KR20030090636A - Three-dimensional microfluidics incorporating passive fluid control structures - Google Patents

Abstract

The present invention discloses a three-dimensional microfluidic device 100 formed from a plurality of substantially planar layers 101, 102 and 103 sealed together.

Description

Three-dimensional microfluidics incorporating passive fluid control structures

This application is a continuation-in-part of patent application Ser. No. 09 / 967,402, filed Sept. 28, 2001 in the United States, the United States application of which is also entitled Provisional Patent Application No. 60 / filed Oct. 13, 1998 Patent Application No. 09 / 417,691 filed on October 13, 1999, filed with Patent Provisional Application No. 60 / 138,092, filed on August 8, 1999 and filed with Patent No. 6,296,020, filed on October 8, 2001 Continued application of the call.

The present application also claims the benefit of the following patent applications:

Patent Application No. 60 / 267,154, filed 7 February 2001;

Patent Application No. 60 / 274,389, filed March 9, 2001;

Patent Application No. 60 / 284,427, filed April 17, 2001;

Provisional Application No. 60 / 290,209, filed May 11, 2001;

Provisional Application No. 60 / 313,703, filed August 20, 2001;

Patent Application No. 60 / 339,851, filed December 12, 2001;

Patent application Ser. No. 09 / 855,870, filed on May 15, 2001, which is based on a claim of priority on Patent Provisional Application No. 60 / 204,306, filed on May 15, 2000;

Patent application 09 / 922,451 filed on August 8, 2001, based on a claim of priority on August 4, 2000, provisional patent application 60 / 223,022; And

June 8, 1999 United States Patent Application No. 10 / 009,674, which is based on the claim of priority on June 8, 2000, based on the provisions of the priority of the 8th Patent Application No. 60 / 138,091. As arcs, each of which is incorporated herein by reference.

Integrated circuits and microelectromechanical systems (MEMS) are typically made using microfabrication processes such as thin film deposition, chemical etching, and micro-lithography on silicon substrates. Microfluidic analysis, or microfluidics, is a subset of MEMS in which microscopic fluid processing structures are constructed, often used to process and / or analyze liquid biochemical samples. Although the microfluidic structure was initially made from silicon, most of the microfluidic devices now consist of plastic, while others are formed of glass. Conventional microfabrication techniques are used as well as new or modified methods such as hot embossing and micro-injection molding. Laser processing using both IR and UV lasers is also performed to form microfluidic structures on the substrate.

Most of the microfluidic system is inde 2 or ₂ ^1/2 -D, this means consisting of a microfluidic structure, such as a channel or a well (wells) lie in one plane. The microfluidic structures of a 2 or 2 ¹ / _2- D system have depths that can vary slightly from structure to structure, but these structures do not differ significantly from each other in front of each other, and one structure does not intersect or overlap with another. Do not. 2 or 2 ¹ / _2- D systems prevail for the simple reason that open channels or wells can be easily formed on the surface of the bulk substrate and consequently can be closed by covering the surface of the substrate with a cover plate or film, the substrate Because the surfaces are typically planar, this approach eventually forms a substantially planar enclosed microfluidic circuit. In contrast, to form structures having overlapping or varying heights on the bulk substrate, it is necessary to form at least one of the structures inside the bulk substrate, which is much more difficult than forming surface structures.

In some cases, it is not possible to form the required microfluidic circuit in a single plane, especially if it involves multiple fluid processing circuits operating in parallel, having numerous inlets and outlets, or having circuits provided with multiple samples or reagents. This is because parts of the circuit must intersect or overlap with other parts of the circuit. In other cases, it may be theoretically possible to form a particular microfluidic circuit in a single layer, but this is not practical in view as the size of the device and the length of the microfluidic channel must be too large. Indeed, although theoretically it may be theoretically possible to form microfluidic circuits in two layers (Anderson et al.), In many cases a two-layer device may not be desirable in practical terms for the reasons described above. For the same reason as above, there has been considerable effort in recent years to develop viable methods of constructing multi-layer or three-dimensional microfluidic devices.

The most common approach taken to form a three-dimensional microfluidic circuit is a multi-layer flat or 2 2 _^1/2 -D one form the microfluidic structure, and then, for transferring the fluid from the circuit of the layer with other layers of the circuit By using vias or connecting channels, these layers are joined to form a three-dimensional structure. By forming a multilayer structure, it is possible to produce microfluidic structures on the surface of the substrate material relatively easily while providing flexibility in forming devices having a theoretically unlimited number of layers.

Published PCT application WO 01/41931 discloses forming a multilayer microfluidic structure by laminating and then firing a ceramic sheet having a channel or other microfluidic structure formed therein. Published PCT application WO 01/25138 discloses a multilayer microfluidic structure formed by laminating together layers of self-adhesive plastic tape together. In both of these methods, the microfluidic channel (or other structure) passes through a relatively thin sheet material such that the sides of the channel are formed by the channeled layer, while the top and bottom of the channel are formed by adjacent layers.

The use of microfabrication techniques has been disclosed, which is used as a mold to form structures on silicon substrates and then to form polymer layers or films comprising microfluidic structures (Anderson et al., WO 01/89788, WO 01/89787). . Another approach disclosed is to form a metal trace that defines a microfluidic structure on a printed circuit board substrate, which can then be stacked to form a three-dimensional fluid circuit, or stacked to form a three-dimensional structure. It can serve as a mold master for replicating polymers (WO 01/25137).

The manufacture of monolithic devices having channel structures overlapped by photoresists, and the etching techniques established in the semiconductor industry and applied for use in MEMS, have also been disclosed in US Pat. No. 6,033,544, issued March 3, 2000.

Indeed, three-dimensional or multilayer microfluidic systems are more complex, more expensive, and more prone to failure than 2-D or single layer systems. A major complication in the manufacture of multilayer microfluidic systems stems from aligning and sealing the various layers together. Geometry systems, where features can belong to more than 1 mm, show fewer problems with alignment. However, in systems with very small features, alignment is a significant problem, especially in systems with small connecting vias that fall below 100 μm.

Providing a leak free seal between the multiple layers remains a challenge. The sealing method chosen depends on the particular substrate material used. Sealing methods include process or use of anodic bonding, adhesives or epoxy, or ultrasonic welding. Most of the plastics, silicon, glass and ceramics used in the construction of microfluidic devices are hydrophilic in nature. Since hydrophilic capillary phenomena generate strong forces that are inversely proportional to the size of the feature, aqueous fluids tend to flow into small gaps within the hydrophilic structure. It is therefore particularly important to form a gap-free seal between the layers in a hydrophilic multilayer system. In some cases, it has a removable seal between certain layers of a multi-layer device, so that the devices can be dismantled, for example certain parts of the device can be disposed of and others can be reused, or certain parts of the device can be cleaned. Or it may be desirable to be able to disinfect. The conduct then finds an adhesive that can form a valid seal, which can be released when needed but not released until needed.

Controlling the movement of fluid within the microfluidic device is an essential aspect of virtually all microfluidic devices, but is more difficult to implement in more complex microfluidic circuits. In microfluidic systems using electro-kinetic or electro-hydrodynamic fluid control, many electrodes attached to flow channels may require complex microfluidic circuitry. Other microfluidic systems utilize pressure driven flow, usually associated with some type of valve to regulate the flow of fluid in the device. Valves can also be used in devices that use interfacial or electrofluidic fluid control. Various types of active and passive microvalves have been disclosed for use in microfluidic structures. However, microscale active valves are relatively complex and difficult to configure, even in 2- or 2 ¹ / _2- D systems. Manual valves, including structures such as capillary valves, capillary brakes, and the like have the advantage of not requiring electrical interfaces or mechanical components and are therefore easier to incorporate into the device. Hydrophilic capillary valves are commonly used in microfluidic devices but tend to be unstable. The hydrophilic capillary valve made of hydrophilic material produces only a local minimum in water pressure, which can easily burst by flow momentum or small disturbances of the fluid, thus losing flow control. In contrast, hydrophobic manual valves as disclosed in US Pat. No. 6,296,020, which is incorporated herein by reference, can cause global minima in water pressure, thereby providing more stable fluid control.

Control elements for microfluidic systems, including electrodes for interfacial or electro-fluidic fluid control, mechanical valves or pumps, and heating elements, all require an electrical interface. If these control elements are to be controlled externally, the electrical traces must come out of the device. Thus, a method of constructing a multi-layer microfluidic device should ideally allow electrical traces to come out of the device and the layers of the device should be sealed together while maintaining the integrity of the electrodes.

While many features have been identified that are important for the implementation of fully functional three-dimensional or multilayer microfluidic devices, and devices have been constructed that include these various features, the need for three-dimensional or multilayer microfluidic devices that truly incorporates these various design considerations Still remains. An ideal multilayer device should be designed in such a way that the layers of the device can be easily and accurately aligned during the construction of the device. Such a device should be constructed in such a way that a reliable leak-free seal can be obtained between the layers. In certain cases, the ability of the device to be dismantled after it has been used to clean and / or reuse all or parts of the device to treat parts of the device that contain waste, or to recover samples / reagents contained within the device. It may be desirable to have. Of course, effective control of fluid motion in the microfluidic device is important. Finally, in order for the device to be commercially produced, it is desirable that the three-dimensional microfluidic device be manufactured by a simple and reliable process of inexpensive and readily available materials, if not essential.

The present invention relates generally to the field of microfluidics and, more particularly, to three-dimensional microfluidic circuits formed in a multilayer structure.

BRIEF DESCRIPTION OF DRAWINGS The above detailed description of the invention, briefly described above, refers to specific embodiments shown in the accompanying drawings so that the above-described and other advantages of the present invention and the methods for achieving the objects of the present invention can be easily understood. Will be done. BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be described and described in more detail and detail with reference to the accompanying drawings, with the understanding that these drawings represent only exemplary embodiments of the invention and should not be considered as limiting the scope of the invention.

1 is an enlarged view of a multi-layer device composed of layers comprising active elements and microfluidic circuitry.

FIG. 2 is an assembly view of the device shown in FIG. 1.

3A shows a multilayer device for performing serial dilution and ELISA.

FIG. 3B is a schematic diagram of the basic microfluidic circuit of the apparatus shown in FIG. 3A.

4 is a multi-layer device that processes a sample and delivers it to a microarray slide with three different hybridization solutions.

5 is a plan view showing overlapping channels in a multilayer microfluidic structure.

6 is a cross-sectional view of the FIG. 5 structure taken along line 6-6 of FIG. 5.

7 is a plan view illustrating overlapping channels in another multilayer microfluidic structure.

FIG. 8 is a cross-sectional view of the FIG. 7 structure taken along line 8-8 of FIG. 7. FIG.

9 is a plan view showing overlapping fluid channels formed in a thin-sheet multilayer structure.

10 is a cross-sectional view of the FIG. 9 structure taken along the 10-10 cut line of FIG.

FIG. 11 is a cross-sectional view of the FIG. 9 structure taken along line 11-11 of FIG. 9. FIG.

12A is an enlarged view of a multilayer structure including wells.

12B is an assembly view of the structure of FIG. 12A.

13 is a perspective view of a fluid channel with a manual valve formed on the substrate surface.

14 is an enlarged view of a manual valve formed in a multilayer structure.

15 is a cross-sectional view of the assembled manual valve of FIG. 14.

16 is an enlarged view of a multilayer structure including another manual valve.

17 is a cross-sectional view of the assembled manual valve of FIG. 16.

18 is a perspective view of a fluid channel formed on opposite sides of the substrate and connected by narrow vias through the substrate.

19 is a cross-sectional view of the FIG. 18 structure along the 19-19 incision line.

20 is a cross-sectional view of the FIG. 18 structure along a 20-20 incision.

FIG. 21 shows the branch structure formed on the surface of the substrate and used to divide the fluid stream.

FIG. 22 is an enlarged view of a multilayer structure including a branch structure similar to the branch structure of FIG. 21.

Figure 23 illustrates another branch structure for dividing a fluid stream formed on the surface of the substrate.

FIG. 24 is an enlarged view of a multi-layer structure for dividing a fluid stream that includes a branch structure similar to the branch structure of FIG. 23.

25A-25D illustrate mixing two fluids flowing in series in the microfluidic mixing element.

FIG. 26 is an enlarged view of a multilayer structure in which mixing elements similar to those shown in FIGS. 25A-25D are implemented.

The present invention relates to a multilayer microfluidic structure comprising a three-dimensional microfluidic circuit. The construction of the device is disclosed, and specific embodiments of the device using specific three-dimensional microfluidic circuits are presented. The device is formed from a multilayer of plastic material having microfluidic circuit elements formed on or through one or more surfaces. Hydrophobic base and / or coating materials are widely used in the present invention because these materials provide improved manual valve performance for control of interlaminar sealing and fluid motion that is desirable for such devices. In a preferred embodiment, the present invention includes a microfluidic circuit based on a passive fluid control structure. Selected layers of the device are removably sealed to one another to clean, separate reusable and disposable parts of the device, and to mount the device such as microarray slides or microtiter plates. Allow the device to be released for reversible mating with the substrate.

It is an object of the present invention to provide a multi-layered three-dimensional microfluidic device that can be simply and easily manufactured from inexpensive and readily available materials.

It is yet another object of the present invention to provide a three-dimensional microfluidic device capable of simple, effective and versatile control of the movement of fluid in the device. This is accomplished by using a pressure-driven flow with a valve that directs the flow of the fluid. The valves used in the present invention do not require complex mechanical structures to be configured in the device.

It is another object of the present invention to provide a method for sealing layers of a multilayer microfluidic device in a reliable and leak-free manner.

Another object of the present invention is to provide a leak-free method of sealing layers of a detachable multilayer microfluidic device. This makes it possible to dismantle the device after use, reusing parts of the device, handling other parts of the device, and also allowing the material contained within the device to be recovered.

Another object of the present invention is to provide a multilayer microfluidic device comprising active components such as electrodes, heating elements or sensors.

It is yet another object of the present invention to provide a multilayer microfluidic device using a mixing technique.

It is yet another object of the present invention to provide a multilayer microfluidic device capable of mating with conventional substrates such as slides or microtiter plates. This provides the advantage of integrating microfluidic pretreatment and post-treatment capabilities with the reaction of microvolumes of fluid on or within conventional substrates.

The presently preferred embodiments of the invention will be best understood by reference to the drawings, wherein like parts are designated by like reference numerals. It will be readily appreciated that the components of the present invention as shown and generally described by the drawings of the present invention may be arranged and designed in a wide variety of configurations. Accordingly, the following more detailed description and examples of the apparatus, system and method of the present invention shown by FIGS. 1 to 26 do not limit the scope described in the claims of the present invention, but simply presently preferred embodiments of the present invention. Just an example.

The basic three-dimensional structure of the present invention consists of multiple thin layers of plastic substrate material that are leak free and (optionally) sealed together in a reversible manner. The layers can be rigid or flexible, but are generally flat and nearly planar when assembled together. The microfluidic structure is formed over the surface of the individual layers or the entire thickness of the individual layers by an easily viable method such as molding, micromachining, laser ablation or die cutting. Thus, the microfluidic structure is formed in a plane that basically corresponds to the plane of the substrate layers, although certain structures pass through the layers. The exact nature of the layers and the method of sealing depend on the specific embodiment of the present invention.

In the first embodiment of the present invention shown in the enlarged view of FIG. 1, the multilayer microfluidic device 100 includes layers 101, 102 and 103 formed of a relatively thick and rigid material. In this exemplary embodiment of the invention, the monolayer 102 comprising the microfluidic circuit 104 comprises a layer 101 comprising a heating element 105 and a layer comprising active valves 106a-106d. Together with 103. The microfluidic circuit 104 consists of microfluidic channels and wells formed in the upper surface 110 and the lower surface 111 of the layer 102 connected by vias 115a-115d and 116a-116d. The depth of the channels and wells of the microfluidic circuit 104 is less than the thickness of the layer 102. Lower surface 118 of layer 101 is sealed relative to upper surface 110 of layer 102 such that microfluidic channels 124, 126a-126d, 128a-128d, 130a-130d, 132a-132d are formed therein. And 133) and top surfaces of wells 127a-127d and 131a-131d. The upper surface of the layer 103 is sealed to the lower surface 111 of the layer 102 to form the lower surface of the channels 129a-129d formed therein, such that the active valves 106a-106d are connected to the channel 129a-. 129d). In FIG. 1, valves 106a-106d are pressure activated valves that, when activated, protrude into channels 129a-129d to impede the flow of fluid in the channel. Alternatively, valves 106a-106d can be various other types of active valves including, but not limited to, mechanical valves, remote valves, and hydraulic valves.

Although the microfluidic circuit shown in FIG. 1 is designed to detect a sequence of interest by conducting a polymerase chain reaction (PCR) with a DNA sample, various biochemical reactions, in particular the sample, have undergone one or more heating steps. It may be used to carry out a biochemical reaction that requires it. Such reactions include various reactions used in DNA processing, eg PCR, which requires multiple heating steps (thermal cycling), ligase chain reaction (LCR), rolling circle amplification (RCA) for DNA amplification. Or, as cycle sequencing, all of which include, but are not limited to, reactions using a single isothermal heating step. PCR cocktails containing DNA samples of interest but without primers are pumped (eg, using a syringe pump) to the inlet 122 of layer 102. From inlet 122 it travels to distribution channel 124 and then to channels 126a-126d leading to wells 127a-127d. Wells 127a-127d contain primer pairs for other amplicons. In general, wells 127a-127d may be considered to be thermal reaction wells because they are adapted to contain reactants during one or multiple heating steps as described above. The valves 106a-106d are in the open position while fluid is injected into the device 100 such that air in the circuit passes through the circuit in front of the sample and exits through the air outlet 135. Once the samples are loaded into the wells 127a-127d, the valves 106a-106d are closed and the heating element 105 heats the wells 127a-127d cyclically to perform the PCR reaction. Because the valves 106a-106d are closed, if the pressure increases during heating, fluid cannot be drawn from the wells 127a-127d to the wells 131a-131d. Upon completion of the PCR reaction, the reacted samples are led into wells 131a-131d, which contain dyes or other compounds used for detection of reaction products formed in the thermal wells. Picogreen dyes, for example, label amplified DNA sequences generated by PCR to generate fluorescent signals, which can be detected to determine the presence or amount of reaction products. The device is disassembled to allow dyes in the wells 131a-131d to be read to quantify the amount of reaction product, or when the device is formed of at least partially transparent material (ie, material transparent to the wavelength to be detected). In this case, the reaction product may be detected without dismantling the device.

The material on which layers 101, 102 and 103 are formed may be hydrophilic or hydrophobic, and the surfaces of the layers and the structures formed therein are treated to be hydrophobic. Suitable hydrophobic materials include PTFE, FEP or PFA. Examples of non-hydrophobic materials are silicone, glass, PET, PMMA or PC. Such materials may be coated with hydrophobic materials such as Teflon or Teflon AF by techniques such as vacuum deposition, spin coating or vapor deposition. Different layers may have different thicknesses. Without limitation, the layers can range in thickness from approximately millimeters to several centimeters.

Layers 101, 102 and 103 of device 100 are held together by a clamp as shown in FIG. 2. The clamp shown in FIG. 2 includes an upper frame 140 and a bottom frame 141. Layers 101, 102 and 103 are maintained in alignment by alignment rods 142 through the alignment holes 136 of each layer. At least two alignment holes and alignment rods must be used to align the layers of the device. Alignment hole 136 can also be seen in FIG. 1. The alignment rod 142 is preferably spring loaded to apply compressive force to hold the layers of the device 100 together and to be screwed into the bottom frame 141. Other various methods of assembling layers with alignment rods may also be devised by those skilled in the art, and the present invention is not limited to any particular method. For example, the alignment rod may only function to align, and the compressive force necessary to seal the layers together may be applied by a separate clamp mechanism. Other features shown in FIG. 2 include electrical traces 144 and 145 connected to heating coil 105, pneumatic drive lines connected to pressure activated valves 106a-106d, fluid inlet 122 and air outlet 135. )to be.

If the outer surface of each substrate layer is coated with hydrophobicity or hydrophobicity, sealing the various layers together is less important than if they were hydrophilic, since there would be no hydrophilic capillary force leading the fluid into the gaps between the layers. Thus, the substrate layers can simply be held together without additional use of a gasket or adhesive. The layers must be held firmly together so that under normal pressure the flow of fluid does not push the layers apart. It is also important that the layers are flat and flat and held together tightly and uniformly enough so that no large gaps occur. This will be sufficient to provide a leak-free bond between the layers, since the amount of pressure required to cause the flow of fluid to cause small gaps (belonging to a few micrometers or less) is significant. A desirable feature for this type of device is that it can be assembled at a biochemically compatible temperature, so that the biochemical reagents can be incorporated into the device during assembly.

3A shows another embodiment of a first “thick layer” version of the present invention, wherein the layer of compliant material 301 is sealed between the layer 302 of the device and the instrument panel 303 to which it is interfaced. It is included as a gasket to improve the. As in the apparatus of FIGS. 1 and 2, a clamp (not shown) will keep the layers together in a sealing relationship. The compliant layer 301 may be deformed plastically or elastically, or a combination thereof. The compliant material may be a sheet material that is cut as shown herein and assembled as a separate layer into the device, or may be applied to layer 302 by printing techniques such as screen printing or stencils. Compliant materials include, for example, various natural or synthetic polymeric materials, rubbers and waxes. The gasket layer can have the same pattern of openings as one of the adjacent layers, and can simply perform a sealing function, the depth of the microfluidic structure being basically defined by the adjacent rigid layer. Alternatively, the gasket may have a pattern of openings that define the microfluidic structure independently of those defined by the openings in adjacent rigid layers, as shown herein, wherein the depth of the microfluidic structure in the gasket layer is dependent upon the thickness of the gasket layer. Is defined by. The gasket layer may be only a few microns thick, or may be considerably thicker than simply performing a sealing function, especially if the gasket layers define a microfluidic structure. The gasket layer will typically be in the range of about 0,1 micron to about 500 microns thick, but may be on the order of a few millimeters thick. In the example of FIG. 3A, openings 310a-310e define wells bordered by bottom surface 312 of layer 301 and bordered by top surface 314 of substrate 303. Regions 318a-318e of substrate 303 contain immobilized capture antibodies contained within wells 310a-310e. The microfluidic circuit of such a device is designed to perform a series of dilution and enzyme linked immuno-absorbent assay (ELISA) and will be described in more detail below.

In another variant of the device (not shown), at least some layers of the device are bonded together with an adhesive by thermal bonding or other bonding technique. Proper selection of adhesive depends on the specific material to which it is bonded.

In another variant of the device, at least some layers are composed of a non-hydrophobic material, and only selected areas of the device are formed of a hydrophobic material or comprise a hydrophobic coating. The non-hydrophobic layers are sealed with other layers by bonding or adhesive, or clamped with a gasket to provide a leak free seal therebetween.

In a second version of the device as shown in FIG. 4, the multilayers 401-408 are comprised of thin, relatively flexible sheet material. Suitable materials include various polymeric materials such as acrylics and polyesters and Myler ^™ . The microfluidic structure is formed over the entire layer thickness and may have one or more layer thicknesses. The sheet material may range in thickness from about 10 microns to about 1 millimeter, with typical sheet materials ranging from about 5 to 500 microns. Layer thicknesses of about 10 to 100 microns are preferred for many microfluidic applications. Different layers may be formed of different materials and may have different thicknesses. The layer material may be hydrophilic or hydrophobic and may be treated over the entire surface or in partial regions to change the surface hydrophobicity / hydrophilicity. The adhesive layer 408 may be used to seal the device to the substrate 409 to be interfaced, or to seal the device to further portions of the device formed of a more rigid material. One example of a suitable adhesive is an acrylic polymer (eg 3M 501FL).

The choice of method of forming the device of the present invention depends on whether the device is a circular or custom device or part of a large scale production run, and whether the structure is formed on the surface of the thicker layer (typically) the overall thickness of the thinner layer. It depends on whether it is formed over. For prototype and on-demand devices, the preferred method of construction is the laser ablation technique as disclosed in PCT publication WO 0074890, incorporated herein by reference. If thin layers are used, the structure is cut over the entire thickness of the layers, which can easily be done with a CO ₂ laser or other infrared laser. If a structure or less features are used with less depth than the thickness of the substrate layer, an excimer laser is preferably used to provide greater depth control. Fluid circuits can be prepared by excimer laser ablation with standard fluorocarbon materials when the fluid circuit is doped with carbon black additives to increase the UV absorption of the material.

For prototype and custom devices, the intralayer microfluidic structure may be formed by micromachining. For large scale production of the device, the layers of microfluidic structure are preferably formed by injection molding. The gasket material can be obtained in sheet form, die cut or laser cut, or applied by silk screen or other printing techniques.

In an embodiment of the present invention formed from thin layers for circular device purposes, the microfluidic structure is individually separated by laser cutting or by manual cutting with a surgical mass or an Exacto ^™ knife (although it is obvious that this method is not particularly reproducible). It can be formed in layers. Plucking is desirable for large scale production, but laser cutting is also a suitable method. Single-sided or double-sided adhesive sheet material, consisting of a plastic sheet material having an adhesive on one or both sides, can be used for some or all of the layers or for the device. These materials may also be plucked or laser cut.

As mentioned above, it is desirable to attach the layers of the multilayer device together in a leak-free manner, but for some applications it is desirable that at least some layers can be released and separated from one another in succession. This may be achieved by, for example, a disposable portion (eg, a fluid processing circuit comprising a sample and a reagent) used once, and then separated from the disposable portion once used and reused with other disposable portions. And reusable parts (including electronics, heaters, etc.) that are retained for the sake of brevity. For example, the apparatus of FIGS. 1 and 2 includes two reusable substrate layers 101 and 103 that include active components.

In some embodiments of the present invention as shown in FIGS. 3A and 4, a multilayered microfluidic device is interfaced with a substrate, such as a glass microscope slide, so that portions of the substrate and biochemicals immobilized thereon are essential. In the microfluidic circuit of the device, the microfluidic treatment can be performed on the material on the substrate. Following processing of the material on the substrate, it may be desirable to separate the microfluidic device from the substrate so that the substrate undergoes further processing or evaluation steps that would not be possible if the microfluidic device is placed.

As mentioned above, a leak-free seal can be formed between two hydrophobic faces by simply clamping. If the devices are simply clamped together, disassembling the device when desired is a simple matter. In order to seal layers formed by weakly hydrophobic or nonhydrophobic materials, or a combination of hydrophobic and hydrophilic materials, a gasket capable of elastic or plastic deformation can be placed between the layers to provide a seal that can be released when needed. have. In addition, certain suitably selected adhesives may be used to provide a removable seal. If two surfaces made of or coated with different materials are to be sealed together with an adhesive, the adhesive should be chosen so that it preferentially adheres to one of the surfaces, which, when the layers are separated, the adhesive adheres to one of the surfaces. But to be able to separate from other surfaces. In this way, the adhesive layer remains intact on one of the flat surfaces and completely separates from the other. It is particularly desirable for the adhesive to be completely removed from the reusable portions of the device or from substrates to be subjected to further processing or evaluation.

Various microfluidic circuit elements can be formed in a multilayer microfluidic device constructed in accordance with the present invention. The most basic structures are channels and wells or chambers, which can be formed by tens of thousands of approaches. As discussed below, this structure can be formed through the entire thickness of a single layer in the surface or in a multi-layer structure, so that it lies substantially in a single plane parallel to the layer. These structures may also be formed such that these structures penetrate multiple layers of a multilayer structure. A typical multilayer structure will include structures formed in many different planes corresponding to different layers, connected by via channels or other structures through multiple layers, where at least some of these form structures are formed. Provides for the exchange of fluid between fluid circuits in different planes.

5-11 illustrate several methods of forming channels in multilayer microfluidic spheres. Channel 500 is formed on the top surface of layer 501, the side and bottom surfaces are formed by layer 501 and the top surface is formed by layer 502. Similarly, channels 503 and wells 504 are formed on the top surface of layer 505 so that the top is blocked by the bottom surface of layer 501. Via holes 506 pass through layers 502 and 501 and are partially cut into layer 505. It is also possible to form the top and bottom portions of the channels and wells in two different layers. That is, as shown in FIG. 8, channel 510 is formed in layers 513 and 514, and channel 511 and well 512 are formed in layers 514 and 515. In this example layer 514 has a structure formed on both sides as well as via channel 516 penetrating through its entire thickness. If the channels do not penetrate through the entire thickness of the layer, a second overlapping channel may be formed in the adjacent layer as shown in FIGS. 5 and 6, or as shown in FIGS. 7 and 8. If it is thick enough it can be formed on the underside of the same layer. Such structures may be formed by laser ablation, machining or injection molding, or by a variety of other microfabrication techniques. The same technique can be used to form the vias (defined as channels moving from one level to another, typically perpendicular to the layers). However, if the layers of the multilayer structure are thin so that channels and other structures are formed over the entire thickness of the sheet, the overlapping channels cannot be formed in adjacent sheets, separated by at least one intermediate layer as shown in FIGS. 9 to 11. Should be. The shape of channel 522 and well 523 is defined by layer 526, and the top and bottom surfaces of this structure are defined by layers 525 and 527, respectively. Similarly, channel 521 is defined by layers 527, 528 and 529. This channel formation method is particularly suitable for multilayer devices formed of thin sheet materials, but can also be used for having thicker layers. Channels and vias can be formed by laser cutting or pruning in the present method.

The chamber or well is also an important component of the microfluidic system. Fluid is typically delivered to a chamber or well to perform various types of reactions or assays. The size, shape and direction of the chamber or well depends on the particular application in which it is designed, the volume of fluid contained in the well, the required flow characteristics of the well, and the orientation of the well relative to other fluid circuit components. A simple way to form a well is to form the bottom and sides of the well in a single layer and the top side in a recognized layer. The top surface may be simply planar as shown in FIG. 6, or may take a form that further defines the size and shape of the well as shown in FIG. 8. The approach shown in FIGS. 5-8 is particularly suitable for devices formed of relatively thick and rigid layers. For an apparatus formed of multiple thin layers, the approach shown in FIGS. 9-11 can be used. In this example, the shape of the well is defined by one layer and the top and bottom surfaces of the well are defined by adjacent layers in the plane. However, the wells thus formed will not have much depth and therefore may have a volume less than the desired volume or may be larger than the desired face-volume ratio. To form a well having a greater depth, a well (such as well 530 in FIGS. 12A and 12B) may be formed over multiple layers 532-537 of multilayer structure 531. . If the layers 532-537 are thick enough to match the dimensions of the well 530, the walls of the well cavities 540-545 in the layer 532-537 each slope to form a well 530 having a smooth inner surface. Can provide. Inclined walls can be formed by both injection and laser cutting manufacturing techniques.

In addition to the channels and wells, the valve consists of a third basic component called a microfluidic circuit. Although various types of valves can be used in the practice of the present invention, in a preferred embodiment of the present invention manual valves and in particular hydrophobic manual valves are used. The configuration of the hydrophobic manual valve is described in detail in US Pat. No. 6,296,020, incorporated herein by reference. Such valves are easily formed in single layers of microfluidic devices in the form of long or short steep channels that narrow or widen. Manual fluid control using hydrophobic materials and hydrophobic capillary valves is desirable for stability. The aqueous fluid does not lead to the hydrophobic channel but needs to be led under pressure. As channels become narrower, more pressure is required to keep the fluid flowing. The resistance to stable flow is almost the same for hydrophilic and hydrophobic channels of the same diameter. However, when the fluid first enters the system and there is an air / fluid interface, the resistance to initial or developing flow is substantially higher. The difference between the flows developed in hydrophobic systems and the resistance to stable flows allows for much more reliable flow control, resulting in much more complex fluid circuits than those possible in hydrophilic capillary systems or interfacial or electrofluidic systems. Make it work.

An example of a channel including a manual valve is shown in FIG. 13. Channel 550 is formed on surface 551 of substrate 552. Manual valve 554 is a short region of channel 550 having a reduced diameter. Manual valve of this type can be carried out in the individual layers of the multilayer system, as shown in the 2, or ₂ ^1/2 -D system. This may also be done between layers as shown in FIGS. 14 and 15. The multilayer structure 562 is constructed from layers 564-568, where the channel 561 is formed by holes 571-575 in each of the layers 564-568. Each of the holes has a diameter D ₁ except hole 573, which has a smaller diameter D ₂ and thus forms a manual valve structure. This approach is particularly suitable for devices composed of relatively thin layers.

In an apparatus composed of thicker layers, narrowing of the channel may be performed between the two layers as shown in FIGS. 16 and 17, or layer thickness and fabrication techniques may be used on both sides of the layer. If so implemented in a single layer as shown in FIGS.

Referring now to FIGS. 16 and 17, multilayer structure 580 is formed from layers 581, 582, and 583. Layers 581 and 583 each include openings 585 and 586 having a uniform diameter throughout each layer thickness. Layer 582 has an opening 587, which includes a large diameter portion 588 and a smaller diameter portion 589 corresponding to the diameters of the openings 585 and 586. The smaller diameter portion 589 functions as a manual valve. 18-20 illustrate a structure 590 formed of a thick central layer 598 with fluid channels 591 and 592 on opposite sides. Channels 591 and 592 are sealed by layers 593 and 594 that are sealed to central layer 598. Narrow channel 595, which is essentially a via channel between channels 591 and 592, has a smaller diameter than these channels and functions as a manual valve. Resistance to fluid flow is obtained not only when fluid enters narrow channel 595 from channel 591 or 592, but also when fluid exits narrow channel 595 to wide channel 596 where the diameter of the channel increases rapidly. It should be noted that it is. The widening of this type of rapid channel is described in the co-filed patent application "Fluid Circuit Components Based on Passive Fluid Dynamics", incorporated herein by reference [Representative Document No. 3153.2. 14, it may be used as a manual valve to control the movement of a fluid in a microfluidic circuit.

Remote valving can be used as a replacement for or in addition to a manual valve to control fluid flow in a three-dimensional microfluidic structure. The remote valve control disclosed in US patent application Ser. No. 09 / 922,451, incorporated herein by reference, controls the movement of air out of the system using a mechanical valve located outside the fluid system and connected to the fluid circuit by an air conduit. When the fluid first enters the fluid circuit, the air in the system must be discharged out as it is replaced by the fluid. If the air cannot escape, it will create a backpressure that will block the flow of fluid. Remote valve regulation controls the discharge of air from the fluid circuit and thus the flow of fluid in the circuit. This simplifies microfluidic circuit fabrication because any expensive and complex valve adjustment and control can be made externally and made reusable. Air conduits are constructed in the same way as fluid channels but typically have a smaller diameter. If the channels are not already filled with fluid, the original air can exit through the fluid channel, which communicates with air pressure.

Remote valve control techniques can be implemented in hydrophilic or hydrophobic systems. However, the use of hydrophobic air conduits is particularly advantageous because the release of air is permitted but the flow of fluid through the air conduits is limited. Air conduits formed of hydrophilic materials can be coated with hydrophobic materials or covered with an air permeable hydrophobic membrane to provide the benefits of hydrophobic air conduits.

In addition to controlling the discharge of air from the microfluidic circuit to regulate fluid flow, positive or negative pressure may be used to control fluid flow or reaction conditions, as disclosed in PCT Publication No. WO 0188204, incorporated herein by reference. It can be applied to air conduits in selected areas of the microfluidic circuit.

Mechanical valves and other types of valves can be used in the practice of the present invention. The mechanical valve can be activated electronically, pneumatically, or hydraulically, for example. The present invention is not limited to any particular type of valve.

Microfluidic channels, chambers and valves can be combined to perform a variety of fluid treatment tasks. One basic task is to divide one fluid stream between multiple channels. This task is easily performed by using a manual valve. For reference, as disclosed in US Pat. No. 6,296,020, which is incorporated herein by reference, the flow of fluid in a network of branch channels provides a set of manual valves each time branches occur, allowing fluid to continue to flow through the branches. Can be controlled. By respectively creating a "stronger" barrier than the previous set, the fluid is allowed to fill all the branches that are currently occurring before moving to generate the next channels. This can be accomplished, for example, by forming each successive capillary barrier narrower than its previous set. The branch circuit formed on the single substrate layer 600 is shown in FIG. 21. The flow of fluid in a single channel 601 is divided into two channels 602 and 603 followed by four channels 604, 605, 606 and 607. Capillary barriers are located at 610, 611 and 612. The branch circuit of FIG. 21 may be implemented within a single layer of a multilayer system, or equivalent circuitry may be configured between the layers, as shown in FIG. Fluid enters the structure of FIG. 22 through an inlet channel 620 in layer 621. From there, fluid enters the central region of primary branch channel 627 in layer 622 and flows to each end. Since the primary via channels 628 and 629 in layer 623 have a smaller cross-sectional area than the primary branch channel 627, the primary branch channel 627 is fluid-filled with the primary via channels 628 and 629. It is completely filled before flowing through it. Primary via channels 628 and 629 thus act as manual valves. Similarly, the secondary via channels 632-635 have a smaller cross-sectional area than the channels or holes in the previous layers, thus providing a higher resistance to the flow of the fluid and allowing the fluid to become secondary via channels ( Fully fill secondary branch channels 630 and 631 before moving through 632-635.

21 and 22 show branch circuits having a binary branch pattern. Other branch patterns may also be used as shown in FIGS. 23 and 24. In the single layer version of FIG. 23, the single inlet channel 640 is branched into four channels 641-644 in one step. In FIG. 24, fluid enters aperture 660 in layer 650 and into branch channel 661 in central region 662 in layer 651. The fluid then flows to the end of the arms 663-666 of the branch channel 661. The outlet channels 670-673 in layer 652 provide greater resistance to the flow of fluid than the arms of the branch channel 661, so that all the arms of the branch channel 661 allow the fluid to exit the outlet channels 670. The fluid is uniformly distributed between the channels since it is filled before moving to any one of -673).

Another basic microfluidic circuit component consisting of a combination of channel and manual valve is a mixer 700 as shown in FIGS. 25A-25D. The function of the mixer 700 is to mix the first fluid 710 and the second fluid 711, which in turn flows into the main channel 701, which bypasses the first fluid 710 to the subchannel 702. The first fluid 710 is then injected back into the downstream portion 706 of the main channel to flow side by side with the second fluid 711 to be diffusely mixed. The operation of the mixing circuit is as follows. As shown in FIG. 25A, the first fluid 710 moves to the main channel 701 under pressure from a pressure source, such as a programmable syringe pump. The first fluid 710 faces the manual valve 703, which stops the flow to the main channel 701. As shown in FIG. 25B, the first fluid 710 is diverted to the subchannel 702 by a manual valve 703, which allows the first fluid 710 to flow more than in the main channel 701. This is because they suffer less resistance. The first fluid 710 flows into the subchannel 702 until it encounters the manual valve 704. The volume of the first fluid 710 is selected to only fill the subchannel 702, with the first fluid 710 following the second fluid 711 entering the main channel 701, followed by the first fluid 710. Push out fluid 710. The manual valve 704 is selected to provide greater resistance to flow than the manual valve 703, so as additional second fluid 711 is directed to the main channel 701, as shown in FIG. 25C. The second fluid 711 first breaks through the manual valve 703 and then flows into the main channel 701. When the second fluid 711 reaches the intersection of the main channel 701 and the subchannel 702, the second fluid 711 wets the downstream surface of the manual valve 704, which prevents the flow of fluid. -Rupture the liquid interface. As shown in FIG. 25D, additional second fluid 711 flows into the main channel 701, with the first fluid 710 from the subchannel 702 and the second fluid from the main channel 701. 711 flows together to the downstream portion 706 of the channel 701.

The mixer circuit shown in FIGS. 25A-25D can also be implemented in multiple thin layers. If multiple thin layers are used such that the fluid circuit structure is cut across the entire layer thickness, the "islands" of the material surrounded by the main and subchannels are not supported, which can complicate the manufacturing process. Thus, the circuit can be formed in multiple layers 720-724 as shown in FIG. 26. Fluid enters the opening 725 in layer 720. Main channel 726 and subchannel 727 are formed in layer 721 like manual valve 728. Small openings 729 in layer 722 form a manual valve just before the intersection of subchannel 727 and main channel 726. The subchannels and main channel are combined in channel 731 in layer 723. As a result, fluid from main channel 726 and subchannel 727 flow together out of opening 732 to a microfluidic device (not shown). Alternatively, such a circuit may be formed in a single thin layer if several position holders are provided to maintain the "island" position relative to the major portion of the substrate material. Such a foresight holder may be, for example, a pin inserted into an island from a top substrate or a bottom substrate, or a backing sheet removed as the “islands” are secured to adjacent layers during the manufacturing process.

Often the microfluidic device preferably includes active elements such as electrodes, mechanical valves, heaters, pumps, various types of sensors, mixing elements and other components. The mixing element may comprise a piezoelectric element, an air or fluid activated bag or membrane, and a structure for circulating or pumping the fluid back and forth to perform the mixing function. Sensors include pressure transducers, optical transducers, flow measurement devices, and the like. Since these components are typically more expensive to fabricate than basic microfluidic circuits, these components are preferably included as part of a reusable device. According to the invention, the microfluidic device can be composed of multiple pieces which can be sealed together during use and then separated for cleaning and reuse (if necessary). As shown in FIG. 1, active elements are included in layers 101 and 103 that are sealed together with microfluidic layer 102.

The following example illustrates how the microfluidic circuit structure and assembly method described above can be implemented. These embodiments represent only a small sampling of the many possible structures that can be constructed in accordance with the present invention, and the implementation of the present invention is not limited to this particular exemplary structure.

Example 1

1 and 2 show a first embodiment of a microfluidic device composed of numerous relatively thick rigid layers, including reusable layers comprising active elements and potentially disposable layers comprising passive microfluidic circuits, according to the present invention. An example is shown. 1 and 2 are used to perform screens based on PCR of DNA samples. As mentioned above this device may also be used for LCR, RCA or other reactions.

Example 2

FIG. 4 shows an apparatus that can be used for hybridization solutions and pretreatment samples for probe- raise or probe-cDNA hybridization on microarrays. Such a device may be used to determine suitable hybridization conditions prior to performing a series of microarray hybridizations. Multiple hybridizations are performed on a single array under different conditions to minimize the slide-to-slide change observed in the microarray hybridization reaction, which obscures subtle differences in significant gene expression experiments during drug discovery. The sample can be combined or dye-labeled with the hybridization solution in the device. The preparation of hybridization solutions having different concentrations of samples or other components can be performed in the apparatus. Salt buffers (SSCs) and formamides are typical components of hybridization solutions that can be adjusted to maximize hybridization sensitivity. This is especially important when studying insufficient genes in probe samples. The device performs the task of labeling the probe solution and delivering the labeled probe along with a hybridization solution of varying selected parameters to several overprinted areas on the surface of a single microarray slide. The best conditions for hybridization can be found by comparing the obtained hybridization results with other hybridization solutions on a single slide.

Referring now to FIG. 4, the purified probe DNA sample enters inlet 410 in layer 402, flows into channels 411 and 412, and is delivered to wells 413 and 414. Manual valves 440 and 441 at the outlet of wells 413 and 414 allow both fluids to fill before further flow downstream. Wells 413 and 414 each comprise one of two different dyes. Probe DNA is cultured in wells 413 and 414 where it is labeled via covalent attachment of dyes. In each of these two microfluidic wells, the labeling probe flows into channels 416 and 418 and collects into separation channel 415, which is in-channel chromatography such as an affinity or sized exhaust matrix. The medium is contained to separate the unlabeled probe from the unreacted dye from the labeled probe. The purified and dye-labeled probe then leaves the separation channel 415 via channel 417 and is collected in chamber 419. These probes pass through channel 420 and pass through holes 420 in layer 403 to layer 404 of the device.

In layer 404, the purified label probe is divided into multiple parts, some of which are further processed to adjust the parameters of interest. The purified marker probe is divided into two parts in split 423. Probe solution in channel 424 travels through via channels 425 and 427 to opening 428 in layer 408, which layer 408 includes an area 429 of monoarray slide 409. To form a hybridization chamber. Probe solution in channel 435 enters well 436 containing a first reagent that is mixed with the probe solution. Reagents in the well 436 modify the probe solution. The modified probe solution exits well 436 and is split at branch point 437. The first portion of the modified probe solution in sieve 438 passes through well 439, where it is modified by the addition of a second reagent from well 439, and then the area of microarray 409 ( 454, and via openings 450 and 459 to opening 453 in layer 408. Probe solution in channel 460 enters subchannel 461 and is subsequently mixed and diluted with additional probe solution. The diluted and modified probe solution travels through the channel 462 to the well 463 where a third reagent is added and then via a chamber formed in the openings 464, 465 and 466 and the opening 467. Delivered to region 468 of microarray 409. As fluid enters the chambers 428, 453, and 467, air is introduced into the via holes 475-477 in the layer 407, the holes 478-480 in the layer 406, and in the layer 405. Exit through holes 482-484, which are connected to an air outlet 486-488 in layer 404, where it contacts the atmosphere.

Reagents in wells 436, 439 and 463 can be any of a number of materials that can alter the hybridization reaction to be performed on the microarray surface. Reagents may include salts for modifying the ionic power of materials such as formamide, SSC, acids, bases or buffers, solutions, detergents, etc., to alter the pH of the solution. The reagents may be loaded into the device in dried form (eg, lyophilized) before use, or in liquid form for at least relatively stable reagents. The functions specified in the different layers of the device can eliminate the significant amount of labor associated with the preparation of different hybridization solutions and the preparation of labeled probe solutions for use in optimization experiments conducted at the beginning of all microarray-based studies. have.

Another feature of the invention as illustrated in FIG. 4 is the ability to stir and mix fluid in chambers 428, 452 and 467 formed on the microarray surface, including air pockets that alternately expand and contract. . Layer 406 includes air pockets 490a, 491a and 492a connected to air line 494 and air pockets 490b, 491b and 492b connected to air line 495. Air lines 494 and 495 are externally connected to positive and negative pressure sources, which in turn inflate and deflate the air pockets to allow fluid to be pushed back and forth within chambers 428, 452 and 467. The use of air pockets to provide pneumatic mixing in a hybridization chamber is described in detail in US patent application Ser. No. 60 / 339,851, incorporated herein by reference. Such mixing mechanisms can be included in various embodiments of the invention and are not limited to use in the specific embodiments described.

Example 3

3A shows a three-dimensional microfluidic device 300, which serially dilutes a sample and delivers sample solutions and a series of appropriate reagents / reactants to the diagnostic surface 303, such as multiple immunoassays such as enzyme binding. Allow immunosorbent assays or "ELISA" to be performed. The microfluidic circuit is designed to interface with a diagnostic surface 303 such as a slide or microtiter plate with small regions 318a-318e containing immobilized capture antibodies specific for the particular analyte (s). The device has been simplified to more clearly illustrate its principle of operation. Indeed, the diagnostic surface 303 may comprise numerous regions that contain capture antibodies, for example in an array consisting of multiple rows, unlike the single row shown here. Device 300 will have numerous microfluidic circuits corresponding to deliver samples and reagents to the numerous regions containing antibodies. A series of dilution steps performed by the device 300 are necessary to ensure that the immunological response used to measure the concentration of the analyte in the sample is performed within the linear range of the assay. Performing an immunological analysis within a straight line is very important in terms of assay accuracy.

An upper circuit layer of the device 300 is formed on the upper surface of the substrate layer 302 and includes a microfluidic circuit 316 that serially dilutes a sample containing one or more analytes of interest at unknown concentrations. Another microfluidic circuit on the top circuit continuously delivers sample solution and reagents to the diagnostic surface 303 so that immunological analysis can be performed. The circuit 316 in the substrate layer 302 is sealed by a lid layer 304 sealed to the top surface of the substrate layer 302. The circuitry of the upper circuit layer is connected to read wells formed on the diagnostic surface by down vias 319a-319e and up vias 320a-320e. The read well is formed by openings 310a-310e in the gasket layer 301 corresponding to regions 318a-318e containing capture antibodies immobilized on the diagnostic surface 303.

Substrate layer 302 includes an inlet 370 which is fed into main channel 321, which leads to a series of sample wells 322a-322e in which various dilutions of the sample solution are applied. Is collected. In series along main channel 321 are shown microfluidic mixing modules 323a-323e of the type shown in FIGS. 25A-25D, which perform serial dilution by mixing sample solution and buffer. Branching of the main channel in each sample well is multiple identical ELISA circuits 324a-324e in which the ELISA reaction is performed on different serial dilutions of the sample. The first ELISA circuit 324a is shown in detail in FIG. 3B, where the operation of the other ELISA circuits is the same. As shown in Fig. 3B, the first ELISA circuit 324a includes a main channel 330a; A conjugation well 333a located on the subchannel 334a and comprising a lyophilized enzyme-antibody conjugate formed with an antibody specific for the analyte (s) of interest; A substrate well 331a located on the subchannel 332a and comprising a lyophilized substrate, the conjugate with the lyophilized substrate results in a detectable reaction product; Read well 310a on diagnostic surface 303; And waste wells 335a, 336a, 337a and 338a. All wells and channels are located on the diagnostic surface 303, except for read wells 310a that are connected to circuitry on layer 302 by down vias 319a and upvia 320a. Positioned on layer 302.

In use, the sample solution containing the analyte (s) of interest is pumped into the inlet 370, passing the sample well 322a over the channel 321 until it is stopped by the manual valves 338a and 339a. Pumped into. The pumping pressure may be provided by a syringe pump or other pumping device. The volume of sample solution sufficient to fill sample well 322a is followed by a larger volume of buffer, which is pumped to inlet 370 immediately after the sample solution. Once the buffer enters the sample well 322a, a portion of the sample solution moves into the ELISA circuit 324a and another portion of the sample solution moves to the mixing module 323a. The manual valve 329a causes the sample to preferentially flow into the subchannel 326 of the mixing module 323a until it encounters the manual valve 328, and the manual valves 340a, 341a, 342a and 343a allow it to be passive. Priority flows into down channel 330a and read well 310a until valves 344a, 345a, 346a and 347a are encountered. Capture antibodies that recognize the analyte of interest in the sample are immobilized in readwell 310a. Samples must be incubated for the time required for immunological analysis to ensure thorough binding in this well.

It is important that the volume of sample fluid loaded into the system is sufficient to fill the subchannels 323a and read well 310a so that once these structures are filled, the sample solution in the sample well 322a is replaced with a buffer solution. . It is also important that the strength of the manual valves be filled to subchannel 323a and readwell 310a to ensure correct distribution of the sample solution without the sample fluid entering the other portion of the microfluidic circuit.

After subchannel 323a and readwell 310a are filled and additional buffers are pumped into channel 321, the buffer may be either ELISA circuit 324a or mixed circuit (depending on the relative strength of valves 329 and 240a). 323a). Once the buffer enters the mixing circuit 323a, the volume of the sample in the subchannel 326 is mixed with the buffer as described in connection with FIGS. 25A-25D and also within the twisted channel 327a leading to the sample well 322b. Are mixed. Manual valves 338b and 339b block the flow of diluted sample after sample well 322b is filled.

Following a sufficient incubation period during which the sample is incubated in readwell 310a, a volume of buffer must be pushed through main channel 330a and readwell 310a to flush out unbound sample. The manual valve 344a must be overcome to allow the sample fluid to move into the waste well 335a while the buffer moves to the read well 310a. The buffer then enters subchannel 334a. The valve 340a at the inlet to the subchannel 334a has a weaker strength than any of the valves 345a, 346a and 347a at the outlet of the read well 310a and at the beginning of the subchannel 332a. With a weaker strength than the valve 341a in place, the fluid should preferentially enter the channel 334a and pass through the junction well 330a once the read well 310a is filled and cleaned. Bonding well 330a includes a deposited bond that is lyophilized in the field or deposited in the form of beads during the manufacturing process of the device. By contacting the buffer, the conjugate is resuspended to form a conjugate-containing buffer solution, which occurs instantaneously. Once the buffer enters channel 334a, air exits through air conduit 350a that is connected (indirectly or directly as shown) to the atmosphere. Channel 334a preferably has a larger diameter than channel 330a such that when both channels are filled with fluid, the fluid preferentially flows into channel 334a so that all the conjugates dissolve and are transported to readwell 310a. . Once the channel 334a is filled, the conjugate-containing buffer solution moves to the read well 310a, whereby the waste well 336a until the rinse buffer solution is blocked by the manual valve 356a past the manual valve 345a. Push it out. The buffer containing the conjugate is incubated in the read well 310a for a period of time so that the conjugate binds to the captured immobilized analyte. Additional buffer is injected through the inlet 370 to flush out the unbound conjugate from the read well and move it to the waste well 337a. The manual valve 357a has a higher strength than the manual valve 341a so that when the waste well 337a is filled, the buffer solution is moved past the manual valve 341a to the subchannel 332a, where it is the well ( Rehydrate the substrate in 331a). Air conduit 354a provides an exit for air as the buffer enters subchannel 332a. The diameter of channel 332a is greater than the diameter of subchannel 332a and main channel 330a such that the buffer preferentially flows through subchannel 332a to allow all of the substrate to move into readwell 310a. have. When the buffer containing substrate is transferred to read well 310a, the previous content of read well 310a is pushed into waste well 338a via manual valve 347a. Sufficient time is allowed for the signal to develop and then detected and quantified by standard laboratory instruments. The layers of the device may be disassembled to send a signal from the read wells (ie, from the diagnostic surface 303), or the signal may be detected through the diagnostic surface 303 if the detected signal can pass through it. It is desirable that the space between adjacent read wells be compatible with existing laboratory instruments.

The dilution steps performed in each of the mixing circuits 323a-323d result in serial dilution of the sample solution into the buffer. A portion of the serially diluted sample solution from each of the sample wells 322b-322e is transferred to each of the ELISA circuits 324a-324e, wherein the above-described process is performed for each diluted sample. Properly selecting the strength and channel diameter of the manual valve can precisely control the movement of the fluid through the fluid circuit of a particular portion.

The method of manufacturing the multilayer structure and fluid circuit components to form the three-dimensional microfluidic circuit described herein may be used to form various microfluidic structures and devices, of which the specific examples provided herein are merely illustrative. . The present invention may be embodied in other specific forms without departing from the structures, methods or other essential features set forth broadly herein and in the claims that follow. The above-described embodiments are to be considered in all cases as illustrative only and not restrictive. Accordingly, the scope of the invention is indicated by the appended claims rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

List of references:

Anderson, J. R., D. T. Chiu, R. J. Jackman, O. Cherniavskaya, J.C. McDonald, H. Wo, S.H. Whitesides, G.M. Whitesides, Fabrication of Topologically Complex Three-Dimensional Systems in PDMS by Rapid Prototyping, Anal. Chem. 2000, 72, 3158-3164

Claims (76)

(a) a plurality of substantially planar layers assembled together in a sealing relationship; (b) a microfluidic structure in at least two planes corresponding to at least two said planar layers of a microfluidic device; And (c) a multi-layer microfluidic device consisting of one or more microfluidic structures that pass through one or more adjacent planar layers and provide fluid flow between microfluidic structures in different planes, Wherein the microfluidic structure is comprised of one or more channels, wells, separators, mixers, valves, air conduits, or air outlets, wherein at least one of the plurality of planar layers has a hydrophobic surface. The multilayer microfluidic device of claim 1 further comprising one or more active elements selected from the group consisting of heating elements, electrodes, sensors, mixing elements, and active valves. The multilayer microfluidic device of claim 2 wherein the active element is comprised of a mixing element selected from the group consisting of a piezoelectric transducer, a pneumatically activated and operated bag, and a hydraulically activated bag. The multilayer microfluidic device of claim 2 wherein the active element is comprised of a sensor selected from the group consisting of an optical sensor, a pressure transducer, a flow transducer, and a temperature sensor. The multilayer microfluidic device of claim 1, wherein the one or more layers are formed of a hydrophobic material. The multilayer microfluidic device of claim 1, wherein the one or more layers are formed of a non-hydrophobic base material and the hydrophobic surface is formed by a hydrophobic coating on the non-hydrophobic base material. The multilayer microfluidic device of claim 1 wherein the layers are aligned in an alignment frame prior to assembly with each other. The multilayer microfluidic device of claim 1, wherein each layer consists of two or more alignment holes formed therethrough, and the layers are aligned by a rod passing through the alignment holes. The multilayer microfluidic device of claim 1, wherein at least two of the layers are assembled together in a sealed relationship by clamping together. 10. The multilayer microfluidic device of claim 9, wherein the fluid-tight seal between the two or more layers is obtained by providing a compressible gasket layer between the non-compressible layers. 10. The multi-layer microfluidic device of claim 9, wherein the fluid-tight seal between the two or more layers is obtained by providing a hydrophobic surface at an interface between the two layers. The multilayer microfluidic device of claim 1, wherein at least two of the layers are assembled together in a sealing relationship by an adhesive. 13. The multilayer microfluidic device of claim 12 wherein the adhesive is removable from one or more of the two or more layers. The multilayer microfluidic device of claim 1, wherein the seal between two or more of the layers can be released to allow the two or more layers to separate. 15. The multilayer microfluidic device of claim 14 wherein the device can be separated into a disposable part and a reusable part between the two or more layers. The multilayer microfluidic device of claim 15 wherein one of the layers is a glass slide. The multilayer microfluidic device of claim 15 wherein one of the layers is a microtiter plate. The multi-layer microfluidic device of claim 15 wherein at least one of the layers consists of at least one region having biomolecules immobilized thereon. The multilayer microfluidic device of claim 1 wherein the planar layers are formed of a hydrophobic base material. The multilayer microfluidic device of claim 1 wherein at least one of said valves is a manual valve. The multilayer microfluidic device of claim 1 wherein at least one of said valves is a remote valve. The microfluidic structure of claim 1, wherein the microfluidic structure in one of the planes is formed over the entire thickness of one or more layers such that a boundary of the microfluidic structures is formed by the one or more layers, and the top and bottom surfaces are defined by adjacent layers. A multilayer microfluidic device, which allows for formation. The multilayer microfluidic device of claim 1, wherein a portion of one or more of the microfluidic structures in one of the planes is formed on the surface of one or more of the layers, but does not pass over the entire thickness of the layer. (a) a plurality of substantially planar layers assembled together in a sealing relationship; (b) a microfluidic structure in at least two planes corresponding to at least two said planar layers of a microfluidic device; And (c) a multi-layer microfluidic device consisting of one or more microfluidic structures that pass through one or more adjacent planar layers and provide fluid flow between microfluidic structures in different planes, One or more portions of the microfluidic structures lying in the two or more planes are formed on the surface of one or more of the layers but do not pass through the entire thickness of the layer, but pass through one or more planar layers within the two or more planes. Wherein the microfluidic structure comprises an additional microfluidic structure selected from the group consisting of channels, wells, separators, mixers, valves, air conduits and air outlets as one or more passive valves and one or more additional microfluidic structures. Fluid devices. The multilayer microfluidic device of claim 24 further comprising one or more active elements selected from the group consisting of heating elements, electrodes, sensors, mixing elements and active valves. The multilayer microfluidic device of claim 25, wherein the active element is comprised of a mixing element selected from the group consisting of a piezoelectric transducer, a pneumatically activated and operated bag, and a hydraulically activated bag. 27. The multi-layer microfluidic device of claim 25, wherein the active element is comprised of a sensor selected from the group consisting of optical sensors, pressure transducers, flow transducers, and temperature sensors. The multilayer microfluidic device of claim 24, wherein the one or more layers have a hydrophobic surface. 29. The multi-layer microfluidic device of claim 28, wherein said at least one layer is formed of a hydrophobic material. 29. The multi-layer microfluidic device of claim 28, wherein the at least one layer is formed of a non-hydrophobic base material and the hydrophobic surface is formed by a hydrophobic coating on the non-hydrophobic base material. The multilayer microfluidic device of claim 24, wherein two or more of the layers are assembled together in a sealed relationship by clamping together. 32. The multilayer microfluidic device of claim 31, wherein the fluid-tight seal between the two or more layers is obtained by providing a compressible gasket layer between the non-compressible layers. 32. The multi-layer microfluidic device of claim 31, wherein the fluid-tight seal between the two or more layers is obtained by providing a hydrophobic surface at an interface between the two layers. The multilayer microfluidic device of claim 24 wherein at least two of the layers are assembled together in a sealing relationship by an adhesive. 35. The multilayer microfluidic device of claim 34 wherein the adhesive is detachable from at least one of the two or more layers. 25. The multi-layer microfluidic device of claim 24, wherein the seal between two or more of the layers can be released to allow the two or more layers to separate. 37. The multi-layer microfluidic device of claim 36, wherein the device can be separated into a disposable portion and a reusable portion between the two or more layers. The multilayer microfluidic device of claim 36 wherein one of the layers is a glass slide. 37. The multilayer microfluidic device of claim 36 wherein one of the layers is a microtiter plate. 37. The multi-layer microfluidic device of claim 36, wherein one or more of the layers consists of one or more regions having biomolecules immobilized thereon. The multilayer microfluidic device of claim 24, wherein at least one of the valves is a remote valve. 25. The microfluidic structure of claim 24, wherein the microfluidic structures in one or more of the planes are formed over the entire thickness of one or more layers such that boundaries of the microfluidic structures are formed by the one or more layers, and the top and bottom surfaces are defined by adjacent layers. A multilayer microfluidic device, which allows for formation. (a) a plurality of substantially planar layers assembled together; (b) one or more sample inlets formed in one or more said layers; (c) one or more thermal reaction wells in fluid communication with the sample inlet; (d) one or more read wells in fluid communication with the thermal reaction well; And (e) one or more active valves positioned between the thermal reaction wells and the read wells to control the flow of fluid between the thermal reaction wells and the read wells, A multilayer microfluidic device for carrying out a biochemical reaction comprising one heating step. The multilayer microfluidic device of claim 43, further comprising a heating element, the heating element being formed in a layer different from the one or more thermal reaction wells and configured to provide heating to the thermal reaction wells. The multilayer microfluidic device of claim 43 wherein the layers are clamped to each other. 46. The multilayer microfluidic device of claim 45, wherein the fluid-tight bonds between the layers are obtained by providing a compressible gasket layer between the non-compressible layers. 46. The multi-layer microfluidic device of claim 45, wherein the fluid- tight bonds between the layers are obtained by providing a hydrophobic surface at the junctions. 48. The multilayer microfluidic device of claim 47 wherein the hydrophobic surface is a surface of a hydrophobic base material. 48. The multilayer microfluidic device of claim 47 wherein the hydrophobic surface is formed by hydrophobic coating a non-hydrophobic base material. (a) loading a solution containing the DNA sample of interest into a multilayer microfluidic device; (b) distributing the solution into one or more thermal reaction wells in the microfluidic device, wherein the one or more thermal reaction wells are provided with additional materials needed to amplify a particular DNA sequence of interest; (c) close the valve beneath the one or more thermal reaction wells to prevent downstream movement of gas or liquid from the one or more thermal reaction wells; (d) heating the solution and additional material in the one or more thermal reaction wells in a manner sufficient to amplify the specific DNA sequence of interest when present in the DNA sample of interest in the thermal well; (e) opening said valve downstream of at least one said thermal reaction well; (f) flushing the contents of the at least one thermal reaction well out of the thermal reaction well, through the channel downstream of the thermal well, into a corresponding read well; (g) detecting the presence or absence of DNA in the read well. 51. The method of claim 50, wherein the DNA solution is adapted to perform PCR analysis, wherein the DNA solution consists of a PCR cocktail without primers, and the additional material consists of primer pairs specific for the particular DNA sequence of interest, Heating the solution and additional material in the one or more thermal reaction wells comprises performing a thermocycle. 51. The method of claim 50, wherein the method of performing DNA processing applied to perform LCR analysis, wherein heating the solution and additional material in the one or more thermal reaction wells comprises an isothermal heating step. 51. The method of claim 50, wherein the method of performing DNA processing applied to perform the RCA analysis, wherein heating the solution and additional material in the one or more thermal reaction wells comprises an isothermal heating step. (a) loading the solution into a multilayer microfluidic device; (b) partitioning the solution into one or more thermal reaction wells in the microfluidic device; (c) close the valve downstream of the one or more thermal reaction wells to prevent downstream movement of gas or liquid from the one or more thermal reaction wells; (d) heating said at least one thermal reaction well in a manner required to effect the biochemical reaction of interest; (e) opening said valve downstream of said at least one thermal reaction well; (f) flush the contents of each thermal reaction well out of each thermal reaction well into a corresponding downstream read well; (g) detecting the presence or absence of the biochemical reaction product in the read well. (a) a plurality of substantially planar layers assembled in a sealing relationship; (b) one or more inlets to receive sample solution in which the analyte of interest may be present; (c) a read well downstream of the inlet, comprising a binding portion adapted for binding with the analyte of interest; (d) one or more waste wells downstream of the read wells for receiving fluid washed from the read wells; (e) one or more manual valves for temporarily stopping the flow of fluid to retain fluid in the read well; And (f) a three-dimensional microfluidic device for conducting a coupling reaction to detect an analyte of interest in a sample, consisting of one or more passive valves for at least temporarily stopping the flow of fluid to retain the fluid in the waste well . The three-dimensional microfluidic device of claim 55, wherein the inlet, read well, waste well and manual valves are located in two or more different planes corresponding to two different planar layers of the microfluidic device. (a) a plurality of substantially planar layers; (b) at least one ELISA circuit having components formed in at least two of said layers, i) a main channel adapted to receive a sample solution in which an analyte of interest may be present; ii) read wells in fluid communication with the main channel and comprising immobilized capture antibodies specific for the analyte of interest; iii) a conjugation well in fluid communication with said main channel and said read well and comprising a quantity of conjugate consisting of an antibody specific for said analyte of interest that is conjugated to an enzyme; iv) a substrate well comprising an amount of an enzyme substrate in fluid communication with said main channel and said read well and capable of reacting with said enzyme to form a detectable reaction product; and v) ELISA consisting of a plurality of manual valves for continuously passing a flow of fluid through the main channel, the read wells, the junction wells and the read wells to sequentially deliver sample solution, conjugate and substrate to the read wells. A three dimensional microfluidic device for performing an ELISA to detect an analyte of interest in a sample, consisting of a circuit. The method of claim 57, wherein (a) one or more additional ELISA circuits having components formed in two or more of the layers, i) a main channel adapted to receive a sample solution in which an analyte of interest may be present; ii) read wells in fluid communication with the main channel and comprising immobilized capture antibodies specific for the analyte of interest; iii) a conjugation well in fluid communication with said main channel and said read well and comprising a quantity of conjugate consisting of an antibody specific for said analyte of interest that is conjugated to an enzyme; iv) a substrate well comprising an amount of an enzyme substrate in fluid communication with said main channel and said read well and capable of reacting with said enzyme to form a detectable reaction product; And v) a plurality of manual valves for continuously passing a flow of fluid through said main channel, said read well, said junction well and said read well to deliver sample solution, conjugate and substrate to said read well in turn. ELISA circuits; (b) a first sample well located upstream of all further sample wells and adapted to receive undiluted samples injected into the microfluidic device; (c) one or more additional sample wells adapted to receive diluted samples from upstream sample wells; And (d) located between each said additional sample well and an upstream sample well and configured to mix a sample from said upstream sample well with a dilution to form a diluted sample solution collected in said one or more additional sample wells Further composed of one or more mixed circuits, Sample solution from the first sample well is delivered to the one or more ELISA circuits, and diluted sample solution from the one or more additional sample wells is delivered to the one or more additional ELISA circuits, each of the ELISA circuits being the sample. A three-dimensional microfluidic device, used to detect an analyte of interest in solution or in the diluted sample solution. (a) a plurality of substantially planar layers assembled in a sealing relationship; (b) an inlet channel through which at least a first portion of the hybridization solution can be loaded into the three-dimensional microfluidic device; (c) a well containing other components or reagents of the hybridization solution coupled with the first portion of the hybridization solution, a separation column for performing a separation step in at least a portion of the hybridization solution, At least one component selected from the group consisting of a mixing circuit for mixing at least a portion of said hybridization solution and a diluent, and a branch circuit for dividing at least a portion of said hybridization solution between two or more channels, A microfluidic processing circuit downstream of the inlet channel; (d) one or more manual valves for regulating the flow of the hybridization solution through the microfluidic processing circuit; And (e) a via channel for delivering at least a portion of the hybridization solution to the surface of the microarray slide, In use, the microarray slide is assembled to the three-dimensional microfluidic device in a sealed relationship such that one or more hybridization chambers are formed at the interface between the microarray slide and the three-dimensional microfluidic device, and the via channel is the hybridization. A three-dimensional microfluidic device for processing a hybridization solution in fluid communication with the chamber and delivering it to the surface of the microarray slide. (a) a plurality of substantially planar layers assembled in a sealing relationship; (b) microfluidic circuits formed in at least two planes defined by the planar layers; (c) one or more microscale channels formed in a plane defined by one or more of said layers; And (d) a manual valve comprising a short sharp narrowing in the one or more microscale channels, And the inner surface of the channel and the manual valve are hydrophobic. 61. The three-dimensional microfluidic structure of claim 60, wherein said layers are formed of a hydrophobic material. 61. The three-dimensional microfluidic structure of claim 60, wherein said layers are formed of a non-hydrophobic base material with a hydrophobic coating. (a) a plurality of substantially planar layers assembled in a sealing relationship; (b) microfluidic circuits formed in at least two planes defined by the planar layers; (c) one or more microscale channels formed through one or more of said layers and providing fluid communication between two or more different in-plane microfluidic circuits defined by said planar layers; And (d) a three-dimensional microfluidic structure consisting of a manual valve comprising a short sharp narrowing in the one or more microscale channels. 64. The three-dimensional microfluidic structure of claim 63, wherein the inner surface of the channel and the manual valve are hydrophobic. 64. The system of claim 63, wherein the channel consists of openings arranged in three or more layers of the microfluidic structure, wherein the manual valve is formed by one or more of the three or more layers, and wherein the openings in the layer are three or more. A three-dimensional microfluidic structure having a smaller cross-sectional area than said opening in other layers. 64. The apparatus of claim 63, wherein the channel consists of openings aligned in at least first and second layers of the microfluidic structure, the first layer having an opening having a narrow portion and a wide portion, wherein the narrow portion is the Narrower than an opening in a second layer, wherein the first and second layers are assembled together such that the narrow portion is positioned adjacent to the second layer, and the manual valve consists of the narrow portion. (a) a plurality of substantially planar hydrophobic layers assembled in a sealing relationship; And (b) a three-dimensional microfluidic structure formed within the microfluidic structure and consisting of wells consisting of a plurality of aligned holes in the plurality of adjacent layers. (a) a plurality of substantially planar layers assembled in a sealing relationship; And (b) as a mixed circuit i) a first channel; ii) a branch point downstream of the first channel where the first channel is divided into a main channel and a subchannel; iii) a first manual valve located downstream of the branch point on the main channel; iv) a junction downstream of the branch point where the subchannel rejoins the main channel; v) a second manual valve located on the subchannel immediately upstream of the junction and stronger than the first manual valve; And vi) a three-dimensional microfluidic structure consisting of a mixing circuit formed in a three-dimensional microfluidic structure, consisting of an outlet channel downstream of said junction. 69. The three-dimensional microfluidic structure of claim 68, wherein said mixing circuit is comprised of components formed in at least two different planes corresponding to at least two said planar layers. (a) a plurality of substantially planar layers assembled in a sealing relationship; (b) a first mixing circuit, comprising: i) a first inlet channel; ii) a branch point downstream of the first channel where the first inlet channel is branched into a first main channel and a first subchannel; iii) a first manual valve located downstream of the branch point on the main channel; iv) a first junction downstream of said branch point at which said first subchannel rejoins said first main channel; v) a second manual valve located on the first subchannel immediately upstream of the first junction and stronger than the first manual valve; And vi) a first mixing circuit formed in a three-dimensional microfluidic structure, the first outlet channel downstream of the junction; And (c) one or more additional mixing circuits formed in the three-dimensional structure downstream of the first mixing circuit, comprising: i) a second inlet channel downstream of the first outlet channel; ii) a second branch point downstream of the second inlet channel where the second inlet channel is branched into a second main channel and a second subchannel; iii) a third manual valve located downstream of the second branch point on the second main channel; iv) a second junction point downstream of the second branch point where the second subchannel rejoins the second main channel; v) a fourth manual valve located on the second subchannel immediately upstream of the second junction and stronger than the third manual valve; And vi) a three-dimensional microfluidic structure adapted to continuously dilute a sample, consisting of one or more additional mixing circuits, consisting of a second outlet channel downstream of the second junction. (a) injecting an amount of a first fluid into the first channel of the mixing circuit of claim 69; (b) inject an amount of a second fluid into the first channel behind the second fluid, wherein the first fluid is moved by the first manual valve when pushed into the mixing circuit by the second fluid; Bypassing into the subchannels, the amount of the first fluid being sufficient to fill the subchannels up to the second manual valve; (c) injecting additional second fluid into the first channel at a pressure sufficient to overcome the first manual valve to move into the main channel until the first fluid reaches the junction; (d) injecting additional second fluid into the first channel to move the first fluid out of the second channel past the junction, where the first fluid is in the outlet channel downstream of the junction 2. A method of mixing two fluids in a microfluidic structure, comprising combining two fluids. (a) a plurality of substantially planar layers assembled in a sealing relationship; (b) an inlet channel through at least the first layer; (c) a primary branch channel formed in a second layer adjacent to the first layer and the inlet channel intersecting at its central region; (d) two primary via channels passing through a third layer adjacent the second layer, one of which intersects the primary branch channel at each end thereof; (e) two secondary branch channels formed in a fourth layer adjacent to the third layer, each of the via channels intersecting in a central region thereof; And (f) passes through a fifth layer adjacent to the fourth layer, one of which consists of four secondary via channels intersecting each of the secondary branch channels at each end thereof; Wherein the two primary via channels have a smaller cross-sectional area than the primary branch channel and the four secondary via channels have a smaller cross-sectional area than the primary via channel. (a) a plurality of substantially planar layers assembled in a sealing relationship; (b) an inlet channel through at least the first layer; (c) a branch channel formed in a second layer adjacent to the first layer, the branch channel in communication with the central region of the branch channel, the branch channel having a plurality of arms extending out from the central region; And (d) formed in a third layer adjacent the second layer, each consisting of a plurality of outlet channels in communication with an end of one of the plurality of arms of the branch channel, Each of said outlet channels provides greater resistance to fluid flow than said arms of said branch channel, such that fluid entering said branch channel fills all of said arms before entering one of said outlet channels. Fluid branches circuit. (a) a plurality of substantially planar layers assembled in a sealing relationship; And (b) a microfluidic circuit comprising a microfluidic structure lying in at least two planes corresponding to at least two said planar layers of a microfluidic device, i) main channel; ii) a subchannel that is a branch of the main channel at a branch point; iii) a first manual valve located in said subchannel immediately downstream of said branch point; iv) at least one microfluidic structure located downstream of the branch point in fluid communication with the main channel and consisting of a well or channel; And v) consisting of a microfluidic circuit, consisting of a second manual valve located downstream of the microfluidic structure, The first manual valve has a strength sufficient to allow fluid first entering the main channel under pressure to preferentially enter the main channel rather than the sub channel at the branch point, and the second manual valve is A three-dimensional microfluidic structure filled to the second manual valve and of sufficient strength to divert the flow of fluid to the subchannels. 75. The system of claim 74, wherein the subchannel is rejoined with the main channel downstream of the branch point but upstream of the second manual valve, and the subchannel is air adjacent to the point at which the subchannel rejoins the main channel. Consisting of a conduit, the subchannel having a diameter sufficiently larger than the diameter of the main channel such that when the main channel and the subchannel are filled with a fluid, additional fluid injected into the main channel is at the branch point. Three-dimensional microfluidic structure, preferentially flowing through subchannels. 75. The apparatus of claim 74, wherein the main channel, the subchannel, the first manual valve, the microfluidic structure, and the second manual valve are in one of the two or more planes, and the microfluidic circuit is in the two or more planes. A three-dimensional microfluidic structure consisting of one or more additional microfluidic structures lying in another of the planes.