WO2009121037A2

WO2009121037A2 - Three-dimensional microfluidic devices

Info

Publication number: WO2009121037A2
Application number: PCT/US2009/038693
Authority: WO
Inventors: Emanuel Carrilho; Andres W. Martinez; Katherine A. Mirica; Scott T. Phillips; Adam C. Siegel; Benjamin Wiley; George M. Whitesides
Original assignee: President And Fellows Of Harvard College
Priority date: 2008-03-27
Filing date: 2009-03-27
Publication date: 2009-10-01
Also published as: AU2009228091B2; WO2009121037A3; EP2257819A2; CA2719320A1; KR20100127301A; US8628729B2; KR101561718B1; AU2009228091A1; EP2257819A4; US20110123398A1; CN102016595A; CN102016595B

Abstract

Three-dimensional microfluidic devices including by a plurality of patterned porous, hydrophilic layers and a fluid-impermeable layer disposed between every two adjacent patterned porous, hydrophilic layers are described. Each patterned porous, hydrophilic layer has a fluid-impermeable barrier which substantially permeates the thickness of the porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions within the patterned porous, hydrophilic layer. The fluid-impermeable layer has openings which are aligned with at least part of the hydrophilic region within at least one adjacent patterned porous, hydrophilic layer. Microfluidic assay device, microfluidic mixer, microfluidic flow control device are also described.

Description

Three-Dimensional Microfluidic Devices Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 61/072,049, filed March 27, 2008, which is hereby incorporated by reference in its entirety. This application claims is related to PCT/US07/081848, which is hereby incorporated by reference in its entirety.

Background

[0002] The disclosed subject matter relates generally to three-dimensional microfluidic devices.

[0003] In recent years, microfluidic systems have attracted increasing interests due to their diverse and widespread potential applications. For example, using very small volumes of samples, microfluidic systems could carry out complicated biochemical reactions to acquire important chemical and biological information. Among other advantages, microfluidic systems reduce the required amount of samples and reagents, shorten the response time of reactions, and decrease the amount of biohazard waste for disposal.

[0004] First developed in the early 1990s, microfluidic devices were initially fabricated in silicon and glass using photolithography and etching techniques adapted from the microelectronics industry. Current microfluidic devices are constructed from plastic, silicone or other polymeric materials, e.g. polydimethylsiloxane (PDMS). Such devices are generally expensive, inflexible, and difficult to construct.

[0005] Lateral-flow fluidic devices are two dimensional (2-D) and are used for applications where fluids need to be transported in a single plane, in series or in parallel. However, fluids in adjacent channels in a 2-D device cannot cross one another without intersecting. This topological constraint ultimately limits the design and applications of 2-D fluidic devices. Thus, there remains a need for three dimensional microfluidic devices that are inexpensive, flexible, and easy to construct. Summary of the Invention

[0006] In one aspect of the invention, a three-dimensional micro fluidic device is described. The three-dimensional microfluidic device includes a plurality of patterned porous, hydrophilic layers including at least a first and second patterned porous, hydrophilic layers. The patterned porous, hydrophilic layer includes a fluid- impermeable barrier which substantially permeates the thickness of the patterned porous, hydrophilic layer and defines a boundary of one or more hydrophilic regions within each patterned porous, hydrophilic layer. The three-dimensional microfluidic device also includes a fluid-impermeable layer disposed between the first and second patterned porous, hydrophilic layers and every two adjacent patterned porous, hydrophilic layers. The fluid-impermeable layer includes one or more openings. The three-dimensional microfluidic device also includes a porous hydrophilic medium disposed in the opening of the fluid-impermeable layer disposed between the first and second patterned porous, hydrophilic layers. The hydrophilic medium contacts at least one of the hydrophilic regions within each of the first and second patterned porous, hydrophilic layers.

[0007] In one embodiment, the porous, hydrophilic medium includes paper. In another embodiment, the porous, hydrophilic medium includes chromatography paper. In yet another embodiment, the porous, hydrophilic medium includes paper and the paper is infused with components selected from the group including chemical or biological reagents, indicators, binders, fluid flow-impeding agents, fluid flow-promoting agents, or a combination thereof. In yet another embodiment, the porous, hydrophilic layer includes paper. In yet another embodiment, the porous, hydrophilic layer includes chromatography paper. In yet another embodiment, the barrier includes polymerized photoresist. In yet another embodiment, the barrier includes polymerized photoresist and the photoresist includes SU-8 photoresist. In yet another embodiment, the fluid-impermeable layer includes a plastic sheet. In yet another embodiment, the fluid-impermeable layer includes a plastic sheet and the plastic sheet includes adhesive tape. In yet another embodiment, the fluid-impermeable layer includes a plastic sheet, the plastic sheet includes adhesive tape, and the adhesive tape includes double-sided adhesive tape. [0008] In yet another embodiment, the hydrophilic regions include a first hydrophilic region including a reservoir for depositing a first fluid; a second hydrophilic region including a distributing region for receiving the first fluid from the reservoir and distributing the first fluid; and an array of hydrophilic regions for receiving the first fluid from the distributing region. In yet another embodiment, each hydrophilic region of the array further includes an assaying reagent for assaying the first fluid. In yet another embodiment, the assay reagent is a protein assay reagent, a glucose assay reagent, a sodium acetoacetate assay reagent, a sodium nitrite assay reagent, or a combination thereof.

[0009] In another aspect, a three-dimensional microfluidic device is described. The three-dimensional microfluidic device includes a plurality of patterned porous, hydrophilic layers. Each patterned porous, hydrophilic layer includes a fluid- impermeable barrier which substantially permeates the thickness of the patterned porous, hydrophilic layer and defines a boundary of one or more hydrophilic regions within each patterned porous, hydrophilic layer. The three-dimensional microfluidic device also includes a fluid-impermeable layer disposed between every two adjacent patterned porous, hydrophilic layers and the fluid-impermeable layer includes one or more openings. The three-dimensional microfluidic device also includes a material plug filling one or more of the openings to provide local temperature control, to assay fluid samples, to filter microfluidic sample, or to modulate microfluidic flow. The material plug is in direct contact with at least of portion of one of the hydrophilic regions within each adjacent patterned porous, hydrophilic layer.

[0010] In one embodiment, the material plug includes an assaying reagent for assaying fluidic samples. In another embodiment, the material plug includes an assaying reagent for assaying fluidic samples and the assay reagent includes a protein assay reagent, a glucose assay reagent, a sodium acetoacetate assay reagent, a sodium nitrite assay reagent, or a combination thereof. In yet another embodiment, the material plug includes a seed. In yet another embodiment, the seed includes soybean seeds, hulls, and powdered soybean, or any other seeds that provide peroxidase to be used as an assay reagent. In yet another embodiment, the material plug includes a filter for filtering solids or blood cells. In yet another embodiment, the filter includes filter papers, any other filtration medium for removing solid contaminants, dextran, or any other materials that can remove red blood cells from a blood sample. In yet another embodiment, the material plug includes a salt which either absorb heat or give off heat when in contact with fluid to provide local temperature control. In yet another embodiment, the salt includes ammonium nitrate or magnesium sulfate. In yet another embodiment, the material plug includes a gel which swells when in contact with fluid to allow microfluidic communication. In yet another embodiment, , the material plug includes a gel which swells when in contact with fluid to allow microfluidic communication and the gel includes agarose, methylcellulose, or hylaronan. In yet another embodiment, the material plug includes a magnetic material which is capable of movement under the influence of a magnetic field to allow microfluidic communication. In yet another embodiment, the magnetic material includes iron oxide. In yet another embodiment, the material plug includes a fluid-mixable material or a mixture of a fluid-mixable material and a fluid-unmixable material; and the fluid-mixable material dissolves when in contact with fluid to allow microfluidic communication and no microfluidic communication is allowed once the fluid-mixable material has been dissolved and flowed away. In yet another embodiment, the fluid-mixable material includes sugar. In yet another embodiment, the sugar includes sucrose. In yet another embodiment, the fluid-unmixable material includes a hydrophobic polymer. In yet another embodiment, the hydrophobic polymer includes polystyrene. In yet another embodiment, the porous, hydrophilic layer includes paper. In yet another embodiment, the porous, hydrophilic layer includes chromatography paper. In yet another embodiment, the barrier includes polymerized photoresist. In yet another embodiment, the photoresist includes SU-8 photoresist. In yet another embodiment, the fluid-impermeable layer includes a plastic sheet. In yet another embodiment, the plastic sheet includes adhesive tape. In yet another embodiment, the adhesive tape includes double-sided adhesive tape.

[0011] In yet another aspect, a three-dimensional microfluidic mixer is described. The three-dimensional microfluidic mixer includes a first patterned porous hydrophilic layer including a fluid-impermeable barrier which substantially permeates the thickness of the patterned porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions. One of the hydrophilic regions within the first patterned porous hydrophilic layer includes a first fluid flowing channel and a second fluid flowing channel, and a combination zone. The first and second fluid flowing channels merge into the combination zone. The three- dimensional microfluidic mixer also includes a second patterned porous hydrophilic layer including a fluid-impermeable barrier which substantially permeates the thickness of the patterned porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions. One of the hydrophilic regions within the second patterned porous hydrophilic layer includes a mixing channel. The three- dimensional microfluidic mixer also includes a fluid-impermeable layer disposed between the first and second patterned porous, hydrophilic layers and including an unfilled opening which is in alignment with the combination zone and at least portion of the mixing channel. The combination zone is capable of moving from a first spaced apart position into a second contact position. In the spaced apart position, the combination zone is not in fluidic contact with the mixing channel. In the contact position, the combination zone is in fluidic contact with the mixing channel.

[0012] In one embodiment, the first and second fluid flowing channels merge into the combination zone and define an interface between the first and second fluid flowing channels. In another embodiment, the mixing channel is perpendicular to the interface. In yet another embodiment, the mixing channel is more than 1 mm long. In yet another embodiment, the porous, hydrophilic layer includes paper. In yet another embodiment, the porous, hydrophilic layer includes chromatography paper. In yet another embodiment, the barrier includes polymerized photoresist. In yet another embodiment, the photoresist includes SU-8 photoresist. In yet another embodiment, the fluid-impermeable layer includes a plastic sheet. In yet another embodiment, the plastic sheet includes adhesive tape. In yet another embodiment, the adhesive tape includes double-sided adhesive tape.

[0013] In yet another aspect, a three-dimensional microfluidic flow control device is described. The three-dimensional microfluidic flow control device includes a plurality of porous, hydrophilic layers including a fluid-impermeable barrier which substantially permeates the thickness of the patterned porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions. The plurality of porous, hydrophilic layers include a first patterned porous hydrophilic layer including a fluid-impermeable barrier which substantially permeates the thickness of the first patterned porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions and of the hydrophilic regions within the first patterned porous hydrophilic layer includes a first flowing channel. The plurality of porous, hydrophilic layers also include a second patterned porous hydrophilic layer including a fluid-impermeable barrier which substantially permeates the thickness of the second patterned porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions and one of the hydrophilic regions within the second patterned porous hydrophilic layer includes a second flowing channel. The three- dimensional microfluidic flow control device also includes a fluid-impermeable layer disposed between every two adjacent patterned porous, hydrophilic layers and the fluid-impermeable layer includes one or more openings. The fluid-impermeable layers includes a first fluid-impermeable layer disposed between the first and second patterned porous, hydrophilic layers and including an unfilled opening which at least is in partial alignment with the first and second flowing channels. At least portion of the first flowing channel is capable of moving from a first spaced apart position into a second contact position. In the spaced apart position, the first flowing channel is not in fluidic contact with the second flowing channel. In the contact position, the first flowing channel is in fluidic contact with the second flowing channel.

[0014] In one embodiment, the porous, hydrophilic layer includes paper. In another embodiment, the porous, hydrophilic layer includes chromatography paper. In yet another embodiment, the barrier includes polymerized photoresist. In yet another embodiment, the photoresist includes SU-8 photoresist. In yet another embodiment, the fluid-impermeable layer includes a plastic sheet. In yet another embodiment, the plastic sheet includes adhesive tape. In yet another embodiment, the adhesive tape includes double-sided adhesive tape. In yet another embodiment, the three-dimensional microfluidic flow control device of further includes a hydrophobic plug disposed in one of the openings within one of the fluid- impermeable layers and the hydrophobic plug includes a low-melting-point hydrophobic material capable of melting and flowing at a pre-selected temperature. One of the patterned porous, hydrophilic layers is below the fluid-impermeable layer containing the hydrophobic plug and includes one of the hydrophilic regions in at least partial alignment with the hydrophobic plug. At the pre-selected temperature the hydrophobic material melts and flows into the hydrophilic region in at least partial alignment with the hydrophobic plug to substantially obstruct the hydrophilic region and prohibits microfluidic flow within the hydrophilic region. In another embodiment, the hydrophobic plug includes a porous hydrophilic medium permeated by the low-melting-point hydrophobic material. In yet another embodiment, the porous hydrophilic medium includes paper. In yet another embodiment, the low-melting-point hydrophobic material includes low-melting- point wax. In yet another embodiment, the wax includes candle wax, cheese wax, packaging wax, paraffin wax, bayberry wax, or other waxes. In yet another embodiment, the wax is bayberry wax. In yet another embodiment, the low-melting- point hydrophobic material has a melting point of less than 200 ⁰C, less than 100 ⁰C, less than 50 ⁰C, or between 45⁰C and 50⁰C.

[0015] In yet another aspect, a three-dimensional microfluidic flow control device is described. The three-dimensional microfluidic flow control device includes a first patterned porous hydrophilic layer and a second patterned porous hydrophilic layer, each including a fluid-impermeable barrier which substantially permeates the thickness of the first or second patterned porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions within the first or second patterned porous, hydrophilic layer. The three-dimensional microfluidic flow control device also includes a fluid-impermeable layer disposed between the first and second patterned porous, hydrophilic layers including one or more openings and a hydrophobic plug disposed in the fluid-impermeable layer. The hydrophobic plug includes a low-melting-point hydrophobic material capable of melting and flowing at a pre-selected temperature. The second patterned porous hydrophilic layer is below the fluid-impermeable layer containing the hydrophobic plug and includes one of the hydrophilic regions in at least partial alignment with the hydrophobic plug. At the pre-selected temperature the hydrophobic material melts and flows into the hydrophilic region in at least partial alignment with the hydrophobic plug to substantially obstruct the hydrophilic region and prohibits microfluidic flow within the hydrophilic region. [0016] In one embodiment, the hydrophobic plug includes a porous hydrophilic medium permeated by the low-melting-point hydrophobic material. In another embodiment, the porous hydrophilic medium includes paper. In yet another embodiment, the low-melting-point hydrophobic material includes low-melting-point wax. In yet another embodiment, the wax includes candle wax, cheese wax, packaging wax, paraffin wax, bayberry wax, or other waxes. In yet another embodiment, the wax includes bayberry wax. In yet another embodiment, the low-melting-point hydrophobic material has a melting point of less than 200 ⁰C, less than 100 ⁰C, less than 50 ⁰C, or between 45 ⁰C and 50⁰C. In yet another embodiment, the porous, hydrophilic layer includes paper. In yet another embodiment, the porous, hydrophilic layer includes chromatography paper. In yet another embodiment, the barrier includes polymerized photoresist. In yet another embodiment, the photoresist includes SU-8 photoresist. In yet another embodiment, the fluid-impermeable layer includes a plastic sheet. In yet another embodiment, the plastic sheet includes adhesive tape. In yet another embodiment, the adhesive tape includes double-sided adhesive tape.

[0017] As used herein, "aperture" and "opening" are used interchangeably. As used herein, "2-D" and "two-dimensional" are used interchangeably. As used herein, "3-D" and "three-dimensional" are used interchangeably.

Brief Description of the Drawing

[0018] Preferred, non- limiting embodiments of the present invention will be described by way of examples with reference to the accompanying figures, in which:

[0019] FIGURE 1 is a schematic illustration of a three-dimensional micro fluidic device fabricated by aligning and stacking alternating layers of patterned paper and double-sided tape;

[0020] FIGURE 2 is a schematic illustration of a three-dimensional micro fluidic device fabricated by aligning and stacking alternating layers of patterned paper and double-sided tape with holes filled with pieces of paper;

[0021] FIGURE 3 is an exploded view of a three-dimensional microfluidic device which enables two aqueous fluids to cross without touching or mixing; [0022] FIGURE 4 illustrates four three-dimensional microfluidic devices which distribute samples into arrays;

[0023] FIGURE 5 illustrates a 16-well three-dimensional microfluidic device for running multiple assays;

[0024] FIGURE 6 illustrates another 16-well three-dimensional microfluidic device for running multiple assays;

[0025] FIGURE 7 illustrates a three-dimensional microfluidic device with reagents added to the paper discs within the openings of the tape layer to detect glucose;

[0026] FIGURE 8 illustrates two configurations of fluids in laminar flow in a paper channel;

[0027] FIGURE 9 is a schematic illustration of a three-dimensional microfluidic mixer made out of patterned paper and double-side tape;

[0028] FIGURE 10 illustrates a three-dimensional microfluidic mixer for mixing a blue dye with a yellow dye;

[0029] FIGURE 11 illustrates two three-dimensional microfluidic mixers with different orientations of the mixing channels with respect to the interfaces of the fluids;

[0030] FIGURE 12 illustrates a three-dimensional microfluidic mixer for mixing four fluids, two by two, in all possible six combinations;

[0031] FIGURE 13 illustrates three-dimensional microfluidic devices with single-use "on" and "off button;

[0032] FIGURE 14 illustrates three-dimensional microfluidic devices with AND and OR logic gates;

[0033] FIGURE 15 illustrates a three-dimensional microfluidic device with

NOT logic gate;

[0034] FIGURE 16 illustrates three-dimensional microfluidic devices with

NAND and NOR logic gates;

[0035] FIGURE 17 illustrates three-dimensional microfluidic devices with XOR and XNOR logic gates;

[0036] FIGURE 18 illustrates a three-dimensional microfluidic de -multiplexer. Detailed Description

[0037] An exemplary multilayer microfluidic device is described here with reference to Figure 1 and demonstrates the principals of vertical and lateral flow. Figure 1 schematically illustrates a perspective view of a three-dimensional microfluidic device 100, which includes two layers of patterned paper 110 and 130, and one separating layer of barrier material 120, e.g., double sided tape, with apertures such as 122, 124, 126, and 128. The three layers 110, 120, 130 are aligned and bonded to each other. Patterned paper layer 110 contains non-intersecting channels 112 and 114. Separating layer 120 includes a set of apertures 122 and 124 that are located to direct fluid from channel 114 in the upper patterned paper 110 to channel 132 in the lower patterned paper 130 and then back up to channel 114' in patterned paper 110. Separating layer 120 also includes a set of apertures 126 and 128 that are located to direct fluid from channel 112 to channel 134 in the lower patterned paper 130 and then back up to channel 112' in patterned paper 110, respectively. The device is one example of multiplayer microfluidic channel that allows the separate channels, through which the dyes are flowing, to cross each other without any mixing taking place between the two fluids.

[0038] The microfluidic device relies on apertures within the separating layer to direct vertical microfluidic flow. The applicants have discovered that vertical flow (or flow through) can be significantly improved by filling the opening within the separating layer with a porous, hydrophilic material that bridges the gap between the adjacent hydrophilic layers. As a result, the two microfluidic regions within two adjacent patterned porous hydrophilic layers that partially overlap with the opening will be in direct contact with the hydrophilic material in the opening, thus allowing optimized microfluidic flow by capillarity. In one or more embodiments, the porous, hydrophilic material substantially fills the void within the separating layer created by the aperture. In one or more embodiments, the porous, hydrophilic material substantially matches the shape of the opening. The porous, hydrophilic material filling the opening can be, for example, porous paper pads or "dots" that have a thickness similar to the thickness of the separating layer. The paper can be cut into shape to match the dimensions of the void space created by the aperture in the separating layer.

[0039] In one aspect, three dimensional (3-D) microfluidic devices are described. In one embodiment, three-dimensional microfluidic devices made out of layers of fluid impermeable separating layers and patterned porous hydrophilic layers are described with reference to Figure 2. The microfluidic devices includes layer(s) of paper, which have been patterned into hydrophilic regions or channels separated by hydrophobic barrier walls, and a fluid impermeable layer, containing apertures or openings filled with a porous, hydrophilic material, e.g. a paper plug, to allow fluids to efficiently move vertically from the hydrophilic regions of one layer of paper into another. The hydrophilic regions or channels in the layers of paper wick fluids in the horizontal plane, while the hydrophobic polymer patterned into the paper dictates where the fluids move within the channels, thus defining the directions of microfluidic lateral flow within the patterned paper. The fluid- impermeable layer is disposed between two adjacent layers of paper and separate the channels within the two adjacent layers of paper from one another. The apertures of the fluid-impermeable separating layers are filled with pieces of paper cut to substantially match the shape of the apertures. The apertures and the pieces of paper within are in alignment with at least a portion of a hydrophilic region within each of the two adjacent patterned porous hydrophilic layers. The paper plug in the openings are thus in contact with at least a portion of a hydrophilic region within each of the two adjacent porous paper layers to improve capillary action and allow fluids to efficiently move in the vertical plane (between adjacent layers of patterned hydrophilic paper) from channels in one layer of paper to channels in an adjacent layer of paper, through the pieces of paper within the opening. Because the layers of paper and tape are thin (-100-200 μm per layer), it is possible to stack several layers of paper and tape, without significantly changing the size of the device. Each layer of paper and tape can also be used to serve a unique function within a device (e.g., storing reagents, assaying fluids, filtering samples, distributing fluids, mixing fluids, and controlling fluidic flow).

[0040] Porous, hydrophilic layers include any hydrophilic substrate that wicks fluids by capillary action. In one or more embodiments, the porous, hydrophilic layer is paper. Non- limiting examples of porous, hydrophilic layers include chromatographic paper, filter paper, nitrocellulose and cellulose acetate, cellulosic paper, filter paper, paper towels, toilet paper, tissue paper, notebook paper, Kim Wipes, VWR Light-Duty Tissue Wipers, Technicloth Wipers, newspaper, any other paper that does not include binders, cloth, and porous polymer film. In general, any paper that is compatible with the selected patterning method may be used. In certain embodiments, porous, hydrophilic layers include Whatman chromatography paper No. 1.

[0041] In one or more embodiments, the hydrophilic layer is patterned following the procedures described in Figure 2. In certain embodiments, the hydrophilic paper is soaked in photoresist, and photolithography is used to pattern the photoresist to form the barriers following the procedures described in PCT/US07/081848. Other hydrophilic material such as nitrocellulose and cellulose acetate are commonly used and well-known membranes for their use in fluid diagnostics, but are not compatible with solvents typically used in photolithography. Other methods, such as screening, stamping or printing, can be more suitable for patterningsuch materials. In addition, the hydrophilic layer and the hydrophobic barrier regions could be prepared using materials that are compatible with the testing conditions, e.g., temperature, pH, and/or ionic strength.

[0042] The photoresist used for patterning porous, hydrophilic material include SU-8 photoresist, SC photoresist (Fuji Film), poly(methylmethacrylate), nearly all acrylates, polystyrene, polyethylene, polyvinylchloride, and any photopolymerizable monomer that forms a hydrophobic polymer.

[0043] The fluid-impermeable player is typically a sheet that is not soluble in the fluid of the micro fluidic device and that provides the desired level of device stability and flexibility. In one or more embodiments, the fluid-impermeable player is a plastic sheet. In certain embodiments, the fluid-impermeable player is an adhesive sheet or tape. Non-limiting examples of fluid-impermeable player includes Scotch^® double-sided carpet tape, water-impermeable barriers include 3M Double Sided Tape, Tapeworks double sided tape, CR Laurence black double sided tape, 3M Scotch Foam Mounting double-sided tape, 3M Scotch double-sided tape (clear), QuickSeam splice tape, double sided seam tape, 3M exterior weather-resistant double-sided tape, CR Laurence CRL clear double-sided PVC tape, Pure Style Girlfriends Stay-Put Double Sided Fashion Tape, Duck Duck Double-sided Duct Tape, and Electriduct Double-Sided Tape. In certain specific embodiments, double- sided tape is used as the fluid-impermeable layer. Double-sided tape adheres to two adjacent layers of patterned paper and can bind to other components of the microfluidic device. It is impermeable to water, and isolates fluid streams separated by less than 200 μm. In addition, it is also sufficiently thin to allow adjacent layers of paper to contact through holes punched in the tape when compressed. It can easily separate from the paper to which it adheres and thus allow disassembly of stacked devices and it is inexpensive and widely available.

[0044] As an alternative to double-sided tape, a heat-activated adhesive can be used to seal the fluid-carrying layers together. Indeed, any fluid-impermeable material that can be shaped and adhered to the pattern hydrophilic layers can be used. In addition, it is also possible to use the same material that is used to pattern the paper layers to join the layers of paper together. In one or more embodiments, a layer of photoresist is disposed between two adjacent patterned porous, hydrophilic layers.

[0045] The porous, hydrophilic material filling the aperture or opening could be the same material as the porous, hydrophilic layers or different material. Non- limiting examples of porous, hydrophilic material include chromatographic paper, filter paper, nitrocellulose and cellulose acetate, cellulosic paper, filter paper, paper towels, toilet paper, tissue paper, notebook paper, Kim Wipes, VWR Light-Duty Tissue Wipers, Technicloth Wipers, newspaper, and any paper that does not include binders.

[0046] In one or more embodiments, the patterned porous hydrophilic layer is Whatman chromatography paper No. 1 (which wicks water well, and is pure cellulose) patterned using SU-8 2010 photoresist, the fluid-impermeable layer is Scotch^® double-sided carpet tape, and the porous, hydrophilic material filling the aperture of opening in the fluid-impermeable layer is Whatman chromatography paper No. 1.

[0047] The advantages of the microfluidic devices described herein include the following: the devices are inexpensive to produce; they are easy to assemble, and do not require specialized equipment; the inner layers are protected from the environment, thus limiting evaporation; the inner layers can be used to store reagents for assays that are sensitive to light; they emphasize short vertical zones corresponding to the thickness of the various types of paper selected for each function; and complex channels can easily be fabricated and easily disassembled. [0048] In one embodiment, a microfluidic device is described with reference to Figure 2 and demonstrates the principals of vertical and lateral flow. As shown in Figure 2, the microfluidic device is assembled by aligning and stacking alternating layers of patterned paper and double-sided tape used as the fluid-impermeable layer. In some embodiments, microfluidic devices having small features (for example, features less than about 1 mm-wide channels) are assembled using automated assembly. In other embodiments, the alignment can be carried out by eye. The assembly is demonstrated in oblique view (on left) and in cross section view (on right). In step 1, paper 200 is patterned into microfluidic regions of hydrophilic paper 220 (white regions) and hydrophobic polymer 210 (black regions) using photolithography. A second hydrophilic paper 205 is then patterned for the lower layer of the device. In this embodiment, the hydrophobic polymer is SU-8 photoresist and the hydrophilic paper is Whatman chromatography paper No. 1. Paper can have a range of thickness; Whatman chromatography paper No. 1 has a thickness of about 160 μm. In step 2, holes 230 were punched within the layer of double-side tape 240 in the locations where contact points between adjacent layers of paper are desired. The separating layer can have a range of thicknesses, but is typically selected to minimize the bulk and volume of the device. In this embodiment, the tape has a thickness of about 75 μm. In step 3, the space within the holes 230 in the tape 240 is filled with a piece of paper 250 cut to match shape of the hole in the tape (2.5 -mm wide). Without filling the space with paper, the fluids would not wick optimally across the small gap (~75 μm) from one layer of paper to another through the holes in the tape. The holes were filled with the same type of paper as the paper layers, such as Whatman chromatography paper No. 1, serves to eliminate the gap, and provides reliable contact between the two layers of patterned paper. Alternatively, the holes could be filled with pieces of any type of hydrophilic material. After the holes in the tape were filled, each side of the tape is attached to a layer of patterned paper to form a sandwich of paper-tape-paper 260 as shown in Figure 2. The overall thickness of the device is quite thin, e.g., less than 0.4 mm. [0049] In another embodiment, a microfluidic device with two channels that cross each other four times without contacting one another is described with reference to Figure 3B to demonstrates the principals of vertical and lateral flow. The device is shown in Figure 3 A and is constructed using similar principle as described in Figure 2, including patterned paper layers containing hydrophilic regions such as 311 and photoresist 312 and tape layers including paper-filled holes such as 313. The microfluidic device 300 enables two aqueous fluids to cross without touching or mixing. As shown in Figure 3B, red and blue aqueous dyes from two sample reservoirs 301 and 310, respectively, were wicked following a path with multiple crossings, without mixing. The fluids flow through the channels in approximately 15 minutes by moving laterally (left to right) a distance of 3 cm (for red dye) and 4.5 cm (for blue dye). As shown in Figure 3A, the device is assembled from two layers of patterned paper (a top layer and a bottom layer) and one layer of double-sided tape. The paper includes Whatman chromatography paper No. 1 which is patterned into hydrophilic regions of exposed paper and hydrophobic regions of SU-8 photoresist using photolithography. As illustrated in Figure 3, 1-mM allura red AC (red dye) and 1-mM erioglaucine (blue dye) were wicked into hydrophilic channels of paper. The middle layer is Scotch^® double-sided carpet tape with 2.5- mm diameter holes in locations that match the patterns of the two pieces of paper. The holes are filled with paper discs, e.g., 302, 304, 306, and 308. As an example, the detailed microfluidic flow of the red dye is described. The red dye is deposited in hydrophilic region 301 in the top patterned paper which is in partial alignment with paper disc 302 in one of the holes in the double-sided tape layer. Red dye is then wicked from 301 through paper disc 302 vertically into the bottom patterned paper, specifically, hydrophilic region 303. The red dye is wicked laterally through channel 303 and then vertically through paper disc 304 to the hydrophilic region 305 in the top layer. The red dye is then wicked again into the bottom layer through paper disc 306 into hydrophilic region 307 in the bottom paper layer, before it is wicked back to the top layer through paper disc 308 into hydrophilic region 309. The blue dye is wicked through the microfluidic device between layers as well and the two dyes cross each other four times without contacting one another. Figure 3B shows a time sequence of photographs of the device. The first image shows the device before the dyes are deposited (t = 0) and successive images show the device at 1 minute, 10 minutes, and 15 minutes after the dyes are deposited. The sequence shows the progressive movement of aqueous red and blue dye within the device. The cross section image shows that the fluids move only between the two layers of paper in the areas where there is a hole in the tape separating the two layers of paper. [0050] Fabrication in 3-D in paper has the advantage of very short path- lengths (i.e., 200-600 μm, depending on the thickness of the paper and the number of layers of paper used) between sample entry points and regions for assays. Short path- lengths lead to rapid distribution of sample and rates of fluid transport by capillarity that are much higher than 2-D, lateral flow paper-based devices, and operationally comparable to open microchannel devices.

[0051] The ability to stack layers of paper brings new capabilities to micro fluidics: i) the ability to build complex, 3-D, multilayer systems by stacking sheets of patterned paper; ii) the ability to fabricate systems with zones of different functionality separated by short distances (a few microns) vertically, rather than much larger distances (mm) horizontally. The microfluidic devices can be used for running multiple assays, adding regents to the intervening layers of paper, controlled mixing of fluids, and programming fluid flow.

Distributing a Single Sample into an Array

[0052] In yet another embodiment, three-dimensional microfluidic devices for distributing samples from reservoirs into arrays of detection zones are described. The microfluidic devices include layers of patterned porous, hydrophilic layers, a fluid-impermeable layer with openings filled with a porous hydrophilic material disposed between every two adjacent patterned hydrophilic layers. In certain embodiments, the microfluidic devices further include a first hydrophilic region that serves as a fluid reservoir, a second hydrophilic region including a distributing region for distributing the first fluid, and an array of hydrophilic regions for receiving the first fluid from the distributing region. The reservoir and distributing regions can be on the same or different layers in the 3-D device. Four examples of 3-D devices that distribute samples in different ways are illustrated in Figure 4 A, 4B, 4C, and 4D, respectively. Each of the devices is constructed using alternated layers of patterned paper with hydrophilic regions conducting lateral flow and fluid- impermeable layers with opening filled with paper disc conducting vertical flow. The microfluidic flow within the device follows the principle of lateral and vertical flow of the microfluidic device described above. The brown regions of the device are hydrophobic SU-8 photoresist, while the regions filled with dye are hydrophilic paper, as indicated in Figure 4A. Each of the devices has the same input consisting of four wells on the top of the device, which are filled with blue, green, red and yellow aqueous dyes, respectively. As shown in Figure 4, each device has different outputs, in which the device distributes the fluids added to the inlets into different patterns in a 4 x 4 array of test zones. The middle layers of the 3-D devices direct the samples into detection zones without mixing or cross-contamination between the samples.

[0053] In the examples shown in Figures 4A and 4B, the detection zones are equidistantly spaced from the sample reservoir to ensure that the same volume of sample is collected in each detection zone. Figures 4A and 4B uses different combinations of linear and star burst hydrophilic distribution regions to accomplish different distribution patterns. In Figure 4A, a blue sample is applied to the application reservoir 421 and is distributed through paper-disc-filled hole 401 to a channel distribution region 402 that distributes the blue sample from a first position 403 directly aligned with hole 401 to a second position 404 that is centrally located in a quadrant of the second paper layer. Liquid within 404 flows vertically through paper-disc-filled hole 405 to a central position 407 of a star-burst shaped hydrophilic distribution region 406. Liquid then flows laterally to termini, e.g., 408 and then vertically through paper-disc-filled hole 409 to the lower receiving layer 410. As shown in Figure 4 A, the receiving layer 410 has a quadrant 425, consisting of four hydrophilic regions filled with blue dyes (as indicated by the hydrophilic regions within the circle). The resultant array has quadrants of one color. Photographs of the top and bottom paper layers, indicating the starting and final positions of each dye is also shown. In Figure 4B, a blue dye is deposited in reservoir 451 at the top of the device. The device in Figure 4B uses a series of channel distribution regions 411 and 412 that produce an array of hydrophilic regions 413 (as indicated by the hydrophilic regions within the box) within the bottom paper layer containing the blue dye. Photographs of the top and bottom paper layers, indicating the starting and final positions of each dye are also shown.

[0054] In the third example, shown in Figure 4C, a blue dye is deposited into reservoir 461 and distributed into a final array contains color zones arranged in a diagonal 415 (as indicated by the hydrophilic regions within the box). The color zones fill at different times because they are not equidistant from sample reservoir. The devices shown in Figures 4A, 4B, and 4C distribute four samples from reservoirs at the top of the device into an array of 16 detection zones. Figures 4A, 4B, and 4C show 3 devices that wicked aqueous blue (1-mM erioglaucine, deposited at reservoirs 421, 451, and 461 in Figure 4A, 4B, and 4C, respectively), green (0.5- mM erioglaucine and 0.5-mM tartrazine, deposited at reservoirs 422, 452, and 462 in Figure 4A, 4B, and 4C, respectively), red (1-mM allura red AC, deposited at reservoirs 423, 453, and 463 in Figure 4A, 4B, and 4C, respectively) and yellow (1- mM tartrazine, deposited at reservoirs 424, 454, and 464 in Figure 4A, 4B, and 4C, respectively) dyes, and filled the 16 detection zones in 5 min. [0055] Figure 4D illustrates the potential to use 3-D devices to distribute samples into large arrays of closely spaced test zones. In this example, four fluids 441, 442, 443, and 444 were deposited on the top of the micro fluidic device as shown in Figure 4D. The four fluids are distributed into an 8 x 8 array of test zones. This device also has four inlets, but it distributes samples into an array of 64 test zones. As shown by the photo of the bottom paper layer of the device, fluid 444 is distributed into 4 hydrophilic regions in the center of the bottom of the layer including hydrophilic region 445. The fluid 443 is distributed into 12 hydrophilic regions surrounding the 4 hydrophilic regions for fluid 444 on the bottom of the layer including hydrophilic region 446. The fluid 442 is distributed into 20 hydrophilic regions surrounding the 12 hydrophilic regions for fluid 443 on the bottom of the layer including hydrophilic region 447. The fluid 441 is distributed into 28 hydrophilic regions surrounding the 20 hydrophilic regions for fluid 442 on the bottom of the layer including hydrophilic region 448. The complexity of the output pattern determines the number of middle layers of paper and tape that are required in the device. In each of the four devices shown in Figures 4A-4D, the holes in the tape are filled with discs of Whatman chromatography paper No. 1. [0056] Other patterns and more complex patterns could be obtained by incorporating additional layers of paper and tape into the device.

3-D Microfluidic Devices for Running Multiple Assays

[0057] In yet another embodiment, 3-D microfluidic devices to run multiple assays on multiple samples simultaneously are described. The microfluidic devices include layers of patterned porous, hydrophilic layers, a fluid-impermeable layer with openings filled with a porous hydrophilic material disposed between every two adjacent patterned hydrophilic layers. The microfluidic devices further include a first hydrophilic region serve as a fluid reservoir, a second hydrophilic region including a distributing region for distributing the first fluid, an array of hydrophilic regions for receiving the first fluid from the distributing region, and the array of hydrophilic regions for receiving the first fluid further include an assay agent for assaying the first fluid. In certain embodiments, the array of hydrophilic regions for receiving the first fluid are pre-spotted with various assay reagents for assaying the fluid.

[0058] In a specific embodiment, an example of a 16-well device used for this purpose is shown in Figure 5. A microfluidic device for detecting protein and glucose in artificial urine is described. The microfluidic device is constructed using alternated layers of patterned paper 510, 520, 530, 540, and 550 with hydrophilic regions conducting lateral flow and fluid-impermeable layers 515, 525, 535, and 545 with opening filled with paper disc conducting vertical flow. Figure 5 A shows the device layers that distribute two samples from inlets 511 and 512 at the top of the device into an array of test zones at the bottom of the device following the principle of lateral and vertical flow of the microfluidic device described above. The samples are distributed so that they arrange in two sets of side -by-side columns. In a first distribution step, sample applied form reservoir 512 is distributed to four termini, 521, 522, 523, and 524 of a star burst shaped distribution region 513 on paper layer 520, while sample applied from reservoir 511 flows vertically in a single aliquot to paper layer 530 where the sample from 511 is distributed from into four termini, 531, 532, 533, and 534 of a star burst shaped distribution region 514 on paper layer 530. The fluids 511 and 512 flow vertically through the paper disc such as filling the holes within tape layer 535. The resulting eight channels of fluid flow (4 from reservoir 511 and 4 from reservoir 512) are further distributed on paper layer 540 using an "elbow" or bent distribution region 515 to produce a 4X4 array of distribution zones on paper layer 550. Columns 1 and 3 contain fluid applied from reservoir 511; and Columns 2 and 4 contain fluid applied from reservoir 512. [0059] Figure 5B shows the top of the assembled device with two labeled inlets for a sample 512 and a control 511. Reagents are spotted for each colorimetric assay in the test zones on the bottom layer of the device and before the assembly of the devices. Figure 5C shows the bottom of the device with reagents for a protein assay and a glucose assay pre-spotted in the test zones. Samples that contained bovine serum albumin (BSA) and glucose are added to the top of the device. The presence of protein is indicated by a color change from yellow to blue. The presence of glucose is indicated by a color change from clear to brown. [0060] The device shown in Figures 5B-5D runs two assays (with four replicates) on a single sample, and runs the same assays simultaneously on a control sample that contains known concentrations of the two analytes (glucose and protein in artificial urine) (Figure 5B). The results of the assays appear side-by-side so that control and sample assays can be compared directly; this feature is particularly important when the samples are to be compared using telemedicine (camera phones and digital imaging software).

[0061] Another 16-well device shown in Figures 6 A-6D runs two assays in duplicate on four different samples, A, B, C, and D, simultaneously. The layout of the various layers of the micro fluidic device is shown in Figure 6A. Four reservoirs for fluids A, B, C, and D distribute four liquids through alternated paper layers 610, 620, 630, and 640 and fluid-impermeable layers 615, 625, and 635. Fluid A, for example, is distributed using distribution region 621 on paper layer 620 and bent distribution region 622 on paper layer 630, and eventually to 4 hydrophilic regions in column 1 of paper layer 640. The resultant test zones include 4 columns, each containing sample from one reservoir. Specifically, columns 1, 2, 3, and 4 of hydrophilic regions contain fluid A, B, C, and D, respectively. In this arrangement, one of the four samples can be a control sample for quantitative detection. The top of a device designed to test four different samples for two analytes is shown in Figure 6B. The bottom of the device with reagents for a protein assay and a glucose assay pre-spotted in the test zones is shown in Figure 6C, and is shown again in Figure 6D at a time 30 minutes after adding 20 μL of sample to the inlets.

[0062] Other non- limiting examples of assay reagents include other protein assay reagents, other glucose assay reagents, sodium acetoacetate assay reagents, sodium nitrite assay reagents, or a combination thereof. Other suitable assay reagents will be apparent to one skilled in the art.

Adding Regents to the Intervening Layers of Paper

[0063] In one aspect, a micro fluidic device includes layers of patterned porous, hydrophilic layers, a fluid-impermeable layer with openings deposited between every two adjacent hydrophilic layers, and material plug filling at least one of the opening. In one or more embodiments, the material plug is a disc of paper containing reagents for assays and release them on contact with fluids. This arrangement has three useful characteristics: (i) the reagents for an assay can be applied to a sheet of paper before cutting the paper into discs of the appropriate size (rather than having to spot reagents in each detection zone); (ii) the discs are protected from light and the surrounding environment (this protection prevents contamination and decomposition of light-sensitive reagents); and, (iii) paper discs containing reagents for different assays can be mixed and matched when assembling a device to enable a large variety of devices using a single design. In this approach, the reagents in the disc need to be mobile and move with the solvent front; if they are not, then they could be spotted into the test zones on the bottom of the device. [0064] In one or more embodiments, an applicator, for example, a pipette man is used to deposit assay reagents onto the hydrophilic material filling the openings within the fluid-impermeable layer. In other embodiments, an ink-jet printer, plotter, or stamp is used to deposit assay reagents onto the hydrophilic material filling the openings within the fluid-impermeable layer.

[0065] The assay reagents embedded in the paper discs include protein assay reagents, other glucose assay reagents, sodium acetoacetate assay reagents, sodium nitrite assay reagents, or a combination thereof. Other suitable assay reagents will be apparent to one skilled in the art.

[0066] In one specific embodiment, Figure 7 shows a three dimensional microfluidic device with paper discs in the opening containing reagents for detecting glucose. The device is constructed using alternated layers of patterned paper layer 710, 720, 730, and 740 with hydrophilic regions conducting lateral flow and distributing fluid from a single fluid reservoir 711 into a 4X4 test zone and fluid- impermeable layers 715, 725, and 735, with opening 712 filled with paper disc conducting vertical flow. Some of the paper discs included in the device are cut from paper that contained the reagents required for the glucose assay (i.e., glucose oxidase, horseradish peroxidase, potassium iodide, and trehalose). The remainder are not spotted with reagent and can be used as controls. The microfluidic flow within the device follows the principle of lateral and vertical flow of the microfluidic device described above. Figure 7 A shows a schematic of the device. An inlet at the top of the device distributes a sample into 16 test zones at the bottom of the device. Before assembling the device, the reagents for the glucose assay are applied to half of the paper discs (right two columns of the device) in holes 750 of layer 735 connecting the test zones in the bottom layer of paper to the rest of the device, as shown in Figure 7B. The left two columns are used as controls, or contains paper disc without added assay reagents. Figures 7C and 7D show the top and the bottom of the device respectively, before adding the sample containing glucose. Figure 7E shows the bottom of the device 30 min after adding 40 μL of a 30-mM glucose solution to the top of the device, wherein the right two columns of detection zones show change of color due to the presence of glucose in the sample. [0067] In another embodiment, the gap between the paper layers is filled with solid reagents for an application, such as bioassays, without using paper discs. The non- limiting examples of solid reagents for bioassay include solid protein assay reagents, solid glucose assay reagents, solid sodium acetoacetate assay reagents, solid sodium nitrite assay reagents, or a combination thereof. Other suitable assay reagents will be apparent to one skilled in the art.

[0068] In yet another embodiment, the opening within the fluid-impermeable layer is filled with seeds. Non-limiting examples of seeds include soybean seeds, hulls, and powdered soybean, or any other seeds that provide peroxidase to be used as a reagent.

[0069] In yet another embodiment, the opening within the fluid-impermeable layer is filled with filters to remove solid contaminants from a sample or to remove red blood cells from a blood sample. Non-limiting examples of filters include filter papers, any other filtration medium for removing solid contaminants, dextran, and any other materials that can remove red blood cells from a blood sample. [0070] In yet another embodiment, the opening within the fluid-impermeable layer is filled with salts that either give off heat or absorb heat when dissolved in fluid to provide localized temperature control. Non-limiting examples of salts that give off heat when dissolved in fluid including magnesium sulfate. Non- limiting examples of salts that absorb heat when dissolved in fluid including ammonium nitrate. This provides a mechanism for local temperature device within the microfluidic devices. In certain embodiments, the microfluidic devices containing salt that give off heat when dissolved in fluid to heat assays. In certain other embodiments, the microfluidic devices containing salt that absorb heat when dissolved in fluid to cool assays. In yet certain other embodiments, the microfluidic devices containing salt that give off heat or absorb heat when dissolved in fluid to provide heating and cooling cycles for polymerase chain reactions. [0071] In yet another embodiment, the opening within the fluid-impermeable layer is filled with gels that swell when fluid is added to them to provide contact between two layers of paper, or provide a mechanical force that activates an 'on' button. Non-limiting examples of gel include agarose, methylcellulose, and hylaronan.

[0072] In yet another embodiment, the opening within the fluid-impermeable layer is filled with magnetic particles. In the presence of a magnetic field, such magnetic particles could function as 'on' buttons in the device. Non-limiting examples of magnetic particles include iron oxide particles. In certain embodiments, the magnetic particles deposited in the openings of the fluid-impermeable layer would exert a force on one patterned paper layer and compress the paper to close a gap between paper and tape layers in the presence of an external magnet, thus resulting in a fluidic connection between two layers of paper. [0073] In yet another embodiment, the opening within the fluid-impermeable layer is filled with materials to control the fluid flow through the device. These materials include a material that is selected for its wicking properties, solid materials such as sugars that dissolve when wet; and mixtures of sugars and polymer beads, such that fluid will only flow when sugar is present, and once all the sugar is dissolved, flow will stop. Non- limiting examples of sugars include sucrose. Non- limiting examples of polymers include polystyrene.

Controlled Mixing of Fluids

[0074] In one aspect, 3-D micro fluidic devices that control the flow and mixing of fluids are described. The micro fluidic devices include layers of patterned porous, hydrophilic layers, a fluid-impermeable layer with openings deposited between every two adjacent hydrophilic layers. In this aspect, one of the hydrophilic regions within a first patterned porous, hydrophilic layer of microfluidic device includes a first fluid flowing channel and a second fluid flowing channel which merge into a combination zone. The microfluidic device further include a mixing channel, formed by one of the hydrophilic regions within a second patterned porous, hydrophilic layer, and an unfilled opening within the fluid-impermeable layer disposed between the first and second patterned porous, hydrophilic layers. The unfilled opening is aligned with the combination zone and at least a portion of the mixing channel. In this aspect, the combination zone is capable of moving from a first spaced apart position, in which the combination zone is not in fluidic contact with the mixing channel, into a second contact position, in which the combination zone is in fluidic contact with the mixing channel.

[0075] In one or more embodiments, the devices can be programmed to mix fluids in specific combinations and sequences, and thus provide platforms for assays that require ordered events. In certain embodiments, 3-D microfluidic devices are used to mix four liquids (two at a time) or more, depending on the configuration of the device, to give six distinct combinations of fluids. [0076] Many colorimetric and immunochromatographic assays require sequential reactions involving the delivery of analytes, reagents, and washing solutions. These assays necessitate either manual mixing of reagents in precise order, or mechanisms for controlling the position and rate that fluids move through a device. Three-dimensional devices may be advantageous in applications where a large number of samples need to be processed, or where a single sample need to be manipulated in a number of different ways.

[0077] Advantages of the 3-D micro fluidic devices described herein include the ability to exert precise control over the dynamics of flow and mixing of fluids in paper, and low cost. Inexpensive, disposable, single-use devices provide a new tool for applications that require multiple tests to be performed quickly at low cost, and for tests that require multiple steps where several reagents need to be combined sequentially. Suitable applications include rapid testing in doctors' offices or emergency rooms; diagnosing disease in the developing world; and home healthcare for monitoring chronic disease.

[0078] Fluids move in microfluidic channels with laminar flow and low Reynolds numbers. The consequence of this behavior is that two fluids flowing next to one another mix by diffusion, not by convection. Diffusion of molecules in laminar flow occurs across the interface of the two fluids perpendicular to the direction of the flow, such as shown in Figure 8 A. The diffusion rate can be estimated using the Stokes-Einstein equation:

_{D =} KL_

6πμR_m (1)

In this equation, D is the diffusion coefficient, μ is the viscosity of the fluid (μ ~ 1x10^" Pa-s for water at 20 ⁰C), and R_m is the radius of the molecule (for molecules of dye R_m ~ 10 A). The diffusion coefficient for small molecules is ~l^χ 10^"6 cm²/s, which means that two fluids (e.g., water and a yellow dye) in a side-by-side configuration, as shown in Figure 8 A, in a paper channel that is 1-mm wide would mix completely in ~5 min. This time scale is too slow for assays, and would require a 5 -cm long channel in a paper-based microfluidic device for thorough mixing of fluids in two dimensions.

[0079] One way to solve this slow mixing step would be to decrease the width of a lateral flow channel. This approach, however, limits the quantity of fluid that can be transported through a channel, and is practical (using current technology) with channels that are only as narrow as 500 μm.

[0080] An arrangement of the fluids that would enable rapid mixing by diffusion would be a double-layer configuration, where one fluid would flow along the top half of the channel, and the second fluid would flow along the bottom half of the channel, as shown in Figure 8B. This arrangement could minimize the distance that molecules need to diffuse to achieve mixing, but would not affect the width of the channel. For a channel that is 180 μm in height (defined by the thickness of Whatman chromatography paper 1), for example, the molecules in the fluids would diffuse across the height of the channel and mix thoroughly in ~5 s. Thus, a 1-mm long channel is sufficient for mixing fluids in a double-layer configuration. [0081] Figure 8 A and 8B illustrate the two configurations of fluids in laminar flow in a paper channel. The height of the channel (h) is defined by the thickness of the paper; the width of the channel (w) is defined by photolithography. The distance molecules need to diffuse in order to achieve complete mixing (x) is proportional to w or h, depending on the configuration of the fluids. Figure 8 A shows the typical side -by- side configuration of two fluids in a channel in laminar flow. The fluids are in contact along the longitudinal section of the channel. Molecules in one fluid diffuse into the other fluid in a direction that is perpendicular to the direction of fluid flow. In this configuration, the molecules need to diffuse across half the width of the channel to mix with the other fluid. In Figure 8B an ideal double-layer configuration for mixing two fluids by diffusion in a channel in laminar flow is shown. The distance the molecules in one fluid needs to diffuse to mix with the other fluid is half the height of the channel.

[0082] In 2-D paper-based microfluidic devices, fluids are introduced side -by- side in a channel as shown in Figure 8 A. 3-D devices, on the other hand, offer the possibility to introduce fluids in a double-layer configuration, as shown in Figure 8B, for rapid and thorough mixing.

[0083] In one aspect, a microfluidic mixer which mix two fluids in a double- layer configuration as they move through the device is described with reference to Figure 9. The microfluidic device is constructed using alternated layers of patterned paper 910, 920, with hydrophilic regions conducting lateral flow and fluid- impermeable layer 915 with opening 925 aligned between the two paper layers. The microfluidic flow within the device follows the principle of lateral and vertical flow of the microfluidic device described above. Figure 9 illustrates in oblique view (left-hand side) and in cross-section (right-hand side) a scheme for fabricating 3-D microfluidic devices out of patterned paper and tape. As shown in Figure 9, each layer of paper is patterned with microfluidic channels comprising hydrophilic paper 911 (white regions) that wick fluids in the horizontal plane and hydrophobic polymer 912 (grey regions) that control the distribution of fluid within each layer of paper. The layers of double-sided tape separate the hydrophilic channels in adjacent layers of paper from one another. A hole is punched into the layer of tape. The device fabrication methods are similar to those described above in Figure 2, but instead of inserting paper discs into the holes in the tape to provide contact between adjacent layers, no paper disc is used. To cause flow, the parts of the hydrophilic regions within the patterned paper that are in alignment with the opening 925 within the tape layers are pressed to bring them into contact with each other at the tape hole location. Specifically, as shown by Figure 9, the hydrophilic region 930 above the hole within the tape is pressed down through the hole so that the hydrophilic region 930 is in direct contact with hydrophilic region 940. The parts of hydrophilic regions of paper in alignment with the hole are pressed to close the 100 μm gap that is created by the tape. The pressed hydrophilic regions contact one another and enable fluids to wick from one layer of paper to the next.

[0084] In certain embodiments, the layers are selected so that the surface tension of the liquid in the channel region prevents the fluid from flowing through the void created by the unfilled aperture in the separating layer. This can be accomplished by controlling the dimensions (e.g., height and diameter of the aperture) and the materials selected for the hydrophobic wall, hydrophilic porous region and separating layer. In certain specific embodiments, more than one fluid-impermeable layer with identical unfilled-openings are disposed between two adjacent patterned porous, hydrophilic layers to provide a wider gap between the two adjacent hydrophilic layers to limit fluidic flow between the two hydrophilic layers. [0085] In one embodiment, a 3-D paper-based microfluidic mixer designed for mixing fluids quickly and completely in a short fluidic path in paper is described with reference to Figure 1OA shown in oblique view (left-hand side) and cross- section (right-hand side). The 3-D mixer has a top layer of paper 1000 with two microfluidic channels 1010, 1012 leading into a central combining zone 1014. As shown in Figure 1OB and 1OC, the two fluids meet in this zone 1014 and form an interface 1018 that is easily discernable in Figure 1OB and 1OC. The direction of the interface is dictated by the line that forms when the two fluids meet as illustrated by 1018. The 3-D microfluidic mixer also include a second layer of paper 1020 that wicks the fluids from the combining zone 1014 into a mixing channel 1022 as shown in Figure 1OA and 1OC. A layer of tape 1024 containing a hole is disposed between the two layers of patterned paper. As shown in Figure 1OA, the part of the hydrophilic region within the top layer which forms the combining zone is pressed down to provide contact with the hydrophilic region in the bottom layer to optimize fluids to wick between paper layers.

[0086] Fluids mix in the 3-D microfluidic mixer are mixed by the mechanism shown in Figure 8B, where they interact with one another over the 180 μm thickness of paper. In order to provide a better visual presentation of the mixing event, a thick 3-D microfluidic device (1.5 mm), instead of the typical 180-μm thick device, is used (shown in Figure 10C). The microfluidic mixer is assembled using a top layer of patterned paper (180-μm thick), a bottom layer of patterned blotting paper (1.5- mm thick), and a tape layer with a hole in between the two paper layers. The thick blotting paper allows for easy visualization of the fluids as they move through the 3- D combining zone (Figure 10C). Figure 1OC shows a longitudinal section of the 3- D mixer and the distribution of yellow and blue dyes as they wick through the device from the combining zone into the mixing channel. [0087] Figure 10 also demonstrates the mechanism of mixing in 3-D microfluidic mixer. Figure 1OA shows oblique and cross-sectional views of a mixer showing the theoretical movement of fluid (blue and yellow aqueous solutions). The blue and yellow solutions form an interface in the combining zone and become layered as they exit the combining zone and enter the mixing channel. Figure 1OB is a top view of the mixer showing the interface between blue and yellow solutions. The solutions are 1 mM erioglaucine (blue) and ImM tartrazine (yellow). Figure 1OC is a cross-sectional view of a mixer comprised of Whatman filter paper 1 (180 μm thick) layered on top of tape (100 μm thick), and connected to a bottom layer of blotting paper (1.5 mm thick). The thick blotting paper is used to facilitate observations of fluid dynamics in the mixer. The photograph in Figure 1OA highlights the double-layer configuration of the blue and yellow solutions as they mix with each other. In this embodiment, the orientation of the mixing channel is perpendicular to the interface of the two fluids within the combining zone. [0088] In another embodiment, a microfluidic mixer that controls the mixing of two fluids is described. As indicated above, the orientation of the mixing channels in the bottom layer of paper in relation to the interface within the combining zone dictates whether fluids mix in the combining zone, or whether they exit the zone with parallel laminar flows. When the mixing channel is parallel to the interface, for example, the fluids form an interface in the combination zone, but do not mix in the mixing channel. Instead, the fluids exit the mixing channel in parallel laminar flows and fill a detection region with well-defined separation between the fluids. In contrast, when the mixing channel is perpendicular to the interface, the fluids become layered on top of one another as they exit the combining zone and enter the mixing channel, resulting in a well-mixed fluid. Since the fluids become layered in the 180-μm thick paper, they quickly mix as they move through the mixing channel. The extent of fluidic mixing in the 3-D microfluidic mixer is independent of the length of the mixing channel when the mixing channel has a length of more than 1 mm.

[0089] Figure 11 illustrates the effect of the orientation of the mixing channel relative to the interface on fluid mixing. Two 3-D microfluidic mixers as shown in Figure 1 IA-11C and 1 ID-I IF that direct two fluids into a single mixing channel are compared. In the microfluidic mixer shown in Figures 1 IA - 11C, the mixer includes patterned paper layer 1110 and 1120, as well as a tape layer 1115 disposed between the two paper layers. Top paper layer 1110 include two hydrophilic channels 1111 and 1112, which merge into a combining zone 1113. Two liquids (blue dye and yellow) are deposited in channels 1111 and 1112, and an interface 1114 of the two fluids (blue dye and yellow) within the combining zone 1113 is shown in Figure 11C. An aperture 1116 in the tape layer 1115 is aligned with combining zone 1113 and is also in alignment with mixing channel 1117 in paper layer 1120. The mixer is pressed so that combining zone 1113 is pressed down into aperture 1116 and is in contact with mixing channel 1117. In this case, the interface 1114 is parallel to the direction of the mixing channel. Thus this mixer as shown does not lead to efficient mixing of fluids.

[0090] In the microfluidic mixer shown in Figures 1 ID - 1 IF, the mixer includes patterned paper layer 1130 and 1140, as well as a tape layer 1135 disposed between the two paper layers. Top paper layer 1130 include two hydrophilic channels 1131 and 1132, which merge into a combining zone 1133. Two liquids (blue dye and yellow) are deposited in channels 1131 and 1132, and an interface 1134 of the two fluids (blue dye and yellow) within the combining zone 1133 is shown in Figure 1 IF. An aperture 1136 in the tape layer 1135 is aligned with combining zone 1133 and is also in alignment with mixing channel 1137 in paper layer 1130. The mixer is pressed so that combining zone 1133 is pressed down into aperture 1136 and is in contact with mixing channel 1137. In this case, the interface 1134 is perpendicular to the direction of the mixing channel. Thus in contrast, the two fluids mix readily as shown in Figures 1 ID - 1 IF according to the mechanism shown in Figure 8B and in the example shown in Figure 1OC. Figures 1 IA and 1 ID are schematics of the three layers in the devices. The difference between the two mixers is the orientation of the mixing channel in the bottom layer with respect to the interface of the fluids in the top layer. In mixer 1 IA, the bottom mixing channel runs parallel to the interface. In the mixer shown in Figure 1 ID, the mixing channel runs perpendicular to the interface. Figures 1 IB and 1 IE are photographs of the mixers (top and bottom) after wicking blue and yellow aqueous dyes into the channels. In Figure 1 IB, the fluids do not mix as they are wicked down the mixing channel. In Figure 1 IE, the fluids mix efficiently. Figures 11C and 1 IF are the same images as in Figures 1 IB and 1 IE respectively, with enhanced contrast and brightness to highlight the differences in mixing between the two devices. In Figure 1 IE, the color of the collection zone is uniform, indicating that the two dyes are mixed efficiently. In 11C, the collection zone shows distinct areas of blue, green and yellow, indicating that this design does not lead to efficient mixing. [0091] In another embodiment, a 3-D microfluidic mixer to control the mixing of more than two fluids is described. In yet another embodiment, a 3-D microfluidic mixer that mix four fluids, two by two, to give all six possible combinations of fluids is described with reference to Figures 12A and 12B. The microfluidic mixers are assembled with a plurality of layers of patterned paper and a tape layer with a hole disposed between every two adjacent paper layers. As described above, the hydrophilic regions within the combining zone are pressed to enable contacts between the two paper layers and optimize vertical microfluidic flow. This design demonstrates the flexibility of the 3-D mixer. The channels in the top layer do not necessarily have to enter the combining zone when oriented 180° from one another; they can enter from any orientation. As shown in Figure 12A and 12B, the mixing channels for fluid A and B, A and C, A and D, B and C, B and D, C and D are perpendicular to their respective interfaces, thus allowing efficient mixing of the fluids. As indicated in Figure 12, fluid A is yellow dye; fluid B is blue dye; fluid C is water; and fluid D is red dye.

[0092] Figures 12C and 12D show a device that does not incorporate 3-D mixers. The microfluidic mixer are assembled with a plurality of layers of patterned paper and a tape layer with a hole disposed between every two adjacent paper layers. As described above, the hydrophilic regions within the combining zone are pressed to enable contacts between the two paper layers. This device illustrates the well- defined interface created when two fluids meet in the combining zone. This interface can be maintained through several layers of paper (not shown), so long as the outlet channels are not perpendicular to the interface of the two fluids. [0093] Figures 12A-12D shows the details of two three-dimensional microfluidic mixers for mixing or combining multiple fluids simultaneously. Both devices have four input zones for adding fluids to the device, though other three- dimensional microfluidic mixers that could accommodate even more fluids are contemplated. The first device (Figures 12A and 12B) mixes fluids, two by two, in all six possible combinations and delivers the products into six output wells on the bottom of the device. The second device (Figures 12C and 12D) combines the fluids — but does not mix them — and delivers the combined fluids into five output wells on the bottom of the device. The sixth combination is left off the device for simplicity in design. Schematics of the layers used in the devices are shown in (Figures 12A and 12C). As shown in Figure 12A, the microfluidic mixer includes patterned paper layers 1210, 1220, 1230, 1240, 1250 and tape layers 1215, 1225, 1235, 1245. Fluid A and D are deposited in hydrophilic region 1211 and 1212, respectively. Fluid A (yellow dye) flows through hole 1213 filled with a paper disc in tape layer 1215 into channel 1216 in paper layer 1220. In a similar manner, Fluid D (red dye) flows through hole 1214 filled with a paper disc in tape layer 1215 into channel 1217 in paper layer 1220. Channels 1216 and 1217 merge into a combining zone 1218, which defines an interface 1219 of the two fluids. The combining zone 1218 is in alignment with an unfilled aperture 1226 within tape layer 1225. The aperture 1226 is in alignment with a mixing channel 1231 within paper layer 1230. The mixer is pressed so that combining zone 1218 is pressed down into unfilled aperture 1226 and is in contact with mixing channel 1231. In this case, the interface 1219 is perpendicular to the direction of the mixing channel 1231 thus allows efficient fluid mixing. The mixed fluid then flows through aperture 1236 filled with a paper disc within tape layer 1235, hydrophilic region 1241 within paper layer 1240, aperture 1246 filled with a paper disc within tape layer 1245, and eventually reaches hydrophilic region 1251 within paper layer 1250, resulting a well-mixed mixture of fluids A and D. The four input wells at the top of the devices are labeled A, B, C and D. The six output wells on the bottom of the device are labeled AB, AC, AD, BC, BD and CD. Following the similar principle, fluids A and B, A and C, B and C, B and D, C and D are also mixed in the mixer as shown in Figure 12A and Figure 12B. As a comparison, the second device (Figures 12C and 12D) combines the fluids — but does not mix them — and delivers the combined fluids into five output wells on the bottom of the device. Figures 12B and 12D show the top and bottom of the devices after allowing water and red, yellow, and blue aqueous dyes to wick through the device. In Figure 12B the dyes mixed with each other as they are wicked through the device to yield six distinguishable colors: red (red dye + water), orange (red dye + yellow dye), yellow (yellow dye + water), green (yellow dye + blue dye), blue (blue dye + water) and purple (blue dye + red dye). In Figure 12D, the four initial colors remain unchanged as they are distributed into the five output wells, even though they are in direct contact with another color. [0094] In yet another embodiment, a 3-D microfluidic mixer could be incorporated into any 3-D microfluidic devices. In yet another embodiment, a 3-D microfluidic mixer requires two layers of patterned paper and one layer of tape with holes disposed between the two paper layers, and occupies ~1 mm x 1 mm x -500 μm of space on a device. An important feature that contributes to mixing is the orientation of the mixing channel to the interface of the two fluids formed in the combining zone. The remaining portions of the 3-D mixer can be modified substantially without affecting its ability to mix fluids.

[0095] The advantages of the described embodiments include the ability to control how fluids interact with one another in the context of an inexpensive, disposable, autonomous paper-based device should enable many applications that require precise handling of fluids. Dipsticks are a ubiquitous example of simple devices that incorporate fluid handling and mixing in a lateral flow format. Three- dimensional microfluidic devices provide a greater level of control than do dipsticks because they enable simultaneous mixing of several fluids, and because they can be programmed to mix fluids in a precise pattern. Other advantages include low cost because the devices are fabricated from paper and tape, and ease of assembly without the need for specialized equipment. In addition, the devices can be used to process many fluids simultaneously.

On and Off Buttons for Programming Fluid Flow

[0096] In one aspect, a microfluidic flow control device with an "on" button is described. The microfluidic flow control device includes a plurality of porous hydrophilic layers including a first and second patterned porous hydrophilic layer which define a first flowing channel and a second flowing channel, respectively. The microfluidic flow control device further includes a fluid-impermeable layer disposed between every two adjacent patterned porous, hydrophilic layers. The fluid-impermeable layer disposed between the first and the second patterned hydrophilic layers contains one unfilled opening that is aligned with at least portions of the first and second fluid flowing channel. At least a portion of the first flowing channel is capable of moving from a first spaced apart position, in which the first flowing channel is not in fluidic contact with the second flowing channel, into a second contact position, in which the first flowing channel is in fluidic contact with the second flowing channel. [0097] In another aspect, a microfluidic flow control device with an "off button is described. The microfluidic flow control device includes a first and second patterned hydrophilic layers with a fluid-impermeable layer disposed in between. The fluid-impermeable has opening and a hydrophobic plug is disposed in one of the openings. The hydrophobic plug includes a low-melting-point hydrophobic material capable of melting and flowing at a pre-selected temperature. The second patterned hydrophilic layer has a hydrophilic region which is in alignment with the hydrophobic plug. In use, the hydrophobic plug is heated to melting and the melted material flows into the hydrophilic region below to obstruct microfluidic flow within the hydrophilic region.

[0098] In one embodiment, a microfluidic flow control device programs the fluid flow within the microfluidic devices made out of patterned paper layers and tape layers using single-use 'on' or 'off buttons. The buttons allow fluids to wick either through a channel ('on' button), or stop the movement of fluid in a channel ('off button). The buttons are formed in three-dimensional (3-D) microfluidic devices. The layered structure of 3-D devices allows for the design of buttons, which add significant function to these devices, without adding complexity to the structure of the device.

[0099] In some embodiment, the 'on' and 'off buttons are formed by compression, and involve either pressing parts of two hydrophilic regions within the patterned paper layer ('on'), or inducing hydrophobic, low melting point wax embedded within the holes of the tape layers into a hydrophilic channel (Off). The buttons are simple and easy to use, and can be compressed for programming the fluidic devices for a variety of functions.

[0100] In other embodiments, microfluidic devices contain the 'on' and 'off buttons which can be configured with fluidic channels to form AND, OR, NAND, NOR, NOT, XOR, and XNOR logic gates, and that these logic gates can be used as the basis for an exceedingly simple, prototype, single use fluidic computer. In another embodiment, a microfluidic de-multiplexer is also described. [0101] Programmable microfluidic devices made out of paper have the desirable feature of allowing the user to have greater control over the function of the device. The function of most microfluidic devices is pre-determined by the design of the device: fluids will flow through the channels in a specific sequence. With a programmable device, the user can choose which channels or areas of the device should be filled with fluid and which areas should not. This can be useful in situations where only a limited quantity of sample is available, or where a generic set of channels (and device) need to be programmed to perform a certain function over another. With programmable devices, the user can also 'turn on' a channel at any arbitrary time. This could be useful in situations where reagents need to be mixed in a timed sequence.

[0102] In other embodiments, three-dimensional microfluidic flow control devices with logic gates based on "on" or "off" buttons are described. The "on" and "off buttons in 3-D microfluidic devices can be used to make very complex devices. The logic gates can also be useful for programming specific function into the devices.

[0103] In yet another embodiment, microfluidic flow control devices with 'on' or 'off buttons are described with reference to Figure 13. The microfluidic flow control device is constructed using alternated layers of patterned paper with hydrophilic regions conducting lateral flow and fluid-impermeable layers with opening. When the layers of patterned paper and tape with unfilled holes are stacked, there is a small gap within the holes in the tape layer between the two adjacent patterned layers of paper that is created due to the thickness of the tape. Thus the parts of the two hydrophilic regions within the two adjacent paper layers in alignment with the hole or opening are not in contact with one another. As a result, vertical fluidic flow from one layer of paper to the other is not optimized unless this gap is filled with a small disc of paper or some other hydrophilic material, or if the gap is closed by pressing the part of the hydrophilic region in alignment with the opening using mechanical force. This mechanical mechanism creates an 'on' button, since vertical fluidic flow is optimized when the layers of paper are compressed and are in contact. The microfluidic flow control device in Figures 13A-13D illustrates this design. The input from the user is the compression of the layers. The output is fluid flowing into a specific channel. The 'on' buttons within the microfluidic flow control device can be pressed at any time, both before the device is used, and while the device is being used. [0104] Figure 13A is a schematic for the fabrication of 'on' buttons. The microfluidic flow control device includes paper layer 1310, 1320, 1330 and tape layer 1315, 1325. Layers of paper are patterned using photoresist. Layers of tape are punched to created holes or openings within. The layers of patterned paper and tape are aligned and stacked. Holes such as 1326 filled by pieces of paper are illustrated by grey ovals in Figure 13 A. Holes 1322 and 1332 are without any fillings and are illustrated by white ovals in Figure 13 A. If the holes in the tape are not filled with a paper disc (white ovals), then there will be a small gap between the layers of paper and fluids will not efficiently wick from one layer of paper to the other through the unfilled holes. When the 'on' button is compressed, i.e., when the parts of hydrophilic regions in alignment with the unfilled opening are compressed, the gap is closed, the parts of the hydrophilic regions within two adjacent paper layers are in contact, and fluids can wick efficiently from one layer of paper to the other vertically. Two fluids are deposited in the hydrophilic regions within the top paper layer 1310 as indicated in Figure 13C. Figure 13B shows an assembled device with two 'on' buttons and button 2 is activated by compression (as indicated by the arrow). Specifically, the two fluids are deposited into hydrophilic regions 1311 and 1312 within paper layer 1310, which reach hydrophilic regions 1321 and 1331 within the bottom paper layer 1330, respectively, by capillarity through the device. Hydrophilic region 1333 within paper layer 1320 is in alignment with unfilled aperture 1332, which is in alignment with a portion of hydrophilic region 1331. The microfluidic flow control device is pressed so that region 1333 is pressed into aperture 1332 and is in contact with a portion of channel 1331. Thus the fluid deposited at 1312 will flow through pressed aperture 1332 and eventually reach hydrophilic region 1314 within the top paper layer 1310, as shown by Figure 13D. On the other hand hydrophilic region 1323 within paper layer 1320 is in alignment with unfilled aperture 1322, which is in alignment with a portion of hydrophilic region 1321. The region 1323 is not pressed and thus is not in contact with any portion of channel 1321. As a result, the fluid deposited at 1311 will not flow through un-pressed aperture 1322 and can not reach hydrophilic region 1313 within the top paper layer 1310, as shown by Figure 13D. Figure 13C and 13D show that a fluid is deposited at two hydrophilic regions, and fluid only reaches the output No 2 where the 'on' button is compressed. Also shown in Figure 13D are two photographs of the cross sections of the uncompressed button 1 and compressed button 2. In the photograph of the compressed button, parts of hydrophilic regions from two layers of paper are in contact with each other and the vertical flow occurred efficiently. In comparison, the photograph of the uncompressed button shows that vertical flow did not occur efficiently.

[0105] In one aspect, a microfluidic flow control device with an 'off button is described. The microfluidic flow control device is constructed using alternated layers of patterned paper with hydrophilic regions conducting lateral flow and fluid- impermeable layers with opening containing low-melting-point hydrophobic materials. In one embodiment, the small gap between the layers of paper that is created due to the thickness of the tape is filled with a small paper disc soaked in a low melting point wax. Suitable waxes include candle wax, cheese wax, packaging wax, paraffin wax, or other waxes. In some embodiments, waxes that melt below 200 ⁰C are used. In other embodiments, waxes with melting points below 100⁰C are used. In a specific embodiment, bayberry wax (mp = 45-50 ⁰C) is used. When the paper disc containing wax is compressed with a hot glass rod, the wax melts and fills the channel beneath it, creating a hydrophilic barrier in the hydrophilic channel that is at least in partial alignment with the paper disc, thus blocking fluid from wicking across the channel (Figures 13E- 13G). Figure 13E is a schematic for the fabrication of microfluidic flow control device with 'off buttons. This microfluidic flow control device includes patterned paper layer 1340 and 1350 with a tape layer 1345 disposed in between. Two fluids are deposited into hydrophilic regions 1341 and 1342 within paper layer 1340, respectively. Through microfluidic flow, the two fluids will reach hydrophilic regions 1349 and 1348, respectively. Aperture 1346 within tape layer 1345 is filled with hydrophilic wax paper and is in alignment with hydrophilic region 1348 within paper layer 1350, and is also in alignment with area 1352 within paper layer 1340. When area 1352 is compressed with a hot object, the wax within aperture 1346 melts, flow into channel 1348 and block the channel. On the other hand, aperture 1347 within tape layer 1345 is filled with hydrophilic wax paper and is in alignment with hydrophilic region 1349 within paper layer 1350, and is also in alignment with area 1351 within paper layer 1340. When area 1351 is not compressed with a hot object, the wax within aperture 1347 does not flow. Thus, fluid deposited at region 1341 flow through channel 1349 and reach hydrophilic region 1343 within the paper layer 1340. Figure 13F shows this device with the two 'off buttons', i.e., apertures 1346 and 1347. Button 2 (aperture 1346) is activated by compressing it with a hot glass stirring rod. Figure 13G shows that two fluids are added to regions 1341 and 1342 and only fluid deposited at 1341 reaches the output 1343 at right where the "off button is not activated.

[0106] In one or more embodiments, 3-D microfluidic flow control devices with both "on" and "off buttons are described. In other embodiments, 3-D microfluidic flow control devices with both "on" or "off are assembled to create the functions of the logic gates such as AND, OR, NOT, NAND, NOR, XOR, or XNOR. Such microfluidic devices and their logic functions are described in Examples. [0107] In addition, the basic logic gates described above could be assembled to form a half adder, as well as other combinations to perform digital computation. [0108] In certain embodiments of the above-mentioned aspects, porous, hydrophilic layers include any hydrophilic substrate that wicks fluids by capillary action. In one or more embodiments, the porous, hydrophilic layer is paper. Non- limiting examples of porous, hydrophilic layers include chromatographic paper, filter paper, nitrocellulose and cellulose acetate, cellulosic paper, filter paper, paper towels, toilet paper, tissue paper, notebook paper, Kim Wipes, VWR Light-Duty Tissue Wipers, Technicloth Wipers, newspaper, any other paper that does not include binders, cloth, and porous polymer film. In general, any paper that is compatible with the selected patterning method may be used. In certain embodiments, porous, hydrophilic layers include Whatman chromatography paper No. 1.

[0109] In certain embodiments of the above-mentioned aspects, the hydrophilic layer is patterned following the procedures described in PCT/US07/081848. In certain embodiments, the hydrophilic paper is soaked in photoresist, and photolithography is used to pattern the photoresist to form the barriers following the procedures described in PCT/US07/081848. Other hydrophilic material such as nitrocellulose and cellulose acetate are commonly used and well-known membranes for their use in fluid diagnostics, but are not compatible with solvents typically used in photolithography. So other methods would be more suitable for patterning them. In addition, the hydrophilic layer and the hydrophobic barrier regions could be prepared using materials that are compatible with the testing conditions, e.g., temperature, pH, and/or ionic strength.

[0110] In certain embodiments of the above-mentioned aspects, the photoresist used for patterning porous, hydrophilic material include SU-8 photoresist, SC photoresist (Fuji Film), poly(methylmethacrylate), nearly all acrylates, polystyrene, polyethylene, polyvinylchloride, and any photopolymerizable monomer that forms a hydrophobic polymer.

[0111] In certain embodiments of the above-mentioned aspects, the fluid- impermeable player is a sheet that is not soluble in the fluid of the microfluidic device and that provides the desired level of device stability and flexibility. In one or more embodiments, the fluid-impermeable player is a plastic sheet. In certain embodiments, the fluid-impermeable player is an adhesive sheet or tape. Non- limiting examples of fluid-impermeable player includes Scotch^® double-sided carpet tape, water-impermeable barriers include 3M Double Sided Tape, Tapeworks double sided tape, CR Laurence black double sided tape, 3M Scotch Foam Mounting double-sided tape, 3M Scotch double-sided tape (clear), QuickSeam splice tape, double sided seam tape, 3M exterior weather-resistant double-sided tape, CR Laurence CRL clear double-sided PVC tape, Pure Style Girlfriends Stay-Put Double Sided Fashion Tape, Duck Duck Double-sided Duct Tape, and Electriduct Double- Sided Tape. In certain specific embodiments, double-sided tape is used as the fluid- impermeable layer. Double-sided tape adheres to two adjacent layers of patterned paper and can bind to other components of the microfluidic device. It is impermeable to water, and isolates fluid streams separated by less than 200 μm. In addition, it is also sufficiently thin to allow adjacent layers of paper to contact through holes punched in the tape when compressed. It can easily separate from the paper to which it adheres and thus allow disassembly of stacked devices and it is inexpensive and widely available.

[0112] In certain embodiments of the above-mentioned aspects, a heat-activated adhesive can be used to seal the fluid-carrying layers together. Indeed, any fluid- impermeable material that can be cut and stuck to the pattern hydrophilic layers can be used. In addition, it is also possible to use the same material that is used to pattern the paper layers to stick the layers of paper together. In one or more embodiments, a layer of photoresist is disposed between two adjacent patterned porous, hydrophilic layers.

[0113] In certain embodiments of the above-mentioned aspects, the porous, hydrophilic material filling the aperture or opening could be the same material as the porous, hydrophilic layers or different material. Non- limiting examples of porous, hydrophilic material include chromatographic paper, filter paper, nitrocellulose and cellulose acetate, cellulosic paper, filter paper, paper towels, toilet paper, tissue paper, notebook paper, Kim Wipes, VWR Light-Duty Tissue Wipers, Technicloth Wipers, newspaper, and any paper that does not include binders. [0114] In one or more embodiments, hydrophobic barriers can be provided in porous, hydrophilic layers using patterning methods that require relatively little equipment, can be performed in nearly any laboratory, and are versatile enough for making many types of patterns and multiple copies of each pattern. Because of the relative ease of fabrication and the ready availability of inexpensive components, bioassay devices can be formed with significantly lower cost than conventional devices such as dipsticks, and thus can be useful, among other things, for detecting disease in remote locations, where resources are limited, and where cost and portability of the devices are useful.

[0115] As noted above, in order to fabricate microfluidic channels in porous, hydrophilic media, such as, but not limited to paper, the patterned hydrophobic polymer generally extends substantially through the entire thickness of the paper in order to confine the liquid within desired areas. This constraint limits the methods that can be used in practice for patterning paper. For example, printing methods using standard inks may not be suitable for making channels in paper because currently available inks are designed to adhere to the surface of paper, not to absorb into the paper. However, it can be envisioned that certain inks could be designed in order to absorb substantially through the thickness of paper. [0116] The composition of the porous medium, e.g., paper, may also limit the patterning methods that can be used in practice. For example, paper typically includes intertwined fibers that are oriented in the x- and y-axes of a sheet of paper and that are stacked on top of one another in the z-direction. The result of this arrangement is the increased spreading of liquids in the x-, y-plane compared to the z-direction, which leads to blurring of the features that were patterned. Appropriate choices of monomers, polymers, and solvents can be made to overcome these properties of paper, and to enable the patterning of distinct features that pass through the entire thickness of paper.

[0117] Some useful methods for patterning paper are based on photolithography, and can be implemented either in a cleanroom or in a laboratory. Cleanroom photolithography works well for making highly defined patterns in paper, but is relatively expensive and slow, possibly making its commercial viability somewhat limited. Other methods, such as laboratory photolithography and soft lithography (also called micro-contact printing), eliminate the need for a cleanroom, and have only modest requirements for equipment and expertise on the part of the manufacturer, while still producing high-quality devices. Laboratory photolithography is useful for making patterns, with well-resolved channels and small feature sizes. Soft lithography is typically less expensive than the photolithographic-based methods, and is useful for making multiple copies of the same pattern relatively quickly.

[0118] For some applications, the feature sizes in paper microfluidic devices are relatively large (e.g., with channels about 1-2 mm wide), so a lower resolution, but faster stamping technique will be sufficient. For other applications, micron-sized features will be used, and so an inexpensive, but higher-resolution method will be useful. For most applications the devices will have features with sizes less than 1.5 mm. It should be recognized however that a wide variety of channel shapes and sizes can be formed using the systems and methods described herein. In both kinds of applications, it is desirable that the patterning method be inexpensive, have high- throughput, and not require a highly technically skilled user to manufacture. [0119] In one or more embodiments, hydrophobic patterns are generated using cleanroom photolithography. For example, chromatographic paper is patterned photolithographically using photoresist to create hydrophobic barriers within the paper. [0120] The following example are provided to illustrate the invention, which is not intended to be limiting of the invention, the scope of which is set forth in the claims which follow.

Example 1. Patterning paper

[0121] The layers of patterned hydrophilic paper are provided following the procedures described in PCT/US07/081848. The patterns for the paper-based microfluidic devices were designed using the layout editor CIe Win. Example 2. Microfluidic flow control devices with logic gates

[0122] Microfluidic flow control devices with logic gates are assembled using layers of patterned hydrophilic paper and layers of fluid-impermeable medium with filled or unfilled openings described above.

[0123] Microfluidic flow control devices with AND and OR Logic Gates are described. These logic gates can be designed in 3-D paper devices using the 'on' button. As shown in Figure 14, for both logic gates, two inputs A and B were designed and contain 'on' buttons designated by circles in the top layer of patterned paper (14A and 14E). When the inputs are compressed, they activate specific channels in the network.

[0124] The logic gates were designed with a source, where fluid is constantly supplied, two inputs A and B that are buttons, and an output, which is a well that either will fill with fluid if the input yields a true value (1), or will not fill with fluid if the input yields a false value (0). Four logic gates were placed on each device so that a complete truth table could be demonstrated for each logic function. The design of the AND gate comprises two 'on' buttons in series. In this configuration, fluid will flow from a source, through the network of channels and into the output, only if both the buttons are compressed. The design of the OR gate comprises two 'on' buttons in parallel. In this configuration, fluid will flow from the source and into the output well, if one or both buttons are compressed.

[0125] Figure 14 shows a design for AND and OR logic gates. Figure 14A is a schematic of the layers in the AND logic gate. Four logic gates were designed on each device so that a complete truth table could be shown for each function. Figure 14B shows the assembled device after compressing the inputs in all possible combinations and adding an aqueous blue dye (1-mM erioglaucine) to the sources. In the described embodiment, the fluid takes 10 minutes to flow from the source to the output. Figure 14C is the AND gate truth table. Compressing A or B is equivalent to an input of 1 , and not compressing is equivalent to an input of 0. An output of 1 is equivalent to fluid filling the output well. An output of 0 is evident by the lack of fluid in the output well. Figure 14D shows a schematic of the cross section of the AND logic gate. The two 'on' buttons are arranged in series so that fluid will reach the output only if both buttons are compressed. Four cross sections are shown, one for each possible combination of inputs.

[0126] Figure 14E is a schematic of the layers in the OR logic gate, and Figure 14F shows the assembled device after compressing the buttons and adding blue dye to the sources. In the described embodiment, the fluid takes 10 minutes to flow from the source to the output. Figure 14G is the OR gate truth table, and Figure 14H shows a schematic of the cross section of the OR logic gate. The two 'on' buttons are arranged in parallel so that fluid will reach the output if either button is compressed.

[0127] A microfluidic flow control device contains the function of a NOT logic gate is described with reference to Figure 15. The 'on' button only allows for the design of AND and OR gates. A NOT gate requires an 'off button. When compressed, an 'off button blocks a channel and prevents fluid from flowing into it. The NOT gate only has one input, in this case one 'off button. When the button is compressed, fluid is blocked from reaching the output. Figure 15 shows a NOT gate. Figure 15A illustrates the schematic of the layers in the device. Two NOT gates were designed on the device to show the two possible combinations of inputs. Figure 15B shows the top of the device after compressing the button in second NOT gate and adding fluid to the source. Fluid only reaches the output if the button is not compressed. Figure 15C shows the bottom of the device. In the second NOT gate, the fluid is blocked by wax in the channel. Figure 15C shows a schematic of the cross section of the not gate. Figure 15E shows the NOT gate truth table. [0128] Microfluidic flow control devices contain the function of NAND and NOR logic gates are described with reference to Figure 16. The NAND and NOR logic gates are the inverse of the AND and OR logic gates. The NAND gate can be achieved by designing two 'off buttons in parallel, such that fluid will reach the output, unless both buttons are compressed. The NOR gate comprises two 'off buttons in series, such that fluid will reach the output, only if both buttons are not compressed. Figure 16 shows how NAND and NOR gates can be made. In Figure 16A we show a schematic of the layers in the NAND logic gate. Figure 16B shows the assembled device after compressing the inputs in all possible combinations and adding blue dye to the sources. In the described embodiment, the fluid takes 5 minutes to flow from the source to the output. Figure 16C is the NAND gate truth table. In Figure 16D we show the schematic of the cross section of the NAND logic gate.

[0129] Figure 16E is a schematic of the layers in the NOR logic gate. The assembled device after compressing the buttons and adding blue dye to the sources is shown in Figure 16F. In the described embodiment, the fluid takes 3 minutes to wick from the source to the output. Figure 16G is the NOR gate truth table and Figure 16H shows a schematic of the cross section of the NOR logic gate. [0130] Microfluidic flow control devices contain the function of XOR and XNOR logic gate is described with reference to Figure 17. The XOR gate can be achieved by a combination of a NAND and an OR gate in series. The XNOR is the combination of a NOR and an AND gate in parallel. Figure 17 show the schematics and implementations of XOR and XNOR gates. Figure 17A is a schematic of the layers in the XOR logic gate. The XOR gate comprises a NAND gate and an OR gate in series. Figure 17B shows the assembled device after compressing the inputs in all possible combinations and adding blue dye to the sources. In the described embodiment, the fluid takes from 5 to 20 minutes to wick from the source to the output. Figure 17C shows the XOR gate truth table, and Figure 17D is a schematic of the cross section of the XOR logic gate.

[0131] Figure 17E is a schematic of the layers in the XNOR logic gate. The XNOR logic gate is comprised of a NOR gate and an AND gate in parallel. Figure 17F shows the assembled device after compressing the buttons and adding blue dye to the sources. In the described embodiment, the fluid takes from 3 to 20 minutes to wick from the source to the output. Figure 17G is the XNOR gate truth table, and Figure 17H is a schematic of the cross section of the XNOR logic gate. Example 3. Microfluidic flow control devices as de-multiplexer

[0132] Microfluidic de-multiplexer is assembled using layers of patterned hydrophilic paper and layers of fluid-impermeable medium with filled or unfilled openings described above.

[0133] One of the applications of 'on' and 'off buttons is the ability to direct fluids into specific channels or zones. A microfluidic de-multiplexer based this principle is described with reference to Figure 18 which includes one inlet for a fluid and six different outputs. The output is controlled by an 'on' button that is placed next to each output. Figure 18 shows a fluidic de -multiplexer. This device can be used to direct fluid from a single source into any number of outputs, in this case six outputs. The device is comprised of a source for fluid, six 'on' buttons, and six output wells. The output is determined by the buttons. Figure 18A is a schematic of the layers in the demultiplexer, which is assembled using layers of patterned paper with defined hydrophilic regions and layers of tape with holes unfilled or filled with discs of paper. The unfilled holes within the devices function as "on" buttons described above. Figure 18B shows an assembled de-multiplexer after compressing the first button (indicated by the arrow) and adding fluid to the source. In the described embodiment, only the first output is filled with fluid even after 30 minutes. Figures 18C - 18J are pictures of de -multiplexers after compressing different buttons or combinations of buttons and adding fluid to the source. Each picture shows a different device.

[0134] A similar approach can be used to construct a multiplexer that has several inputs and allows the user to choose which one will be directed to a single output.

Claims

1. A three-dimensional microfluidic device comprising: a plurality of patterned porous, hydrophilic layers including at least a first and second patterned porous, hydrophilic layers, wherein each said patterned porous, hydrophilic layer comprises a fluid-impermeable barrier that substantially permeates the thickness of said patterned porous, hydrophilic layer and defines a boundary of one or more hydrophilic regions within each said patterned porous, hydrophilic layer; a fluid-impermeable layer disposed between said first and second patterned porous, hydrophilic layers, wherein said fluid-impermeable layer comprises one or more openings; and a porous hydrophilic medium disposed in said opening of said fluid-impermeable layer ; wherein said hydrophilic medium is in fluidic contact with at least one of said hydrophilic regions within each of said first and second patterned porous, hydrophilic layers.

2. The three-dimensional microfluidic device of claim 1, wherein said porous, hydrophilic medium comprises paper.

3. The three-dimensional microfluidic device of claim 1, wherein said porous, hydrophilic medium comprises chromatography paper.

4. The three-dimensional microfluidic device of claim 1 , wherein said paper is infused with components selected from the group comprising chemical or biological reagents, indicators, binders, fluid flow-impeding agents, fluid flow- promoting agents, or a combination thereof.

5. The three-dimensional microfluidic device of claim 1, wherein said porous, hydrophilic layer comprises paper.

6. The three-dimensional microfluidic device of claim 1, wherein said porous, hydrophilic layer comprises chromatography paper.

7. The three-dimensional microfluidic device of claim 1, wherein said barrier comprises polymerized photoresist.

8. The three-dimensional microfluidic device of claim 7, wherein said photoresist comprises SU-8 photoresist.

9. The three-dimensional microfluidic device of claim 1, wherein said fluid- impermeable layer comprises a plastic sheet.

10. The three-dimensional microfluidic device of claim 9, wherein said plastic sheet comprises adhesive tape.

11. The three-dimensional microfluidic device of claim 10, wherein said adhesive tape comprises double-sided adhesive tape.

12. The three-dimensional microfluidic device of claim 1, wherein said hydrophilic regions comprise a first hydrophilic region comprising a reservoir for depositing a first fluid; a second hydrophilic region comprising a distributing region for receiving said first fluid from said reservoir and distributing said first fluid; and an array of hydrophilic regions for receiving said first fluid from said distributing region.

13. The three-dimensional microfluidic device of claim 12, wherein each hydrophilic region of said array further comprises an assaying reagent for assaying said first fluid.

14. The three-dimensional microfluidic device of 13, wherein said assay reagent comprises a protein assay reagent, a glucose assay reagent, a sodium acetoacetate assay reagent, a sodium nitrite assay reagent, or a combination thereof.

15. A three-dimensional microfluidic device comprising: a plurality of patterned porous, hydrophilic layers; wherein each said patterned porous, hydrophilic layer comprises a fluid-impermeable barrier which substantially permeates the thickness of said patterned porous, hydrophilic layer and defines a boundary of one or more hydrophilic regions within each said patterned porous, hydrophilic layer; a fluid-impermeable layer disposed between every two adjacent said patterned porous, hydrophilic layers; wherein said fluid-impermeable layer comprises one or more openings; and a material plug filling one or more said openings to provide local temperature control, to assay fluid samples, to filter microfluidic sample, or to modulate microfluidic flow; wherein said material plug is in direct contact with at least of portion of one of said hydrophilic regions within each said adjacent patterned porous, hydrophilic layer.

16. The three-dimensional microfluidic device of 15, wherein said material plug comprises an assaying reagent for assaying fluidic samples.

17. The three-dimensional microfluidic device of 16, wherein said assay reagent comprises a protein assay reagent, a glucose assay reagent, a sodium acetoacetate assay reagent, a sodium nitrite assay reagent, or a combination thereof.

18. The three-dimensional microfluidic device of 15, wherein said material plug comprises a seed.

19. The three-dimensional microfluidic device of 18, wherein said seed comprises soybean seeds, hulls, and powdered soybean, or any other seeds that provide peroxidase to be used as an assay reagent.

20. The three-dimensional microfluidic device of 15, wherein said material plug comprises a filter for filtering solids or blood cells.

21. The three-dimensional microfluidic device of 20, wherein said filter comprises filter papers, any other filtration medium for removing solid contaminants, dextran, or any other materials that can remove red blood cells from a blood sample.

22. The three-dimensional microfluidic device of 15, wherein said material plug comprises a salt which either absorb heat or give off heat when in contact with fluid to provide local temperature control.

23. The three-dimensional microfluidic device of 22, wherein said salt comprises ammonium nitrate or magnesium sulfate.

24. The three-dimensional microfluidic device of 15, wherein said material plug comprises a gel which swells when in contact with fluid to allow microfluidic communication.

25. The three-dimensional microfluidic device of 24, wherein said gel comprises agarose, methylcellulose, or hylaronan.

26. The three-dimensional microfluidic device of 15, wherein said material plug comprises a magnetic material which is capable of movement under the influence of a magnetic field to allow microfluidic communication.

27. The three-dimensional microfluidic device of 26, wherein said magnetic material comprises iron oxide.

28. The three-dimensional microfluidic device of 15, wherein said material plug comprises a fluid-mixable material or a mixture of a fluid-mixable material and a fluid-unmixable material; and wherein said fluid-mixable material dissolves when in contact with fluid to allow microfluidic communication and no microfluidic communication is allowed once said fluid-mixable material has been dissolved and flowed away.

29. The three-dimensional microfluidic device of 28, wherein said fluid- mixable material comprises sugar.

30. The three-dimensional microfluidic device of 29, wherein said sugar comprises sucrose.

31. The three-dimensional microfluidic device of 28, wherein said fluid- unmixable material comprises a hydrophobic polymer.

32. The three-dimensional microfluidic device of 31 , wherein said hydrophobic polymer comprises polystyrene.

33. The three-dimensional microfluidic device of claim 15, wherein said porous, hydrophilic layer comprises paper.

34. The three-dimensional microfluidic device of claim 15, wherein said porous, hydrophilic layer comprises chromatography paper.

35. The three-dimensional microfluidic device of claim 15, wherein said barrier comprises polymerized photoresist.

36. The three-dimensional microfluidic device of claim 35, wherein said photoresist comprises SU-8 photoresist.

37. The three-dimensional microfluidic device of claim 15, wherein said fluid-impermeable layer comprises a plastic sheet.

38. The three-dimensional microfluidic device of claim 37, wherein said plastic sheet comprises adhesive tape.

39. The three-dimensional microfluidic device of claim 38, wherein said adhesive tape comprises double-sided adhesive tape.

40. A three-dimensional microfluidic mixer comprising: a first patterned porous hydrophilic layer comprising a fluid-impermeable barrier which substantially permeates the thickness of said patterned porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions; wherein one of said hydrophilic regions comprises a first fluid flowing channel and a second fluid flowing channel, and a combination zone; wherein said first and second fluid flowing channels merge into said combination zone; a second patterned porous hydrophilic layer comprising a fluid-impermeable barrier which substantially permeates the thickness of said patterned porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions; wherein one of said hydrophilic regions comprises a mixing channel; and a fluid-impermeable layer disposed between said first and second patterned porous, hydrophilic layers and comprising an unfilled opening which is in alignment with said combination zone and at least portion of said mixing channel; wherein said combination zone is capable of moving from a first spaced apart position into a second contact position; wherein in said spaced apart position, said combination zone is not in fluidic contact with said mixing channel; and wherein in said contact position, said combination zone is in fluidic contact with said mixing channel.

41. The three-dimensional microfluidic mixer of claim 40, wherein said first and second fluid flowing channels merge into said combination zone and define an interface between said first and second fluid flowing channels.

42. The three-dimensional microfluidic mixer of claim 41 , wherein said mixing channel is perpendicular to said interface.

43. The three-dimensional microfluidic mixer of claim 40, wherein said mixing channel is more than 1 mm long.

44. The three-dimensional microfluidic mixer of claim 40, wherein said porous, hydrophilic layer comprises paper.

45. The three-dimensional microfluidic mixer of claim 40, wherein said porous, hydrophilic layer comprises chromatography paper.

46. The three-dimensional microfluidic device of claim 40, wherein said barrier comprises polymerized photoresist.

47. The three-dimensional microfluidic mixer of claim 46, wherein said photoresist comprises SU-8 photoresist.

48. The three-dimensional microfluidic mixer of claim 40, wherein said fluid-impermeable layer comprises a plastic sheet.

49. The three-dimensional microfluidic mixer of claim 48, wherein said plastic sheet comprises adhesive tape.

50. The three-dimensional microfluidic mixer of claim 49, wherein said adhesive tape comprises double-sided adhesive tape.

51. A three-dimensional microfluidic flow control device comprising: a plurality of porous, hydrophilic layers comprising a fluid-impermeable barrier which substantially permeates the thickness of said patterned porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions, including: a first patterned porous hydrophilic layer comprising a fluid-impermeable barrier which substantially permeates the thickness of said first patterned porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions; wherein one of said hydrophilic regions comprises a first flowing channel; and a second patterned porous hydrophilic layer comprising a fluid- impermeable barrier which substantially permeates the thickness of said second patterned porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions; wherein one of said hydrophilic regions comprises a second flowing channel; and a fluid-impermeable layer disposed between every two adjacent said patterned porous, hydrophilic layers comprising one or more openings, including: a first fluid-impermeable layer disposed between said first and second patterned porous, hydrophilic layers and comprising an unfilled opening which at least is in partial alignment with said first and second flowing channels; wherein at least portion of said first flowing channel is capable of moving from a first spaced apart position into a second contact position; wherein in said spaced apart position, said first flowing channel is not in fluidic contact with said second flowing channel; and wherein in said contact position, said first flowing channel is in fluidic contact with said second flowing channel.

52. The three-dimensional microfluidic mixer of claim 51 , wherein said porous, hydrophilic layer comprises paper.

53. The three-dimensional microfluidic mixer of claim 51 , wherein said porous, hydrophilic layer comprises chromatography paper.

54. The three-dimensional microfluidic device of claim 51 , wherein said barrier comprises polymerized photoresist.

55. The three-dimensional microfluidic mixer of claim 54, wherein said photoresist comprises SU-8 photoresist.

56. The three-dimensional microfluidic mixer of claim 51 , wherein said fluid-impermeable layer comprises a plastic sheet.

57. The three-dimensional microfluidic mixer of claim 56, wherein said plastic sheet comprises adhesive tape.

58. The three-dimensional microfluidic mixer of claim 57, wherein said adhesive tape comprises double-sided adhesive tape.

59. The three-dimensional microfluidic flow control device of claim 51 , further comprising a hydrophobic plug disposed in one of said openings within one of said fluid- impermeable layers; wherein said hydrophobic plug comprises a low-melting-point hydrophobic material capable of melting and flowing at a pre-selected temperature; wherein one of said patterned porous hydrophilic layers is below said fluid- impermeable layer containing said hydrophobic plug and comprises one of said hydrophilic regions in at least partial alignment with said hydrophobic plug; wherein at said pre-selected temperature said hydrophobic material melts and flows into said hydrophilic region in at least partial alignment with said hydrophobic plug to substantially obstruct said hydrophilic region and prohibits microfluidic flow within said hydrophilic region.

60. The three-dimensional microfluidic flow control device of claim 59, wherein said hydrophobic plug comprises a porous hydrophilic medium permeated by said low-melting-point hydrophobic material.

61. The three-dimensional microfluidic flow control device of claim 59, wherein said porous hydrophilic medium comprises paper.

62. The three-dimensional microfluidic flow control device of claim 59, wherein said low-melting-point hydrophobic material comprises low-melting-point wax.

63. The three-dimensional microfluidic flow control device of claim 62, wherein said wax comprises candle wax, cheese wax, packaging wax, paraffin wax, bayberry wax, or other waxes.

64. The three-dimensional microfluidic flow control device of claim 63, wherein said wax comprises bayberry wax.

65. The three-dimensional microfluidic flow control device of claim 59, wherein said low-melting-point hydrophobic material has a melting point of less than 200 ⁰C, less than 100 ⁰C, less than 50 ⁰C, or between 45 ⁰C and 50⁰C.

66. A three-dimensional microfluidic flow control device comprising a first patterned porous hydrophilic layer and a second patterned porous hydrophilic layer, each comprising a fluid-impermeable barrier which substantially permeates the thickness of said first or second patterned porous, hydrophilic layer and defines boundaries of one or more hydrophilic regions within said first or second patterned porous, hydrophilic layer; a fluid-impermeable layer disposed between said first and second patterned porous, hydrophilic layers comprising one or more openings; and a hydrophobic plug disposed in one of said openings within said fluid-impermeable layer; wherein said hydrophobic plug comprises a low-melting-point hydrophobic material capable of melting and flowing at a pre-selected temperature; wherein said second patterned porous hydrophilic layer is below said fluid- impermeable layer containing said hydrophobic plug and comprises one of said hydrophilic regions in at least partial alignment with said hydrophobic plug; wherein at said pre-selected temperature said hydrophobic material melts and flows into said hydrophilic region in at least partial alignment with said hydrophobic plug to substantially obstruct said hydrophilic region and prohibits microfluidic flow within said hydrophilic region.

67. The three-dimensional microfluidic flow control device of claim 66, wherein said hydrophobic plug comprises a porous hydrophilic medium permeated by said low-melting-point hydrophobic material.

68. The three-dimensional microfluidic flow control device of claim 66, wherein said porous hydrophilic medium comprises paper.

69. The three-dimensional microfluidic flow control device of claim 66, wherein said low-melting-point hydrophobic material comprises low-melting-point wax.

70. The three-dimensional microfluidic flow control device of claim 69, wherein said wax comprises candle wax, cheese wax, packaging wax, paraffin wax, bayberry wax, or other waxes.

71. The three-dimensional microfluidic flow control device of claim 70, wherein said wax comprises bayberry wax.

72. The three-dimensional microfluidic flow control device of claim 66, wherein said low-melting-point hydrophobic material has a melting point of less than 200 ⁰C, less than 100 ⁰C, less than 50 ⁰C, or between 45 ⁰C and 50⁰C.

73. The three-dimensional microfluidic flow control device of claim 66, wherein said porous, hydrophilic layer comprises paper.

74. The three-dimensional microfluidic flow control device of claim 66, wherein said porous, hydrophilic layer comprises chromatography paper.

75. The three-dimensional microfluidic device of claim 66, wherein said barrier comprises polymerized photoresist.

76. The three-dimensional microfluidic flow control device of claim 75, wherein said photoresist comprises SU-8 photoresist.

77. The three-dimensional microfluidic flow control device of claim 66, wherein said fluid-impermeable layer comprises a plastic sheet.

78. The three-dimensional microfluidic flow control device of claim 77, wherein said plastic sheet comprises adhesive tape.

79. The three-dimensional microfluidic flow control device of claim 78, wherein said adhesive tape comprises double-sided adhesive tape.