BACKGROUND OF THE INVENTION [0002] Microfluidic devices are becoming more important in a variety of fields, from biochemical analysis to medical diagnostics and to fields as diverse as environmental monitoring to chemical synthesis. There has been a growing interest in the manufacture and use of microfluidic systems for the acquisition of chemical and biological information. In particular, microfluidic systems allow complicated biochemical reactions to be carried out using very small volumes of liquid. These miniaturized systems increase the response time of the reactions, minimize sample volume, and lower reagent cost.

[0003] Traditionally, these microfluidic systems have been constructed in a planar fashion using silicon fabrication techniques. Representative systems are described, for example, in some early work by Manz et al. (Trends in Anal. Chem.

(1990) 10 (5): 144-149; Advances in Chromatography (1993) 33: 1-66). These publications describe microfluidic devices constructed using photolithography to define channels on silicon or glass substrates and etching techniques to remove material from the substrate to form the channels. A cover plate is bonded to the top of this device to provide closure.

[0004] More recently, a number of methods have been developed that allow microfluidic devices to be constructed from plastic, silicone or other polymeric materials. In one such method, a negative mold is first constructed, and plastic or silicone is then poured into or over the mold. The mold can be constructed using a silicon wafer (see, e. g., Duffy et al., Analytical Chemistry (1998) 70: 4974-4984; McCormick et al., Analytical Chemistry (1997) 69: 2626-2630), or by building a traditional injection molding cavity for plastic devices. Some molding facilities

have developed techniques to construct extremely small molds. Components constructed using a LIGA technique have been developed (see, e. g., Schomburg et al., Journal of Micromechanical Microengineering (1994) 4: 186-191). Other approaches combine LIGA and a hot-embossing technique. Imprinting methods in polymethylmethacrylate (PMMA) have also been demonstrated (see, Martynova et al., Analytical Chemistry (1997) 69: 4783-4789). However, these techniques do not lend themselves to rapid prototyping and manufacturing flexibility.

Additionally, these techniques are limited to planar structures. Moreover, the tool- up costs for both of these techniques are quite high and can be cost-prohibitive.

[0005] Generally, it is difficult to construct flow control elements such as pumps or valves in microfluidic devices using traditional construction techniques.

Rigid silicon fabrication, for example, does not lend itself to the construction of flexible or otherwise moveable parts. Several different manufacturing techniques may be required to fabricate microfluidic devices having integrated valves or pumps. In light of these limitations, there exists a need for improved microfluidic flow control devices. It would be further desirable if microfluidic devices having integrated flow control elements could be fabricated quickly and inexpensively. It would be further desirable if such device could be fabricated from a variety of different materials.

SUMMARY OF THE INVENTION [0006] In a first separate aspect of the invention, a microfluidic device includes a first microfluidic channel defined through a first stencil layer, a second microfluidic channel defined through a second stencil layer, a flap layer positioned between the stencil layers and having a moveable flap, and a sealing surface positioned adjacent to the flap layer. The first flap is capable of intermittently engaging the sealing surface to affect fluid communication between the first microfluidic channel and the second microfluidic channel.

[0007] In another aspect of the invention, multiple flaps may be provided in a single microfluidic device. Fluid displacement means in fluid communication with a first flap and a second flap may be provided.

[0008] In another aspect, a filter material may be disposed between the first microfluidic channel and the second microfluidic channel.

[0009] In another aspect, one or more layers of the device or surfaces thereof may be formed with self-adhesive tapes or coated with adhesives.

[0010] In another aspect, any of the foregoing aspects may be combined for additional advantage.

[0011] The foregoing and other aspects and objects of the invention will be apparent to one skilled in the art upon review of the appended description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS [0012] FIG. 1A is an exploded perspective view of a microfluidic device having a microfluidic valve disposed therein. FIG. 1B is a top assembled view of the device of FIG. 1A. FIG. 1C is a side cross-sectional view of a portion of the device of FIGS. 1A-1B in a first operating state, with a large internal arrow showing the direction of fluid flow. FIG. 1 D is a side cross-sectional view of a portion of the same device in a second operating state, with large arrows showing the direction of fluid flow reversed within the device.

[0013] FIG. 2A is an exploded perspective view of a microfluidic device capable of being used to provide fluid pumping utility. FIG. 2B is a top view of the assembled device of FIG. 2A. FIGS. 2C and 2D are cross-sectional views of a portion of the same device along section lines"A"-"A"in FIG. 2B in two different operating states. FIG. 2C shows the device with negative pressure applied to an actuation channel 111, while FIG. 2D shows the device with a positive pressure applied to the actuation channel 111.

[0014] FIGS. 3A and 3B are cross-sectional views of a portion of a microfluidic device having a microfluidic diversion valve therein where the flap portion is usually in the down position. In FIG. 3A, the device is show in operation with fluid flowing according to the single arrows through channels 189 and 192. In FIG. 3B, fluid is flowing in the reverse direction through channels 191 and 189 as indicated by the single arrows. In both cases, fluid flows through filter region 190.

[0015] FIGS. 3C and 3D show cross-sectional views of a microfluidic device having a microfluidic diversion valve therein where the microflap portion is usually in the up position. In FIG. 3C, the device is show in operation with fluid flowing according to the single arrows through channels 192 and 189. In FIG. 3D, fluid is

flowing in the reverse direction through channels 191 and 189 as indicated by the single arrows. In both cases, fluid flows through filter region 190.

[0016] FIG. 4A is a top view of a microfluidic device having a flap valve contained therein. FIGS. 4B-4C are a side cross-sectional views of a portion of the device of FIG. 4A in two different operating states. In FIG. 4B, the flap is deformed toward and into channel 220, while in FIG. 4C the flap is closed against a sealing surface of layer 224.

[0017] FIGS. 5A-5C are a side cross-sectional views of a portion of a microfluidic device having a flap valve therein in three different operating states.

In FIG. 5A, the flap is not subject to any external forces. In FIG. 5B, the flap is subject to an external force that deforms the flap valve upward into channel 87. In FIG. 5C, the flap is subject to an external force that deforms the flap valve downward into channel 86.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION Definitions [0018] The term"channel"as used herein is to be interpreted in a broad sense. Thus, the term"channel"is not intended to be restricted to elongated configurations where the transverse or longitudinal dimension greatly exceeds the diameter or cross-sectional dimension. Rather, the term is meant to include a conduit of any desired shape or configuration through which liquids may be directed. A channel may be filled with one or more materials, or may contain a moveable element or structures.

[0019] The term"microfluidic"as used herein refers to structures or devices through which one or more fluids are capable of being passed or directed and having at least one dimension less than about 500 microns.

[0020] The term"microfluidic flap"as used herein is to be understood to refer to a portion of a layer or sheet bounding a microfluidic channel wherein the portion is not connected at all points to other portions of the structure forming the channel. A microfluidic flap is thus only partially restrained from movement, and may move within said channel when certain physical characteristics of the channel change, such as pressure, temperature, flow rate of fluid, type of fluid, etc.

[0021] The term"stencil"as used herein refers to a material layer or sheet that is preferably substantially planar through which one or more variously shaped and oriented portions have been cut or otherwise removed through the entire thickness of the layer, and that permits substantial fluid movement within the layer (e. g., in the form of channels or chambers, as opposed to simple through-holes for transmitting fluid through one layer to another layer). The outlines of the cut or otherwise removed portions form the lateral boundaries of microstructures that are formed when a stencil is sandwiched between other layers such as substrates or other stencils.

Microfluidic device fabrication [0022] Microfluidic flow control devices of various designs according to the present invention may be built with different fabrication techniques. In an especially preferred embodiment, microfluidic devices are constructed using stencil layers or sheets to define channels and/or other microstructures. For example, a computer-controlled plotter modified to accept a cutting blade may be used to cut various patterns through a material layer. Such a blade may be used either to cut sections to be detached and removed from the stencil layer or to fashion slits that separate certain regions of a layer without removing any material.

Alternatively, a computer-controlled laser cutter may be used to cut portions through a material layer. While laser cutting may be used to yield precisely- dimensioned microstructures, the use of a laser to cut a stencil layer inherently involves the removal of some material. Further examples of methods that may be employed to form stencil layers include conventional stamping or die-cutting technologies. The above-mentioned methods for cutting through a stencil layer or sheet permits robust devices to be fabricated quickly and inexpensively compared to conventional surface micromachining or material deposition techniques that are conventionally employed to produce microfluidic devices.

[0023] After a portion of a stencil layer is cut or removed, the outlines of the cut or otherwise removed portions form the lateral boundaries of microstructures that are completed upon sandwiching a stencil between substrates and/or other stencils. The thickness or height of the microstructures such as channels or chambers can be varied by altering the thickness of the stencil layer, or by using

multiple substantially identical stencil layers stacked on top of one another. When assembled in a microfluidic device, the top and bottom surfaces of stencil layers are intended to mate with one or more adjacent layers (such as stencil layers or substrate layers) to form a substantially enclosed device, typically having at least one fluidic inlet port and often having at least one fluidic outlet port.

[0024] Various means may be used to seal or bond layers of a device together. For example, adhesives may be used. In a preferred embodiment, one or more layers of a device may be fabricated from single-or double-sided adhesive tape, although other methods of adhering stencil layers may be used. A portion of the tape (of the desired shape and dimensions) can be cut and removed to form channels, chambers, and/or apertures. A tape stencil can then be placed on a supporting substrate with an appropriate cover layer, between layers of tape, or between layers of other materials. In one embodiment, stencil layers can be stacked on each other. In this embodiment, the thickness or height of the channels within a particular stencil layer can be varied by varying the thickness of the stencil layer (e. g., the tape carrier and the adhesive material thereon) or by using multiple substantially identical stencil layers stacked on top of one another.

Various types of tape may be used with such an embodiment. Suitable tape carrier materials include but are not limited to polyesters, polycarbonates, polytetrafluoroethlyenes, polypropylenes, and polyimides. Such tapes may have various methods of curing, including curing by pressure, temperature, or chemical or optical interaction. The thickness of these carrier materials and adhesives may be varied.

[0025] Notably, stencil-based fabrication methods enable very rapid fabrication of devices, both for prototyping and for high-volume production. Rapid prototyping is invaluable for trying and optimizing new device designs, since designs may be quickly implemented, tested, and (if necessary) modified and further tested to achieve a desired result. The ability to prototype devices quickly with stencil fabrication methods also permits many different variants of a particular design to be tested and evaluated concurrently.

[0026] Still further embodiments may be fabricated from various materials using well-known techniques such as embossing, stamping, molding, and soft lithography.

[0027] In addition to the bonding methods discussed above, other techniques may be used to attach one or more of the various layers of microfluidic devices, as would be recognized by one of ordinary skill in attaching materials.

For example, attachment techniques including thermal, chemical, or light-activated bonding steps; mechanical attachment (such as using clamps or screws to apply pressure to the layers) ; and/or other equivalent coupling methods may be used.

Microfluidic flow control devices [0028] In various embodiments of the present invention, microfluidic devices contain valves for controlling fluid flow. Within these devices, certain sections of microfluidic channels are flaps that are partially restrained, that is, not connected at all points. These flaps can be used to control the flow of fluid.

[0029] Preferably, a flap has at least one dimension, typically width, that is smaller than that of the channel into which it is deflected. A flap typically may have a closed position sealed against a sealing surface formed by the second layer and can open into the first microfluidic channel.

[0030] Microfluidic devices constructed according to one or more of the methods described above may be provided with one or more fluid control valve regions disposed therein. In one embodiment, such a valve is a one-way valve or a check valve. A microfluidic valve may include a first channel formed within a first layer of a microfluidic device and a second channel formed within a second layer of the microfluidic device substantially coplanar with the first channel. The first channel is preferably smaller than the second channel in at least one dimension within the plane of the channels such that a seating surface is formed.

A third layer disposed between the first and second layers has a flap that is movable within the device but remains attached to the third layer. In a preferred embodiment, the flap and the third layer are formed from the same material, with material removed from the third layer to form the flap.

[0031] The flap is movable, such that in a closed position it seals with the seating surface. As used herein, the term"seals"refers to contact of a flap against a seating surface. Sealing of a flap includes both the formation of a fluid- tight junction and junction that allow restricted fluid flow through the device.

Mobility of the flap can be achieved by any suitable modification of the flap

material or dimensions, including, for example, altering the material of the flap, the dimensions of the flap (e. g., thickness), the degree of connection of the flap to the third layer and combinations thereof. For example, a flap can be formed from a substantially rigid material, with a hinge region to allow movement. A hinge region can be formed in a rigid material by reducing its thickness at the desired hinge region. The flap also can be formed from a pliable material. A material is suitably pliable if, at the desired operating pressures of the device, the material will bend or deform. The degree of pliability will depend on the nature of the material used and on the thickness of the material used. A flap can have any shape such that the flap can deform within the device towards the second channel. In certain embodiments, the flap will seal against the first channel within normal operating pressures of the device. In other embodiments, the flap will seal against the second channel within normal operating pressure of the device. In one embodiment, a flap has one side separated from the membrane from which it is formed. In another embodiment, the flap is formed by cutting three sides of a rectangle into the membrane material to form a flap with a substantially rectangular shape.

[0032] The flap layer or membrane in which the flap resides can be made of any suitable material. A suitable material can be chosen by one of skill in the art, depending on the type of construction used to make the microfluidic device.

Either flexible or substantially rigid materials may be used to form flaps. When the flap is substantially rigid, the flap can have a hinge region. A hinge region can be a portion of the material that has a reduced thickness relative to the movable portion of the flap. In another embodiment, the hinge region is constructed from a different material from the movable portion of the flap.

[0033] For repeated usage, it is preferred that the material chosen has a degree of elasticity allowing it to rebound into the seated position. For example, the material can be a metal foil, paper or polymer or combinations or laminates thereof. When the device is to be constructed from layered stencil layers, and the flap is integral with the flap layer, the flap and flap layer are preferably fabricated from a polymeric material. Suitable polymeric materials include, for example, polytetrafluorethylenes, polystyrenes, polypropylene, polyethylene, polyimides, polyacrylates, rubbers and silicones. In certain embodiments, one or both sides of

the flap region may be covered or coated with a material to enhance adhesion to the upper or lower channel surface. The adhesion materials can be permanent or reversible. In a preferred embodiment, adhesive materials are coated onto the surface of the flap portion prior to the construction of the device.

[0034] As mentioned previously, microfluidic devices may be fabricated with substrates and stencil layers that define channels therein. Such devices can be constructed from discrete layers of material or can be fabricated as an integral unit. When a coupling device is constructed as an integral unit, layers refer only to positions within a device rather than to individual components. When the device is to be constructed by assembling stencil layers with adhesive separating the layers or using self-adhesive tape materials, the material forming the sealing surface and the side of the flap region interacting with the sealing surface are both preferably non-adhesive. Alternatively, the area of the flap interacting with the sealing surface and/or the seating surface can be adhesively coated, and the adhesive strength can be chosen to prevent permanently closing the valve. Any suitable adhesive can be used to assemble a device from stencil layers.

[0035] The material chosen for use as a valve is preferably substantially impermeable to the fluid to be used in the device. A device can, however, use a material that is permeable to a fluid. In one embodiment, the flap layer is formed from a material that is impermeable to the fluid for which the device is designed, but may be permeable to a gaseous fluid, such as air.

[0036] A flap-containing device can be constructed such that the movable flap can contact one or more surfaces of the first channel, and restrict fluid flow therein. Such a microfluidic device also can have a sealing layer adjacent to the flap layer with holes or apertures disposed therein to allow fluid communication through the flap region. A sealing layer can be formed with substantially rigid material. The sealing layer may also contain an aperture disposed adjacent to the movable flap. In a preferred embodiment, the flap region may be used to seal a hole or via that connects one level of the device to another level. In certain embodiments, the flap portion may be more effective at blocking fluid flow if it covers a hole or via rather than a channel portion.

[00371 The height of the outlet channel also can be varied to change the operation of the flap. A large flap relative to the height of the outlet channel will

allow the flap to seal against a (e. g., the upper or lower) surface of the channel into which the flap is deflected. For example, referring to FIG. 1D, a flap 30 defined in a central membrane layer 22 is capable of opening into a channel 26 defined in an adjacent layer 23. The thickness of the layer 23 (the height of channel 26) and the length of the flap 30 can be varied such that the deflected flap may or may not come in contact with the lower surface of an upper layer 24 that bounds the channel 26 from above.

[0038] In certain embodiments, microfluidic devices having flaps can be used as one-way or check valves. Referring to FIGS. 1A-1B, a microfluidic device 32 is constructed from five layers 20-24 (including two stencil layers 21,23) that define channels 25,26, vias 27, and inlet/outlet ports 28,29. Additionally, one portion or flap 30 in layer 22 is cut so that the flap 30 is still attached to the intermediate layer 22 but is only partially restrained from moving. The assembled device 32, including flap valve region 31, is shown in FIG. 1B. FIGS. 1C and 1D provide cross-sectional views of a portion of the device 32, focusing on the flap valve region 31 and illustrating two different operating states. In one state shown in FIG. 1C, fluid is injected into the device 32 through port 29. The fluid passes through channel 26 until it reaches the flap valve region 31. Here, the flap 30 is shown contacting against a sealing surface 21A of stencil layer 21. Fluid pressure within channel 26 helps force the flap 30 closed against the surface 21A. In the closed state, the flap 30 prevents fluid from flowing from channel 26 into channel 25. If the fluid injected into port 29 is a liquid, then in actuality this liquid may never contact the closed flap 30 as shown in the first operating state, since this liquid will compress any air present within the channel 26, thus building a high- pressure region that will prevent further liquid flow.

[0039] FIG. 1 D shows operation of the device 32 with fluid flow in the reverse direction. When fluid is injected into the device through port 28, it passes through the vias 27 into channel 25. When the fluid encounters the flap 30 from below, the flap 30 is capable of being displaced upward since it is only restrained along one end, and the distal end is bounded above by an open channel 26. The advancing fluid displaces the flap 30 upward and passes from the lower channel 25 into the upper channel 26, from which it is free to exit the device 32 through port 29. The device 32 thus serves as a passive one-way valve or check valve,

since it permits fluid flow in one direction but disallows fluid flow in the reverse direction.

[0040] In a microfluidic device, multiple one-way flap valve regions may be arranged in fluid communication with a fluid displacement means to provide pumping utility. A basic configuration of such a device includes a first inlet channel, a first microfluidic one-way valve having a first flap opening into a chamber in fluid communication with the first inlet channel, and an outlet channel in fluid communication with the chamber through the second microfluidic one-way valve. The second one-way valve has a second flap opening into the second channel.

[0041] Various fluid displacement means may be used. In one embodiment, a pumping chamber includes a cylinder and piston assembly. In another embodiment, a pumping chamber includes a deformable membrane forming one side of the chamber. The deformable membrane may be deformed or otherwise moved to alter the pressure within the chamber, thus inducing fluid movement. The deformable membrane can be moved by various means, including mechanical force. For example, the membrane can be deformed using a mechanical actuator. In one embodiment, the mechanical actuator includes a piston. In another embodiment, the mechanical actuator includes an electromechanical material, such as, for example, a piezoelectric device or a Ti-Ni device. In another embodiment, the membrane can include a magnetic material, and a magnetic field can be fluctuated to force the membrane up and down. In another embodiment, the material can be driven up and down using an asymmetrical cam. In another embodiment, force to deform the membrane is supplied by having an additional chamber opposite the pumping chamber, the pressure of which can be varied, for example, by an external pressure pump or vacuum pump. In another embodiment, the temperature of the chamber 111 can be cycled up and down to induce movement of the membrane.

[0042] Referring to FIG. 2A, a microfluidic pumping device 120 was constructed from nine layers 100-108 (including stencil layers 101, 102,107).

Starting from the top, the first layer 108 defines two fluid ports 113,114 and an actuation port 115. The second through sixth layers 107-103 define vias 113A, 114A, with a further via 114A defined in the seventh layer 102. The second layer

107 defines an actuation channel 111. The fourth layer 105 defines a channel 112. The fifth layer 104 defines a small aperture 118 and a large aperture 119.

The sixth layer 103 defines two flaps 116,117. Preferably, these flaps are only partially restrained, such that they can be deflected upward or downward, as permitted by the surrounding structures. The seventh layer 102 defines a channel 110 and a small aperture 121. The eighth layer 101 defines a channel 109. The ninth layer 100 serves as a cover layer to define the lower boundary of the channel 109 defined in the eighth layer 101. The assembled device 120 is shown in FIG. 2B.

[0043] A side cross-sectional view of a portion of the pumping device 120 (along section lines"A"-"A"in FIG. 2B) is shown in FIGS. 2C and 2D. In use, the device 120 worked as follows. Fluid was injected into the device 120 through an inlet port 114 and vias 114A, through channel 109 and into the aperture 121 to stop against the first flap 117. An external pressure/vacuum source (not shown) was connected to the actuation port 115. When the external source applied a slight vacuum to the actuation channel 111, a central portion of layer 106 flexed up towards the actuation channel 111. This upward deformation of the layer 106 created a slight negative pressure in a displacement channel 112 disposed immediately below the deformable portion of the membrane 106. In response to the reduced pressure in channel 112, the flap 117 was lifted up into the channel 112, thus permitting fluid to flows into the channel 112. Notably, the second flap 116 did not open since its upward travel was blocked by the sealing surface 104A of layer 104.

[0044] Upon equilibration of the channel 111 (or prior to equilibration), the pressure on the external source at actuation port 115 was reversed to supply positive pressure to the actuation channel 111. Referring to FIG. 3D, the positive pressure within channel 111 caused the deformable portion of layer 106 to push down into the channel 112, thus increasing the pressure. In order to adjust to the new pressure, the second flap 116 opens downward towards an outlet channel 110 to admit fluid into the outlet channel 110. When the pressure at the inlet 115 oscillates up and down, a net fluid flow occurs from channel 109 to channel 110.

This pumping mechanism can be used to push fluid throughout additional channels within a microfluidic structure, or to push fluid off-board. The pumping

speed and amount can be altered in a number of ways. The size of the pressure change at the actuation port 115 as well as the period of the oscillation can have an effect. The geometry and size of the channels themselves can also alter the pumping parameters. For example, the size of the flaps will determine the amount of fluid transferred per stroke. Alternatively, the size of the actuation channel 111 just below the deformable portion of layer 106 can also be altered to change the parameters. Likewise, the material used to construct deformable portion of membrane 106 will determine the change in volume of pumping chamber 119, as will the composition of the fluid itself.

[0045] Fluid control valves according to the present invention also can be used to direct fluid flow among layers of a microfluidic device. These valves can be incorporated into a system in such as way that a particular microfluidic device can perform a variety of functions depending on how the chip is used.

Additionally, channels within a particular device can be used more than once for different functions when using these valves.

[0046] Microfluidic devices of the invention also can have filter materials embedded within the channels. A filter is any material that partially blocks or selectively alters fluid flow within a channel. A filter is typically a porous material.

The material also can have surface chemical properties that alter its interaction with various fluids to be used in the device.

[0047] Further embodiments in two similar configurations that perform in two distinct manners are shown in FIGS. 3A-3D. These figures are cross- sectional views of a portion of a microfluidic device having flap regions to provide flow control utility. In one particular embodiment, a microfluidic device contain filter material for performing ultra-filtration of biological or chemical molecules.

Referring to FIG. 3A, a device 179 is shown with an input channel 189 for loading a biological sample. Fluid injected into channel 189 from the left will encounter filter material 187. When fluid is injected, it enters the filter material 187 below from region 190 and flows upward through the filter 187. In a preferred embodiment, the filter 187 is chosen so that biological targets within the sample of choice will become stuck at the lower surface of the filter 187 adjacent to region 190. In certain embodiments, the filter 187 can be chosen so that large nucleic acid targets will be blocked at the entrance of the filter and other non-specific

biomass can pass through. The flap layer 182 in this device is composed of a flexible material so that a flap 188 rests against the upper surface of a lower layer 184 in an unactuated position. Additionally, semi-permanent adhesive material may be used on the lower surface of the flap portion 193 and/or the upper surface of the lower layer 184. In the normal, unactuated position, channel 191 within layer 183 is blocked and the fluid flowing through the filter 187 from below is diverted into the upper channel 192.

[0048] Once the fluid sample is fully injected and the filter material is washed, an extraction fluid is injected into channel 191. Referring to FIG. 3B, the pressure from the extraction fluid being injected at 191 pushes the moveable flap 188 up into a closed position against stencil layer 181. The extraction fluid passes down through the filter 187 and carries the nucleic acid off the filter and into the solution. The extraction fluid then proceeds down channel 189 to another portion of the device or off-board for further analysis.

[0049] A similar configuration of the device 179 is shown in FIG. 3C. In this example, the flap layer 182 is composed of a material such that flap region 188 is normally in the closed position as shown in FIG. 3D. In use, a pressurized sample containing wash buffer is loaded through channel 192, thus deforming the flap downward (as shown in FIG. 3C) to permit the sample to pass through the filter region 187. A nucleic acid material in this example is stuck on the top surface of the filter area 190. Once the wash buffer has been passed across the filter 187, elution buffer can be added in the reverse direction (see FIG. 3D) and be directed to the exit channel 191. Other configurations are possible, as are other types of sample materials and filters.

[0050] In further embodiments, microfluidic devices containing one or more internal flaps can be used to direct or divert fluid among different levels of a three- dimensional device. One such embodiment of the present invention is shown in FIGS. 4A-4C. In this embodiment, a separate control channel is used to alter the fluid being pumped. Referring to FIG. 4A, a top view of a microfluidic device 219 having a flow channel 221 and control channel 220 is provided. The flow channel 221 is defined in a first layer 229, and the control channel 220 is defined in a second layer 228. Also shown are a via (or aperture) 227 formed in a spacing layer 224 and a flap region 226 that operates as a valve. Cross-sectional views of

the valve region are shown in FIGS. 4B and 4C. In use, if no pressure is applied to control channel 220, then fluid flowing through channel 221 reaches the valve region and the pressure of the flow opens the flap valve 226 that covers the via 227. A portion of the fluid then passes into the upper channel 220 as well as the flow channel 221. In an alternative use, pressure is applied to the control channel 220 during use. If the pressure in control channel 220 is higher than the pressure in flow channel 221, then the flap 226 completely covers the via 227 and all of the fluid flowing in channel 221 continues to flow in channel 221. The pressure in channel 220 can be adjusted as desired to serve as a pseudo flow regulator for channel 221. If the flap 226 is partially open, then only a smaller portion of fluid will flow up into channel 220. In a similar manner, channel 220 could be used as the flow channel and channel 221 as the control channel. In this embodiment, all of the fluid would remain in channel 220 and the movement of the flap region 226 would act as a flow constrictor.

[0051] A further embodiment depicting an externally controllable flap is shown in FIGS. 5A-5C. In this embodiment, a flap 88 is composed in whole or in part of a magnetic or magnetizable material, such as a ferromagnetic, paramagnetic, or diamagnetic material. The device has an associated magnetic actuator (not shown), preferably external to the device, for deforming and therefore controlling the position of the flap 88. Referring to FIG. 5A, a portion of a microfluidic device 90 is shown in side sectional view with no magnetic field is applied to the device. Both a flap 88 and a first channel 85 are defined in a central layer 82. An upper channel 87 is defined in layer 83, and a lower channel 86 is defined in layer 81. Outer layers 80,84 further define the boundaries of the upper and lower channel 87,86, including inner surfaces 84A and 80A. Inner layers 81,83 assist with directing the flow when the flap 88 is deflected from a substantially linear position aligned with central layer 82, such as shown in FIG.

5B. In FIG. 5B, an external magnetic force is applied to the flap 88 in the direction of the dark arrow (upward) using a magnetic actuator (not shown). This upward force causes the flap 88 to deflect, either just a small amount or sufficiently to contact the lower surface 84A of the outer layer 84. In the situation where fluid is flowing from channel 85 and being split into both downstream channels 86, 87, then a small deflection in the position of the flap 86 may alter the flow

characteristics of the liquid in the downstream channels 86,87. If sufficient force is applied, then the flap 88 may deflect sufficiently to divert all the flow into channel 86. FIG. 5C is substantially the same as FIG. 5B, but the direction of the magnetic force, represented by the dark arrow, is reversed (downward). In this manner, a microfluidic device responsive to the application of magnetic force may be constructed to control the flow of liquid.

[0052] The particular devices and construction methods illustrated and described herein are provided by way of example only, and are not intended to limit the scope of the invention. The scope of the invention should be restricted only in accordance with the appended claims and their equivalents.