoi, this generally produces an electro-osmotic flow, which draws fluid along the field axis and ejects perpendicular to the field axis, foi both AC and DC fields. Weaker flows of the same type can be produced around dielectrics, relying upon polarization by the orientation of bound dipoles rather than the separation of flee charges. [0067] Electro-osmotic flows around an uncharged and charged conducting cylinder. The induced-charge electro-osmotic flow around an uncharged conducting cylinder conducting cylinder can arise either from an applied background DC field aftei the charging time λa/D or from an applied field AC field with a frequency less than ωc=D/λa Using Eq. 1. one can identify the geneial sense of the electro-osmotic flow On the side of the conductor facing the field source, the diffuse charge q is positive, so the fluid slips in the direction of the tangential field Ei, forwaid toward the equator. On the other side, away from the field source, the diffuse charge is negative, so the fluid slips opposite the tangential field direction, toward the equator Therefore, the electro-osmotic flow for any uncharged conductor generally pulls fluid in along the field axis toward both poles and expels it, radially from the equator.

[0068] In weak AC fields, if the field direction is reversed, then so are the signs of the induced charges, and thus the flow remains unchanged. Theiefore, this electro-osmotic flow will persist even in an AC applied field For example, it can be shown that the time averaged slip velocity for a conducting cylinder in a weak background AC field Eo cos(ωt) is given by,

[0069] where ωc=D/λa (»103-105 for a ~l-10 μm and λ^l-10 nm) is the characteristic double-layer charging frequency, above which the average electro-osmotic slip velocity vanishes because ions cannot relax quickly enough to keep up with the oscillating field. [0070] Note that the typical pumping velocities in weak fields are of the order of microns per second or more, depending on the applied field, which is comparable to other existing electroldnetic phenomena of potential use for microfluidic pumping, and much greater velocities can be achieved with strong fields Note that the induced-charge electro-osmotic fluid velocity grows with the square of the applied field. This favorable nonlinear response can be exploited in our miciofluidic devices to achieve much largei pumping velocities than with "normal electio- osmosis "

[0071] If there are no electrochemical reactions at the electrodes, the same diffuse-layei chaiging effect occurs at the electiode surfaces It can be shown that following a suddenly imposed DC voltage, the electrode diffuse layers become charged and screen out the bulk electric field at the time scale, τL=λL/D, where L is the distance between the electrodes. Similarly, foi an AC field with applied voltage VO cos(ωt), the bulk electric field amplitude is given by

[0072] which decays to zeio above the characteristic frequency ωi^D/λh^l -l Q Hz for L~100- 10,000 μm and λ^l-10nm

[0073] Therefore, strong induced-charge electro-osmotic flows driven by AC applied voltages can persist only in a certain band of driving f equencies, ω_L<ω<ω_a~ For example, if a = lOum and lambda = IOnm, w_a = 300Hz.

[0074] Induced-charge electio-osmotic flows around a charged cylinder, where the cylinder is electrically isolated with a non-zero charge, produces the flow described hereinabove combined with the normal electro-osmotic flow, which simply wraps aiound the object. Since the latter flow is proportional to the field and the total charge, it changes direction if the electric field is reversed, and therefore, it averages to zero in an AC field, leaving only a quadrupolar induced- charge electro-osmotic flow, regardless of the total charge of the conductor. [0075] Induced-charge distributions and slip velocities for various asymmetric conducting objects in a DC or AC field, are envisioned as well By manipulating the fore-aft symmetry of a conductor in a DC or AC applied field, a net osmotic flow along the field axis or a net phoretic swimming velocity can be produced. For example, a conducting cylinder whose fore-aft syminetiy is broken lhiough the application of a metallic coating with a higher Stem compact layer capacitance, which absorbs ions and prevents them fiora producing electro-osmotic slip, reduces the pumping effect on the coated side relative to the uncoated side, iesulting in a net flow past the object

[0076] In another embodiment, a directed electro-osmotic osmotic flow, even in an AC field, can be obtained, In one embodiment, the arrangement includes a cylinder, which is partially insulated with a dielectric coating used to suppress double-layer charging (for example using with a layered strip). Following a time-dependent diffuse-layer charging, the effect of the dielectric coating is, in one embodiment, to bring the negative ions towards the sides of the cylinder and the positive ions on the bottom region of the cylinder The slip velocity produced by the negative charges is directed downward past the equatorial region of the cylinder, towards the uncoated side The positive charges also produce a slip velocity directed upward toward the equatorial region of the cylindei Note that the magnitude of the slip velocity formed by the negative charges is larger in magnitude than the slip velocity formed by the positive charges, due to the stronger tangential field near the equator compared to that near the pole. The net osmotic flow would thus be directed downward, toward the uncoated side. It is important to note as well mat a conducting cylinder, which is entirely coated with a dielectric layer has a greatly i educed induced-charge electro-osmotic fluid flow; and clean conductor/electrolyte suifaces obviate this issue

[0077] In another embodiment, an asymmetric, using a tear-drop asymmetric shaped conductor — or more generally, any asymmetrically-shaped body, can produce a directed induced- charge electro-osmotic flow under the influence of an AC electric field When a background field is applied, the tear-drop asymmetric shaped conductor, for example, produces positive and negative charge regions. According to this aspect of the invention, and in one embodiment, the negative charge regions include the most curved legion, of the tear-drop shaped conductor. The positive regions include the less curved portion of the tear-drop shaped conductor The direction of the slip velocity foimed by the negative charge regions is directed downward, and the direction of the slip velocity formed by the positive charge regions is upward along the teai-drop shaped conductor The magnitude of the slip velocity produced by the negative chaigc regions is larger than the magnitude of the slip velocity produced by the positive charge regions. Therefore, the net electro-osmotic flow is diiected towards the region of lower curvature, downward along the tear-drop shaped conductor. [0078] In anothei embodiment, the direction of the background field changes, such that the charge distribution also changes. For example, the negative regions will include the bottom regions of the tear-diop shaped conductor, and the positive charge regions will include the upper most curve regions of the tear-drop shape conductor In one embodiment, however, the field driving the induced-charge electro-osmotic flow is also reversed, so that the net electro-osmotic flow remains a net downward, away from the pointed edge. Thus, net flow persists in an AC field, which is veiy different from normal electro-osmosis, which averages to zero in an AC field, [0079] hi some embodiments, the conductor configurations described have a symmetry, which is broken in the fore-aft sense, measuied relative to the applied field direction The left-right symmetry of the conductor may also be broken, in another embodiment, leading to induced- charge electro-osmotic flows which are driven perpendicular to the applied field, and which peisist even in AC fields

[0080] In one embodiment, electio-osmotic miciopumps may be positioned in a device to form, or conduct into cross. T, Y and/or elbow junctions Using the principles hereinbcfoie iegarding electro-osmotic flow, one can design different junction pump aiiangements By using a working conductor in conjunction with an applied electric field, the induced-charge electro- osmotic flow generally drives fluid flow in along the field axis and ejects it out ftom the "equator', perpendicular to the field axis. This effect can be used to pump fluid at right angles, by simply placing a cylindrical conducting wiie in the junction, perpendicular to the field axis and the plane of flow.

[0081] For example, in Figure 3A, a microfluidic cross-shaped micropump design 3-10 is shown The cross-shaped micropump design 3-10 includes fom junction walls 3-32, 3-34, 3-36. and 3-38, four electrodes 3-12, 3-14, 3-16, and 3-18, and a cylindrical conductor 3-30 The cylindrical conductor 30 has transient surface charges in the applied field, which drive the electro-osmotic flow In the configuration of FIG 3 A, electrodes 3-12 and 3-14 have the same polarity wheieas elecUodes 3-16 and 3-18 have the opposite polarity, which sets up a field in the vertical direction, causing a pumping of fluid from the veitical channels into the horizontal channels. By switching electrode polarity so that electiodes 3-12 and 3-16 have the same polarity and electrodes 3-14 and 3-18 have the opposite polarity, the field can be switched from vertical to horizontal, and the pumping direction can be reversed Also, the cylindrical conductor is strategically placed at the intersection point between the microchannels 3-20, 3-22, 3-24, and 3- 26 [0082] FIG, 3B demonstrates a T-junction miciopump arrangement 3-58 The T-junction micropump arrangement 3-58 includes junction walls 3-40, 3-42, and 3-44, a paii of electrodes 3- 46 and 3-50, and a conducting plate 3-48 placed on the junction wall 3-40 between the pair of electrodes 3-46 and 3-50 The flow is directed into the miciochannel 3-52. In this embodiment, the polarities of the pair of electiodes 3-46 and 3-50 cannot be reversed, thus preventing the reversal of the pump. However, a reveisible T-junction can also be designed with four electrodes and a conduction post, like in FIG, 3A with one channel closed. This allows the flow direction to be driven either into or out of microchannel 3-52.

[0083] FIG 3C demonstrates an elbow junction arrangement 3-78 This arrangement includes four electrodes 3-66, 3-68, 3-70, and 3-72, a cylindrical conductor 3-73, and junction walls 3-60, 3-62, and 3-64 The electiodes 3-66, 3-68, 3-70, and 3-72 are aligned on the junction walls 3-60, 3-62, and 3-64, The cylindrical conductor 3-73 is strategically placed in the center of intersection point between microchannels 3-74 and 3-76 By placing the cylindrical conductoi 3-73 in the junction, peipendiculai to the field axis and the plane of flow, the fluid is driven around a corner to microchannel 3-76. In this embodiment, the electrodes 3-66 and 3-70 have the same polarity and the electrodes 3-68 and 3-72 have the opposite polarity, and the direction of the pumping is fiom miciochannel 3-74 toward microchannel 3-76. However, by driving eiectiodes 3-66 and 68 with the same polarity, and 70 and 72 with polarity opposite to that of electrodes 3-66 and 3-68, the direction of flow is reversed, pumping fluid into microchannel 3-74. [0084] The junction pumps shown in FIGS. 3A-3C and described above can be operated using a DC electric field or an AC electric field, oi a pulsed AC electric field Furthermore, the 'working' conductor in each of these devices can be electrically isolated from the electrodes, which drive the electric field; or the working conducting element can be held at a fixed potential or grounded. Holding the working conductor at a fixed potential induces an additional induced- charge electro-osmotic flow that is pioportional to the square of the applied field, and is directed away from the nearest wail This additional flow can be incorporated into any of the devices described herein, enhancing the fluid flow driven into certain channels in the micropumps, or providing an additional mixing flow in the mixers described below

[0085] In one embodiment, the devices and/or methods of this invention make use of a micromixer. In one embodiment, the miciomixers of this invention comprise a passageway for transmitting an electrolyte fluid; a source providing an electric field in the microfluidic channels; an array of conductor elements placed in an orientation that is perpendicular to the axis of the electric field, at a location within or proximal to the microfluidic channels, whereby interactions between the electric field and each conductor element produce electro-osmotic flows with varied trajectories, and the electrolyte fluid is driven across the microfluidic channels. This results in the electrolyte fluid being mixed in the microfluidic channels

[0086] In one embodiment, micromixer function is as depicted in Figure 4 In one embodiment, FIG, 4A, demonstrates a design for a fast induced-charge electro-osmotic mixer 4- 80. The mixer 4-80 includes a pair of microelectiodes 4-82 and 4-84 and an array of conducting posts 4-88. The electrode 4-82 is positive and the electrode 84 is negative, and their polarities can be reversed The conducting posts 4-88 include metallic wires, as in the junction pumps described herein. A background flow passes through the array of conducting posts 4-88. Also, an AC field in the appropriate frequency range (ωi.<ω<ω,,) is applied perpendicular to the posts 4-88 and to the mean flow diiection, which generates an array of persistent convection iolls via the same electro-osmotic mechanism used in the junction pumps, described herein. The particles in the background flow aic advected through convection rolls along complicated trajectories, which stretch fluid elements This enhances diffusive mixing. Using pulsed AC fields to produce chaotic flows can also further enhance the degiee of mixing.

[0087] FIG. 4B demonstrates another embodiment for the design for a fast electro-osmotic mixer 4-90. The mixer 4-90 includes four electrodes 4-98, 4-100. 4-102, and 4-104 and metal strips 4-92 embedded in the interior of the channel walls 4-94 and 4-96. This design produces the same kind of convective mixing pioduced by the mixer 4-80, By applying an AC or DC field along the channel with the metal strips 4-92 embedded within channel walls 4-94 and 4-96 in between electrodes 4-98, 4-100, 4-102, and 4-104. Various arrows illustrate the convection mixing. As with posts 4-88 described herein, are electrically isolated from the electrodes 4-98, 4- 100, 4-102, and 4-104 If the metal strips 4-92 were grounded or held at a fixed potential, an additional induced-charge electro-osmotic flow would result, in addition to the flow described here,

[0088] Figure 5 A and 5B depict other embodiments of pumps driven by electro-osmotic flows generated at asymmetric conducting posts. As described herein, a conductor in AC or DC applied fields with broken fore-aft or left-right symmetry generally pioduce net electro-osmotic pumping along the direction of broken symmetry. Therefore, it is possible to produce linear channel pumps using conducting posts, which possess broken asymmetry. Triangular conducting posts 5-120 are shown in FIGS. 5A-5B and represent embodiments for methods of breaking the symmetry of the conducting array, of which three examples are shown in FIGS. 3A-3C, Furthermore, the applied field can eithei be along the direction of the channel as shown in FIG. 5 A or across the channel, perpendicular to it as shown in FIG 5B In all cases, fluid flow is driven along the channel. [0089] FIG. 5 A demonstrates a linear-channel pump 5-106, The linear-channel pump 5-106 includes electrodes 5-108, 5-1 10, 5-1 12, and 5-114, asymmetric conducting posts 5-120, and a microchannel 5-122. FIG 5B demonstrates a linear-channel pump 5-107. The linear -channel pump 5-107 includes electrodes 5-1 16 and 5-118, asymmetric conducting posts 5-121, and a microchannel 5-123 The posts 5-120 and 5-121 are schematically represented by triangles to indicate any oi the general symmetry-breaking mechanisms, of which three are shown in FIGS. 3A-3C, The linear channel pumps 5-106 and 5-107 are driven by electro-osmotic flows geneiated by posts with symmetry broken in the channel direction, and an AC or DC field directed along or across the microchannels 5-122 and 5-123 Other broken symmetry conducting posts, such as conducting posts having a cross-section of a tear-drop oi triangle, dielectric or metallic partial coatings, can also be used. In the case of a broken fore-aft spatial symmetry, as shown in FIG. 5A, the sharpest point of the cross section is directed opposite to the desired flow direction of induced-charge electro-osmotic pumping In the case of a broken left-right spatial symmetry, as shown in FIG 5B, the sharpest point of the cioss section is directed in the desired direction of induced-charge electro-osmotic pumping. Another embodiment for preparing such posts (5-120 and 5-121) may be to simply place two or more wiies of different cross sections against each other to approximate the triangle's shape. In this way, an AC electro-osmotic linear-channel pump can be built out of ordinary metal micro-wires of circular cross-section [0090] Unlike the junction pumps described herein, which are driven by a single electro- osmotic source that cannot drive flows across very large distances, the asymmetric posts can be arranged in extended arrays to provide the distributed forcing needed to drive fluid quickly along lengthy channels.

[0091] Figure 6 A and B are schematics of embodiments of linear-channel pump-mixers driven by electro-osmotic flows. The design of the linear-channel pump can be altered to produce microfluidic devices, which can simultaneously pump and mix fluids FIG. 6A demonstrates a pump-mixer arrangement 6-124 that includes electrodes 6-126, 6-128, 6-130, and 6-132, asymmetric conducting posts 6-136 associated with a cylinder covered with a dielectric or metallic coating, and a microchannel 6-134. The electrodes 6-126, 6-128, 6-130, and 6-132 permit reversing their polarities and producing AC or DC fields. Instead of four electrodes, two electrodes, as in FlG, 5B, placed on either side of the channel and driving an AC or DC electric field perpendicular to the channel direction can also be used. The coatings of the conducting posts 6-136 are directed opposite the flow direction, in an AC or DC field directed along the microchannel 6-134 Given that each of the conducting posts 6-136 produces flows that are directed in along the field axis and out perpendicular to the field axis, this provides an overall mixing pattern shown in FIG. 6A. Also, the asymmetric shape provides the necessary force to pump fluid through the microchannel 6-134. Of course, any broken symmetry will be sufficient to produce a pump/mixer, as discussed above.

[0092] FIG 6B demonstrates another arrangement of a linear-channel pump-mixer 6-138, The pump-mixei 6-138 includes four electrodes 6-140, 6-142, 6-144, and 6-146 and asymmetric metal ridges 6-152 patterned on the walls 6-148 and 6-150 of a microchannel 154 between the electrodes 6-140, 6-142, 6-144, and 6-146 The electrodes 6-140, 6-142, 6-144, and 6-146 allow reversing their polarities and producing AC or DC fields. The asymmetric ridges 6-152 are designed to lean in the direction of the flow, in an AC oi DC field directed along the microchannel 6-154. The suiface of the asymmetric ridges 6-152 is a grooved metallic surface, not connected in any way to the external circuit, which includes noimal electrodes positioned in the channel walls 6-148 and 6-150 on either side of the grooved surface. [0093] In one embodiment, the conductor element is an array of conducting elements, as will be appreciated by one skilled in the art Some embodiments of arrays of such elements are described hereinabove In some embodiments, the airays may comprise a lattice, which may have a variety of geometries, such as a square, hexagon, etc , as will be appreciated by one skilled in the ail. Such orientation oi arrays may be particularly useful in the micromixers of this invention In one embodiment, a single unit functions as both miciopump and micromixcr, as will be appreciated by one skilled in the art. In one embodiment, the teπn "mixing" as used herein iefers to circulation of materials to promote their distribution in a volume of space, for example, a mixture of 2 species, in a device of this invention, refers, in one embodiment, to a random distribution of the 2 species within a given volume of space of the device, e.g , in a microchannel of the devices of this invention. In one embodiment, the term "circulation" and "mixing" are interchangeable, In one embodiment, mixing refers to a change in a particular distribution which is not accompanied by agitation of the sample, in one embodiment, or in anothei embodiment, minimal agitation and/or formation of "bubbles" in the liquid medium in which the species are conveyed.

[0094] In some embodiments, the conducting element may be fashioned to assume a variety of geometries, as described and exemplified herein. In one embodiment, such design will reflect a consideration of a desired trajectory for a particular application. In some embodiments, the geometry may approximate an arrowhead, a teardrop, oi elliptical shape, or one related thereto. [0095] While the electrode and field polarities as "+" and "-^*' signs throughout, all fields can also be AC or DC corresponding to electrode polarities oscillating between + and -, giving rise to the same induced-charge electio-osmotic flow Thus all of the devices of the invention can operate in AC or DC.

[0096] In some embodiments, the present invention provides for the operation of the device in AC with DC offset, as will be understood by one skilled in the art, for example, as described in U. S. Patent Number 5,907,155, In another embodiment, asymmetric driving signals may be used,

[0097] The invention provides a number of designs for microfluidic devices taking advantage of induced-charge electro-osmotic flows around conductors Although these devices can operate with DC voltages, the invention also woilcs with AC applied voltages

[0098] In one embodiment, the device is adapted such that analysis of a species of interest may be conducted, in one embodiment, in the device, or in another embodiment, downstream of the device In one embodiment, analysis downstream of the device refers to removal of the obtained product from the device, and placement in an appiopriate setting for analysis, or in another embodiment, construction of a conduit fiom the device, for example, from a collection port, which relays the material to an appropriate setting for analysis Ln one embodiment, such analysis may comprise signal acquisition, and in another embodiment, a data processor, In one embodiment, the signal can be a photon, electrical current/impedance measurement or change in measurements It is to be understood that the devices of this invention may be useful in various analytical systems, including bioanalysis microsystems, due to the simplicity, performance, robustness, and integrabilty to other separation and detection systems, and any integration of the device into such a system is to be considered as part of this invention. In one embodiment, this invention provides an appaiatus comprising a device of this invention, which in some embodiments, comprises the analytical modules as described herein

[0099] Device geometiy can take a variety of shapes and sizes In nearly all devices, there are at least two electrodes. These electrodes can be raised off the base substrate or flat. Electrodes can be integrated into microchannels, microchannel walls, or can be external to the channel and the channel walls, [00100] In some embodiments, there can be a center electrode. This center electrode can be cylindrical, peai-shaped, elliptical, arrow-shaped, etc. There may be multiple center electrodes. In devices with multiple center electrodes, all electrodes can be the same shape, different shapes, different sizes, etc. Center electrodes can be electrically insulated form one another, electrically connected, or electrically connected in gioups. External circuitry can be used to control electrical connections. External circuitry can be used to fix the voltage/potential of any or all of the center electrodes. Center electrode potential can be controlled relative to the two outer electrodes in magnitude, frequency, and phase lag.

[00101 j In some embodiments, the total charge on the center electrodes can also be controlled.

Charge can be controlled relative to the outer electrodes in magnitude, frequency, and phase lag, as above.

[00102] In some embodiments, a raised center electrode and flat outer electrodes can be employed to maximize out-of-plane mixing (and the out-of-plane electric field components).

Various shapes of outer electrodes can also be employed to effectively tailor mixing and/or pumping.

[00103] In some embodiments, additional electrode geometries can include rounded portions, which can be fabricated for instance, by evaporating through a narrow slit, or by wet etching a vertical, electroplated electrode.

[00104] hi some embodiments, the outer electrodes can be arranged in a variety of geometries relative to the center electrode. The outer electrodes can be parallel to one anothei and transverse to a background fluid flow, or in other embodiments, they can be parallel to one another and parallel to background fluid flow. In some embodiments, they can have an angle between them, resulting in some electric field gradients, which may enhance fluid mixing on the front or¹ backside of the center electrode. In some embodiments, they can be substantially circular, nearly smrounding the center electrode. In some embodiments, the spacing of the outer electrodes can be controlled to tailor the driving and control voltage/charge requirements, i.e within a particular range of voltages or at a particular frequency.

[00105] The electrical connections between electrodes and external circuitiy (the distinction is made between internal comprising the fluid channel and electrodes, and the external comprising all electrical connections, etc.), can, in some embodiments, be as simple as planar wires connecting the center posts to the external circuits. The electrical connections can be electroplated, in some embodiments. The electrical connections can be buried beneath an insulating material, in some embodiments.

[00106] Driving and control electronics can be manufactured on-chip along with the electrodes, in some embodiments. The driving and control electronics can be a separate electronics module, in some embodiments, an external stand-alone unit or microfabricated electronics The miciofabricated electronics module, in some embodiments, can be wire-bonded to the chip containing the electrodes or can be flip-chip bonded.

[00107] In another embodiment, a center electrode is formed by attaching a pre-fabricated metal rod to a metal post deposited or patterned on the surface of the substrate The attachment can be via chemical bonds, welded, oi any other means.

[00108] Fluidic channels can be fabricated by a variety of means, including soft-lithogiaphic molding of polymers on rigid or semi-rigid molds. Channels can also be fabricated in glass via wet etching, plasma etching or similar means Channels can be formed in plastics via stamping, hot embossing, or other similar machining piocesses. The channels can then be bonded to the substrate containing the electrode stiuctures Alignment marks can be incorporated onto the substrate to facilitate assembly. In some instances, metal surfaces can be exposed on substrate and channels to enable metal-to-metal bonding. Glass-to-glass bonding can be done at elevated temperatures and with applied potential Plastic-to-glass can be facilitated with cleaning of glass surfaces prior to bonding, or fabrication of the fluidic portion of the device can be accomplished by any means known in the art,

[00109] Raised obstacles of an insulating or semiconducting nature can be fabricated on the substrate as well, in some embodiments, to provide obstacles to impede or change fluid flow The fluidic channel geometries can also be tailored to affect dispersion or mixing. [00110] In some embodiments, the conductor element will have a defined radius. In some embodiments, the conductor clement will have a radius of about 5-500 μm. In one embodiment, the conductor element will have a radius of about 5 μm, or in another embodiment, about 10 μm, or in another embodiment, about 15 μm, or in another embodiment, about 20 μm, or in another embodiment, about 25 μm, or in another embodiment, about 50 μm, or in another embodiment, about 75 μm, or in another embodiment, about 100 μm. or in another embodiment, about 125 μm, oi in anothei embodiment, about 150 μm, or in another embodiment, about 175 μm, or in another embodiment, about 200 μm, or in another embodiment, about 225 μm, or in anothei embodiment, about 250 μm, or in another embodiment, about 275 μm, or in another embodiment, about 300 μm, oi in another embodiment, about 350 μm, or in another embodiment, about 400 μm, or in another embodiment, about 450 μm, or in another embodiment, about 500 μm. [001 1 1] In some embodiments, this invention provides for analysis, detection, concentration, processing, assay, production of any material in a microfluidic device, whose principle of operation comprises electro-osmotically driven fluid flow, for example, the incorporation of a source providing an electric field in a miciochannel of the device, and provision of an electrokinetic means for generating fluid motion whereby interactions between the electric field and induced-charge produce electro-osmotic flows. Such flows may in turn, find application in fluid conductance, mixing of materials, or a combination thereof, and any application which makes use of these principles is to be considered as part of this invention, representing an embodiment thereof.

In some embodiments, this invention provides a miciofluidic device comprising one or moie inlet ports, at least one outlet port, and microfluidic channels in fluid communication with said ports, where the channels comprise one or more rnicropumps oi one or more micormixers, or a combination thereof. According to this aspect of the invention, the micropumps comprise a passageway for transmitting an electrolyte fluid, a source providing an electric fleld in the microchannel and an electrokinetic means for generating fluid motion, whereby interactions between the electric field and induced- charge produce electro-osmotic flows. In another embodiment, according to this aspect of the invention, AC driven electro-osmosis may be accomplished without use of a conductor element positioned perpendicularly to the electric field, yet another electrokinetic means for generating fluid motion is provided in the device, as will be understood by one skilled in the art, and such a permutation is to be considered as part of this invention, as well.

[00112] In another embodiment, this invention provides a method of cellular analysis, comprising the steps of: a, introducing a buffered suspension comprising cells to a first inlet port of a microfluidic device; b introducing a reagent for cellular analysis to said first inlet port or a second inlet port of said microfluidic device, said microfluidic device comprising: i. one or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or more micropumps, one oi more micromixers, or a combination thereof, wherein: o said micropumps comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microchannel; at least one conductor element in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and said at least one conductor element produce electro-osmotic flows so that said suspension and said reagent are driven across said microfluidic channels; and e said micromixers comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microfluidic channels; at least one conductor element in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and said conductor element produce electro-osmotic flows with varied trajectories, and said suspension and said reagent are driven across said microfluidic channels so that said suspension and said reagent are mixed in said microfluidic channels; and d. analyzing at least one parameter affected by contact between said suspension and said reagent.

[00113] In one embodiment, the reagent is an antibody, a nucleic acid, an enzyme, a substrate, a ligand, or a combination thereof.. In another embodiment, the reagent is coupled to a detectable marker, which in another embodiment is a fluorescent compound, In another embodiment, the device is coupled to a fluorimeter or fluorescent microscope, In another embodiment, the device is comprised of a transparent material,

[00114] In another embodiment, the method further comprises the step of introducing a cellular lysis agent in an inlet port of said device. According to this aspect of the invention, and in one embodiment, the reagent specifically interacts or detects an intracellular compound. [00115] In one embodiment, this invention provides a method of cellular analysis using a device or apparatus of this invention. In one embodiment, the method of cellular analysis comprises the steps of: a, introducing a buffered suspension comprising cells to a first inlet port of a microfluidic device; b, introducing a reagent for cellular analysis to a second inlet port of said microfluidic device, said miciofluidic device comprising: i. two or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or moie micropumps, one or more micromixers, or a combination thereof, wherein: β said rnicropumps comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microchannel; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or pioxirnal to said microchannel, whereby interactions between said electric field and said at least one conductor element produce electro-osmotic flows so that said suspension and said ieagent are driven across said microfluidic channels; and β said micromixers comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microfluidic channels; an array of conductor elements placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microfluidic channels, whereby interactions between said electric field and each conductor element produce electro-osmotic flows with varied trajectories, and said suspension and said reagent are driven across said microfluidic channels so that said suspension and said reagent are mixed in said miciofluidic channels; and c. analyzing at least one parameter affected by contact between said suspension and said reagent.

[001 16] One embodiment of carrying out such cellular analysis is exemplified herein, in Example 2.

[001 17] In one embodiment, the surface of the microchannel may be functionalized to reduce oi enhance adsorption of species of interest to the surface of the device. In another embodiment, the surface of the microchannel has been functionalized to enhance or reduce the operation efficiency of the device.

[00118] In one embodiment, the device is further modified to contain an active agent in the microchannel, or in another embodiment, the active agent is introduced via an inlet into the device, or in another embodiment, a combination of the two is enacted. For example, and in one embodiment, the microchannel is coated with an enzyme at a region wherein molecules introduced in the inlet will be conveyed past, according to the methods of this invention. According to this aspect, the enzyme, such as, a protease, may come into contact with cellular contents, oi a mixture of concentrated proteins, and digest them, which in another embodiment, allows for fuither assay of the digested species, for example, via introduction of a specific protease into an inlet which conveys the enzyme fuither downstream in the device, such that essentially digested material is then subjected to the activity of the specific protease. This is but one example, but it is apparent to one skilled in the art that any number¹ of other reagents may be introduced, such as an antibody, nucleic acid probe, additional enzyme, substrate, etc. [00119] In one embodiment, processed sample is conveyed to a separate analytical module. For example, in the protease digested material described hereinabove, the digestion products may, in another embodiment, be conveyed to a peptide analysis module, downstream of the device. The amino acid sequences of the digestion products may be deteimined and assembled to generate a sequence of the polypeptide. Prior to delivery to a peptide analysis module, the peptide may be conveyed to an interfacing module, which in turn, may perform one or more additional steps of separating, concentrating, and or focusing.

[00120] In another embodiment, the microchannel may be coated ■ with a label, which in one embodiment is tagged, in order to identify a particular protein or peptide, or other molecule containing the recognized epitope, which may be a means of sensitive detection of a molecule in a large mixture, present at low concentration.

[00121] For example, in some embodiments, reagents may be incorporated in the buffers used in the methods and devices of this invention, to enable chemiluminesceπce detection. In some embodiments the method of detecting the labeled material includes, but is not limited to, optical absorbance, refractive index, fluorescence, phosphorescence, chemiluminescence, electrochemiluminescence, electrochemical detection, voltametry or conductivity. In some embodiments, detection occurs using laser-induced fluorescence, as is known in the ail-, [00122] In some embodiments, the labels may include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, fiuorescamine, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbcne, Lucifer Yellow, Cascade Blue™, Texas Red, 1 ,1 -[1,3- piOpanediyIbis[(dirnetliylimino-3,l-propanediyl]]bis[4-[(3-methyl-2(3H)- benzoxazolylidene)melhyl]]-,tetraioide, which is sold under the name YOYO-I , Cy and Alexa dyes, and others described in the 9Ui Edition of the Molecular Probes Handbook by Richard P Haυgland, hereby expressly incorporated by reference, Labels may be added to 'label' the desiied molecule, prior to introduction into the devices of this invention, in some embodiments, and in some embodiments the label is supplied in a miciofluidic chamber In some embodiments, the labels are attached covalently as is known in the art, or in other embodiments, via non-covalent attachment.

[00123] In some embodiments, photodiodes, confocal microscopes. CCD cameras, or photomultiplier tubes maybe used to image the labels thus incorporated, and may, in some embodiments, comprise the apparatus of the invention, representing, in some embodiments, a "lab on a chip" mechanism.

[00124] In one embodiment, detection is accomplished using laser-induced fluorescence, as known in the art. In some embodiments, the apparatus may further comprise a light source, detector, and other optical components to direct light onto the microfluidic chamber/chip and thereby collect fluorescent radiation thus emitted. The light source may comprise a laser light source, such as, in some embodiments, a laser diode, or in other embodiments, a violet or a red laser diode. In other embodiments, VCSELs, VECSELs, or diode-pumped solid state lasers may be similarly used, In some embodiments, a Brewstei's angle laser induced fluorescence detector may used. In some embodiments, one or more beam steering mirrors may be used to direct the beam to a desired location for detection. [0001] ??In one embodiment, a solution or buffered medium comprising the molecules for assay are used in the methods and for the devices of this invention. In one embodiment, such solutions or buffeied media may compiise natural or synthetic compounds. In another embodiment, the solutions oi buffered media may comprise supεrnatants oi culture media, which in one embodiment, are harvested from cells, such as bacterial cultures, or in another embodiment, cultures of engineered cells, wherein in one embodiment, the cells express mutated piotcins, or overexpress proteins, or other molecules of interest which may be thus applied. In another embodiment, the solutions or buffered media may comprise lysates or homogenates of cells or tissue, which in one embodiment, may be otherwise manipulated for example, wherein the lysates are subject to filtration, lipase or collagenase, etc, digestion, as will be understood by one skilled in the art, In one embodiment, such piocessing may be accomplished via introduction of the appropriate reagent into the device, via, coating of a specific channel, in one embodiment, oi introduction via an inlet, in another embodiment.

[00125] It is to be understood that any complex mixture, comprising two or more molecules, whose assay is desired, may be used foi the methods and in the devices of this invention, and represent an embodiment thereof.

[00126] In one embodiment, a device for use in such cellular analysis may comprise rnicromixers, rnicropumps, or a combination thereof There are many conceivable means of placement of the micromixers and/oi micropumps in a given device, their respective placement, will be a function of the assay to be conducted, the reagents introduced, the nature of the sample being assayed, etc., as will be appieciated by one skilled in the art

[00127] In another embodiment, this invention piovides a method of analyte detection or assay. comprising the steps of: a. introducing a fluid comprising an analyte to a first inlet port of a microfluidic device, said micro fluidic device comprising: i. one or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or moie micropumps, one or more micromixers, or a combination thereof, wherein: a, said micropumps comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said raicrochannel; al least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and said at least one conductor element produce electro- osmotic flows so that said suspension and said reagent are driven across said microfluidic channels; and b.. said micromixers comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microfluidic channels; an array of conductor elements placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microfluidic channels, whereby interactions between said electric field and each conductor element produce electro-osmotic flows with varied trajectories, and said suspension and said reagent are driven across said microfluidic channels so that said suspension and said reagent are mixed in said microfluidic channels; c. said microchannels being coated with a reagent for the detection, assay, or combination thereof of said analyte; and β detecting, analyzing, or a combination thereof, of said analyte.

[001.28] Analyte refers in some embodiments, to any material whose detection or other analysis is desired, or in some embodiments, analyte refers to a molecule, upon interaction with another molecule, provides a means for detection or assay of the second molecule. For example, and in some embodiments, an analyte is a probe, which upon binding to a target molecule provides a means for the identification, assay, processing, or other manipulation, or the target molecule, In another embodiment, the analyte is the target molecule, which upon interaction with a probe, ligand, receptor, antibody, or other desired molecule, which in one embodiment, is coated on, or found within a microchannel of the device, may be detected, assayed, processed, or otherwise manipulated, One skilled in the art will readily appreciate the multitude of permutations and applications, which make use of the devices, methods, and/or principles of this invention, whereby electrokinetic flow causes a sample to circulate over a target, or vice versa, or, in other embodiments, whereby eleclrokinetic flow causes a sample to be mixed with a target, or vice versa. Some of these applications may find use in the development of biosensor and/or bioassay applications In some embodiments of this invention, such applications include the ability to move fluids in a contained cavity for assays where this principle is useful, for example, in protein crystallization methods, and others, as will be understood by one skilled in the art. [00129] In another embodiment, this invention provides a method of high-throughput, multi- step product formation, the method comprising the steps of: a. introducing a first liquid comprising a precursor to a first inlet port of a microfluidic device; b. introducing a reagent, catalyst, reactaπt, cofactor, or combination thereof to a second inlet port of said microfluidic device, said miciofluidic device comprising: i. two or more inlet ports, at least one outlet port and miciofluidic channels in fluid communication with said ports, said channels comprising one or more micropumps, one or more micromixers, or a combination thereof, wherein:

© said micropumps comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microchannel; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and said at least one conductor element produce electro-osmotic flows so that said suspension and said reagent are driven acioss said micro fluidic channels; and o said micromixers comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microfluidic channels; an array of conductor elements placed in an orientation that is perpendicular to the axis of said electric field;, at a location within or proximal to said microfluidic channels, whereby interactions between said electric field and each conductor element produce electro-osmotic flows with varied trajectories, and said suspension and said reagent are driven across said microfluidic channels so that said suspension and said reagent aie mixed in said microfluidic channels; and c. collecting the pioduct formed from an outlet port of said device [00130]

[00131] In another embodiment, this invention provides a method of high-throughput, multi- step product formation, the method comprising the steps of: a. introducing a first liquid comprising a precuisor to a first inlet port of a miciofluidic device; b. introducing a second liquid comprising a reagent, catalyst, reactant. cofactor. or combination thereof to said first inlet port or a second inlet port of said microfluidic device, said microfluidic device comprising: i one or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or more miciopumps, one or more micromixers, or a combination thereof, wherein: β said micropumps comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microchannel; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and said at least one conductor element produce electro-osmotic flows so that said first liquid and said second liquid are driven across said microfluidic channels; and

© said micromixers comprise a passageway for tiansmitting said suspension and said leagent; a source providing an electric field in said microfluidic channels; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and each conductor element produce electro-osmotic flows with varied trajectories, and said first liquid and said second liquid are driven across said microfluidic channels so that said fust liquid and said second liquid are mixed in said microfluidic channels; and c, collecting said mixed liquid formed from an outlet port of said device. [00132] In one embodiment, the method further comprises the step of carrying out iterative introductions of said second liquid, as in (b), to additional inlet poits In one embodiment, the reagent is an antibody, a nucleic acid, an enzyme, a substrate, a ligand, a reactant or a combination thereof.

[00133] In another embodiment, this invention provides a method of drug processing and delivery, the method comprising the steps of: a, introducing a first liquid comprising a drug to a first inlet port of a raicrofluidic device; b. introducing a second liquid comprising a buffer, a catalyst, or combination thereof to said first inlet port or to a second inlet port of said microfluidic device, said microfluidic device comprising: i. two or more inlet ports, at least one outlet port and miciofluidic channels in fluid communication with said ports, said channels comprising one oi more micropumps, one or more micromixeis, or a combination thereof, wherein: o said micropumps comprise a passageway for transmitting said first and said second liquids; a source providing an electric field in said microcharmel; at least one conductor element that is placed in an orientation that is perpendiculai to the axis of said electric field, at a location within or proximal to said microchannel, whereby inteiactions between said electric field and said at least one conductor element produce electro-osmotic flows so that said first liquid and said second liquid are driven across said microfluidic channels; and

® said micromixers comprise a passageway for transmitting said first liquid and said second liquid; a source providing an electric field in said microfluidic channels; one or more conductor elements placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microfluidic channels, whereby interactions between said electric field and each conductor¹ element produce electro-osmotic flows with varied trajectories, and said first liquid and said second liquid are driven across said microfluidic channels so that said first liquid and said second liquid are mixed in said microfluidic channels; and elivering the product of (b) to a subject, through an outlet port of said device. [00134] In one embodiment, the method further comprises carrying out iterative introductions of said second liquid to said inlet ports. In another embodiment, the second liquid serves to dilute the drug to a desired concentration.

[00135] In one embodiment, the method further comprises carrying out iterative introductions of reagent, catalyst, reactant, cofactor, or combination thereof as in (b) to additional inlet ports [00136] Metabolic processes and other chemical processes can involve multiple steps of reactions of precursors with an enzyme, or catalyst, or mimetic, etc., in some embodiments, with or without the involvement of cofaclors, in oilier embodiments, to obtain specific products, which in turn are reacted, to form additional products, etc, until a final desired product is obtained. In one embodiment, the devices aπd/oi methods of this invention are used for such a puφose. In one embodiment, such methodology enables use of smaller quantities of reagents, or precursors, which may be limiting, in othei embodiments, such methodology enables isolation of highly reactive intermediates, which in turn may promote greater pioduct formation. It will be apparent to one skilled in the art that a means for stepwise, isolated or contiolled synthesis provides many advantages, and is amenable to any number of permutations, [00137] It is to be understood that any of the embodiments described herein, with regards to samples, reagents and device embodiments are applicable with regard to any method as described herein, representing embodiments thereof.

[00138] In another embodiment, the induced-charge electroosmotic mixers of this invention are incorporated in a device, which in turn circulates solutions containing probe molecules over target surfaces. In one embodiment, the probe may be any molecule, which specifically interacts with a target molecule, such as, for example, a nucleic acid, an antibody, a ligand, a τeccptor, etc. In another embodiment, the probe will have a moiety which can be chemically cross-linked with the desired target molecule, with reasonable specificity, as will be appreciated by one skilled in the art. According to this aspect of the invention and in one embodiment, a microchannel of the device may be coated with a mixture, lysate, sample, etc., comprising a target molecule of interest

[00139] In one embodiment, such a device provides an advantage in terms of the time needed for assay, the higher sensitivity of detection, lowei concentration of sample/reagents needed, since the sample may be recirculated over the target surface, or combination thereof. [00140] In another embodiment, this invention provides a method of drug processing and delivery, the method comprising the steps of: a. introducing a first liquid comprising a drug to a first inlet port of a microfluidic device; b. introducing a second liquid comprising a buffer, a catalyst, or combination thereof to said first inlet port or to a second inlet port of said microfluidic device, said microfluidic device comprising: i. two or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or more micropumps, one or more micromixers, or a combination thereof, wherein:

© said micropumps comprise a passageway for transmitting said first and said second liquids; a source providing an electric field in said microchannel; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby intei actions between said electric field and said at least one conductor element produce electro-osmotic flows so that said first liquid and said second liquid aie driven across said microfluidic channels; and

«> said micromixers comprise a passageway for transmitting said first liquid and said second liquid; a source pioviding an electric field in said microfluidic channels; one or more conductor elements placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microfluidic channels, whereby interactions between said electric field and each conductor element produce electro-osmotic flows with varied trajectories, and said first liquid and said second liquid are driven across said microfluidic channels so that said first liquid and said second liquid are mixed in said microfluidic channels; and elivering the product of (b) to a subject, through an outlet port of said device, [00141] In one embodiment, the method further comprises carrying out iterative introductions of said second liquid to said inlet poits, In anothei embodiment, the second liquid serves to dilute the diug to a desiied concentration In one embodiment, the device comprises valves, positioned to regulate fluid flow through the device, such as, for example, for iegulating fluid flow through the outlet of the device, which in turn prevents depletion from the device, in one embodiment. In another embodiment, the positioning of valves provides an independent means of regulating fluid flow, apart fiom a relay from signals from the subject, which stimulate fluid flow through the device.

[00142] In another embodiment, this invention provides a device for use in drug delivery, wherein the device conveys fluid from a reservoir to an outlet port- In one embodiment, drug deliveiy according to this aspect of the invention, enables mixing of drug concentrations in the device, or altering the flow of the drug, oi combination thereof, or in another embodiment, piovides a means of continuous deliveiy. In one embodiment, such a device may be implanted in a subject, and piovide drug delivery in situ. In one embodiment, such a device may be prepaied so as to be suitable for transdermal drug deliveiy, as will be appreciated by one skilled in the art. [00143] Although the present invention has been shown and described with respect to several preferred embodiments thereof, various changes, omissions and additions to the form and detail thereof, may be made therein, without depaiting from the spirit and scope of the invention.

EXAMPLES

EXAMPLE l:

Device Comprising Multiple Induced-Charge Electro-Osnwtically Driven

Microβuidic Mixers

[00344] It is possible to create many permutations of a device comprising multiple induced- charge electro-osmotically driven microfluidic mixers or pumps, or a combination thereof, as will be appreciated by one skilled in the art, and as described hereinabove. One embodiment of such a device is a microfluidic two-stage mixer, as depicted in Figure L The device will comprise inlet ports foi the introduction of a sample, a reagent, a detecting moiety, a catalyst, or a combination thereof, or any agent whose introduction is desired. Such inlet ports may be constructed as depicted in the figure (1-10, 1-20, and 1-30). The inlet ports, in turn, may lead to channels (depicted at 1-40, 1-50, and 1-80 respectively, in the figure), which in this example serve to convey the introduced matter into another region of the device. Channels 1-40 and 1-50 may merge, for example as a Y-junction (1-60) and lead to a channel (1-70), which contains a nonlinear elecliokinetic mixer], as described herein In a two-step mixing process, for example, matter introduced into 2 channels are first mixed, and the mixture (from Channel 1-70) is then contacted with matter introduced into a third channel (1-80) at, for example, the Y-junction (1- 90), which conveys both materials to channel 1-100, which contains a nonlinear electrokinetic mixer. Channel 1-100 connects to channel 1-110, which serves to convey the mixed product to outlet 1-120.

EXAMPLE 2:

Analysis of Cellular Components

[00145] It will be appreciated by one skilled in the art that the device described in Example 1 may be useful in a wide array of applications In one application, for example analysis of expiessed proteins or nucleic acids in a single cell, inlet 1-10 is used to introduce a single cell in a buffer solution into the device. It will be appreciated by one skilled in the ait that the inlet will be so constructed as to facilitate entry of singular cells, which are then conveyed to channel 1-40 Inlet 1-20 conveys a lysing agent The lysing agent mixes with the cell and buffer solution in 1- 70, causing cell lysis, and ielease of cellular contents. Inlet 1-80 conveys a fluorescent-tagged probe oi antibody which mixes with the cellular contents in 1-100, resulting in specific labeling of cellular components. It is also envisioned that wash solutions are introduced in the inlet following a period of time of exposure to the labeled agent, oi. another inlet which conveys the solution may be constructed, whereby non-specific labeling may be diminished. Fluorescent detection may be carried out by imaging the material conveyed to channel 1-110. Again, it will be apparent to one skilled in the art, that the construction of the device will accommodate imaging of the appropriate channel, thus the material used for construction of at least this region of the device may be transparent.

EXAMPLE 3:

Construction of a Device Comprising Induced-Charge Electro-Osmotically Driven Microfluidic Mixers

[00146] Many methods for fabricating devices as described heiein will be apparent to one skilled in the ait One embodiment for such a means of construction is depicted in Figure 2. The figuie specifically describes fabrication of the device outlined in Example I₅ Figure 1, at the region of the device labeled as section A-A.

[00147] The stalling substrate 2-10 is a 0,5mm thick four-inch diameter double-side polished borofloat glass wafer. Substrate 2-10 is cleaned in 3:1 sulfuric acid:hydrogen peroxide to remove any organic material from the surface, The substrate 2-10 is then oxygen plasma ashed for 5 minutes to roughen the surface.

[001481 Following prepar ation of the substrate, a gold layer 2-20 is e-beam evaporated onto the substrate. Polymer photoresist is deposited onto the gold layer 2-20 and patterned using a chromium photomask The patterned photoresist is then used as a masking material for a wet etch of gold layer 2-20A. Masking material 2-30 such as SU-8 cpoxy-based photoresist is deposited onto substrate 2-10 and patterned. Gold structure 2-20B is electroplated into mold 2- .30 and subsequently removed leaving an isolated metal post 2-20B Substiate 2-40 is a sacrificial substrate for casting the fluidic channel. Photoresist material 2-50 is deposited and patterned on substrate 2-40 PDMS or similar polymer material is then cast onto pattern 2-50 forming channel 2-60. Channel 2-60 is peeled off of substrate 2-40 and used to form the channels on substrate 2-10

[00149] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. ϊt is, therefore, to be understood that the appended claims are intended to cover all sucli modifications and changes as fall within the tiue spirit of the invention

Claims

CLAIMS[00150] What is claimed is:

1. A microfluidic device comprising one or more inlel ports, one or more outlet ports and microfluidic channels in fluid communication with said ports, said channels comprising one or more micropumps, one or more micromixers, oi a combination thereof, wherein: β said micropumps comprise a passageway for transmitting an electrolyte fluid; a source providing an electric field in said microchannel; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said field and said at least one conductor element produce electro-osmotic flows so that said electrolyte fluid is driven across said microfluidic channels; and

» said micromixers comprise a passageway foi transmitting an electrolyte fluid; a somce providing an electric field in said microfluidic channel; at least one conductor element that is placed in an orientation that is perpendiculai to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said field and said conductor element produce electto-osmotic flows with varied tiajectories, and said electrolyte fluid is driven acioss said microfluidic channels so that said electiolyte fluid is mixed in said microfluidic channels.

2. The microfluidic device of claim 1, wherein said electric field is comprised of a DC electric field.

.

3. The microfluidic device of claim 1, wherein said electric field is comprised of an AC oi pulsed AC electric field.

4. The microfluidic device of claim 1, wherein said electric field is comprised of an AC or pulsed AC electric field with a DC offset.

5. The microfluidic device of claim 1, wherein said field source is comprised of electrodes of different polarities.

6. The microfluidic device of claim 1, wherein said conductor element is comprised of a symmetric cylinder of a defined radius.

7. The microfluidic device of claim 5, wherein said radius ranges from about 5 to about 250 μm.

8. The microfluidic device of claim 1 , wherein said conductor element is comprised of an asymmetric conductor element, with either non-uniform surface composition or non-circular¹ cross section,

9. The microfluidic device of claim 8, wherein said conductor element is comprised of a conducting strip

10. The microfluidic device of claim 9, wheiein at least one wall of said microfluidic channels comprises said conducting strip.

11. The microfluidic device of claim 8, wherein the shape of said conductor element approximates an arrowhead, teardrop, or ellipse,

12. The microfluidic device of claim 1 , wherein said microfluidic channels form a cross- junction, an elbow-junction, a T-junction, a Y-junction, or a combination thereof

13. The microfluidic device of claim L wherein said conductor clement is comprised of a symmetric conductor element.

14. The microfluidic device of claim 1, wherein said microfluidic channels ate comprised of a transparent material

15. The microfluidic device of claim 1, wherein said microfluidic channels are comprised of a metal.

16. The microfluidic device of claim 15, wherein said metal is a metal bilayer.

17. The microfluidic device of claim 16, wherein an exposed layer of said bilayer is functionalized to minimize adherence of material conveyed through said device.

18. The microfluidic device of claim 1, wherein said device comprises an array of conductor elements.

19. An apparatus comprising the microfluidic device of claim 1 ,

20. A method of cellular analysis comprising the steps of: a introducing a buffered suspension comprising cells Io a first inlet port of a microfluidic device; b introducing a reagent for cellular analysis to said first inlet or to a second inlet port of said microfluidic device, said microfluidic device comprising:

L one or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels compiising one or more micropumps, one or more micromixers, oi a combination thereof, wherein: a said micropumps comprise a passageway for transmitting said suspension and said ieagent; a source providing an electric field in said microchannel; at least one conductor element that js placed in an orientation that is perpendiculai to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and said at least one conductoi element produce electro- osmotic flows so that said suspension and said reagent are driven across said microfluidic channels; and b. said micromixers comprise a passageway for transmitting said suspension and said reagent; a source providing an electic field in said microfluidic channels: an array of conductor elements placed in an orientation that is perpendicular to the axis of said electric field, at a location within oi proximal to said microfluidic channels, whereby interactions between said electric field and each conductor element produce electro-osmotic flows with varied trajectories, and said suspension and said reagent are driven across said microfluidic channels so thai said suspension and said reagent are mixed in said microfluidic channels; and o analyzing at least one parametei affected by contact between said suspension and said reagent.

21. The method of claim 20, wherein said reagent is an antibody, a nucleic acid, an enzyme, a substrate, a ligand, or a combination thereof

22. The method of claim 21 , wherein said reagent is coupled to a detectable marker.

23. The method of claim 22, wherein said marker is a fluorescent compound.

24. The method of claim 23, wherein said device is coupled to a fluorimetei or fluoτescent microscope

25. The method of claim 24. wherein said device is comprised of a tianspaient material.

26. The method of claim 20, further comprising the step of introducing a cellular lysis agent in an inlet port of said device.

27. The method of claim 26, wherein said ieagent specifically interacts oi detects an intracellular compound

28. A method of high-throughput, multi-step product foimation, the method comprising the steps of: a, introducing a first liquid comprising a precursor to a first inlet port of a microfluidic device; b introducing a second liquid comprising a reagent, catalyst, reactant, cofactor, or combination thereof to a second inlet port of said miciofluidic device, said microfluidic device comprising: i. two or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or moie micropumps. one or more micromixers, or a combination thereof, wherein; o said micropumps comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microchannel; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and said at least one conductor element produce electro-osmotic flows so that said fiist liquid and said second liquid are driven across said microfiuidic channels; and β said micromixers comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microfiuidic channels; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and each conductor element produce electio-osmotic flows with varied trajectories, and said first liquid and said second liquid are driven across said microfiuidic channels so that said first liquid and said second liquid are mixed in said miciofluidic channels; and c. collecting said mixed liquid from an outlet port of said devjce.

29. The method of claim 28, further comprising carrying out iterative introductions of said second liquid, as in (b), to additional inlet poits.

30. The method of claim 28, wherein said reagent is an antibody, a nucleic acid, an enzyme, a substrate, a iigaπd, a reactant or a combination thereof

31. A method of drug processing and delivery, the method comprising the steps of: a, introducing a first liquid comprising a drug to a first inlet port of a microfiuidic device; b. introducing a second liquid comprising a buffer, a catalyst, or combination theieof to said first inlet port or to a second inlet port of said microfluidic device, said microfiuidic device comprising: i. two or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one oi more micropumps, one or more miciomixers, or a combination thereof, wherein:

© said micropumps comprise a passageway foi transmitting said first and said second liquids; a source providing an electric field in said microchannel; at least one conductor element that is placed in an orientation that is perpendicular to the axis of said electric field, at a location within or proximal to said microchannel, whereby interactions between said electric field and said at least one conductor element produce electro-osmotic flows so that said first liquid and said second liquid are driven across said microfluidic channels; and

* said miciomixers comprise a passageway for transmitting said first liquid and said second liquid; a source providing an electric field in said microfluidic channels; one or more conductoi elements placed in an orientation that is perpendicυlai to the axis of said electric field, ai a location within oi proximal to said microfluidic channels, whereby interactions between said electric field and each conductoi element pioduce electro-osmotic flows with varied trajectories, and said first liquid and said second liquid are driven acioss said microfluidic channels so that said first liquid and said second liquid aie mixed in said microfluidic channels; and c. delivering the product of (b) to a subject, through an outlet port of said device

32. The method of claim 31 , further comprising carrying out iterative introductions of said second liquid to said inlet ports. .

33. The method of claim 31 , wherein introduction of said second liquid serves to dilute said drug to a desiicd concentration

34. A method of analyte detection or assay, comprising the steps of: introducing a fluid comprising an analyte Io a first inlet port of a micro fluidic device, said miciofluidic device comprising: i one or more inlet ports, at least one outlet port and microfluidic channels in fluid communication with said ports, said channels comprising one or more micropumps, one or more micromixers, oi a combination thereof, wherein: a. said micropumps comprise a passageway for transmitting said suspension and said ieagent; a source providing an electric field in said microchannel; at least one conductor element that is placed in an orientation that is perpendicular' to the axis of said electric field, at a location within or proximal to said microchannel, λvhereby interactions between said electric field and said at least one conductor element pioduce electro- osmotic flows so that said suspension and said reagent are driven across said microfluidic channels; and b. said micromixers comprise a passageway for transmitting said suspension and said reagent; a source providing an electric field in said microfluidic channels; an array of conductor elements placed in an orientation that is perpendiculai to the axis of said electric field, at a location within or proximal to said microfluidic channels, whereby interactions between said electric field and each conductor element produce electro-osmotic flows with varied trajectories, and said suspension and said reagent are driven across said microfluidic channels so that said suspension and said reagent are mixed in said microfluidic channels; c said microchannels being coated with a reagent for the detection, assay, oi combination thereof of said analyte; and o detecting, analyzing, or a combination thereof, of said analyte