CN105964316A - Programmable microfluidic digital array - Google Patents

Abstract

The invention relates to a microfluidic device, and more specifically relates to a programmable microfluidic digital array; the microfluidic device includes a pressure source and a control line in fluid communication with the pressure source. The microfluidic device also includes a plurality of valves operated via the control line and an independent valve positioned adjacent the control line and between the pressure source and the plurality of valves.

Description

Programmable Microfluidic Digital Array

This application is a divisional application of the Chinese patent application with application number 201510158081.2. The original application is a divisional application of the Chinese patent application with application number 200980154761.X. The original application 200980154761.X is a PCT international application filed on December 7, 2009 PCT/US2009/067037 entered the Chinese national phase application on July 15, 2011.

technical field

The present invention relates to programmable microfluidic digital arrays.

Background technique

Microfluidic devices enable analysis, preparation, measurement, and other manipulation functions at scales that were unimaginable until recently. Advantages of microfluidic devices include saving precious reagents and samples, high density and throughput of sample analysis or synthesis, fluidic precision and accuracy at a level barely visible to the naked eye, and replacement of corresponding devices operating on a macrofluidic scale The resulting space reduction. Associated with the reduced size and increased density of microfluidic devices is higher complexity and greater engineering and manufacturing costs as well as more elaborate device configurations.

Despite these advances in microfluidic design and applications, it would be useful to reduce the complexity of microfluidic chips and simplify their operation. In addition, there is a need for an enhanced ability to control fluid flow and the associated reaction processes that occur in microfluidic devices. Accordingly, there is a need in the art for improved methods and systems related to microfluidic devices.

Contents of the invention

The present invention relates to microfluidic devices. More specifically, the present invention relates to programmable microfluidic digital arrays and methods of operation thereof. By way of example only, the methods and apparatus have been applied in systems providing asynchronous logic functions in microfluidic chips. Additionally, some embodiments of the present invention integrate check valves into digital arrays to provide lockout control lines. However, it should be realized that the present invention has a much broader scope of applicability.

According to one embodiment of the present invention, a microfluidic device is provided. The microfluidic device includes a pressure source and a control line in fluid communication with the pressure source. The microfluidic device also includes a plurality of valves operated by the control line, and an independent valve located adjacent the control line and between the pressure source and the plurality of valves.

According to another embodiment of the present invention, a method of operating a microfluidic device having a valve and a control line having a valve assembly connected thereto is provided. The method includes closing the valve and applying pressure to the control line. The closed valve renders the valve set connected to the control line inoperable.

According to yet another embodiment of the present invention, a microfluidic device is provided. The microfluidic device includes a first valve, a second valve, and a control line in fluid communication with the first valve and the second valve. The microfluidic device also includes a pressure accumulator in fluid communication with the control line and a one-way valve located adjacent the control line between the pressure accumulator and the second valve.

According to an alternative embodiment of the present invention, a microfluidic device is provided. The microfluidic device comprises a plurality of reaction chambers arranged in an array configuration. Each of the plurality of reaction chambers has a first valve in fluid communication with one of the plurality of reaction chambers and a second valve in fluid communication with the one of the plurality of reaction chambers. The microfluidic device also includes a first control line operable to actuate the first valve and the second valve and a set of input lines in fluid communication with the plurality of reaction chambers. The microfluidic device also includes a plurality of sample inlets in fluid communication with the set of input lines and a one-way valve disposed in the first control line.

According to another alternative embodiment of the present invention, a microfluidic device is provided. The microfluidic device includes a plurality of reaction chambers and a plurality of first input ports. Each of the first plurality of input ports is in fluid communication with one or more of the plurality of reaction chambers via one of the first plurality of input lines. The microfluidic device also includes a plurality of second input ports. Each of the second plurality of input ports is in fluid communication with one or more of the plurality of reaction chambers via one of the second plurality of input lines. The microfluidic device also includes a first pressure accumulator in fluid communication with the first control line. The first control line is configured to close the first plurality of input lines. Additionally, the microfluidic device includes a second pressure accumulator in fluid communication with a second control line. The second control line is configured to close the second plurality of input lines. Additionally, the microfluidic device includes a first one-way valve disposed in the first control line between the first pressure accumulator and the first plurality of input lines, and a first check valve disposed in the second A second one-way valve in the second control line between the pressure accumulator and the second plurality of input lines.

According to yet another alternative embodiment, a method of operating a microfluidic device having a plurality of valves and a one-way valve is provided. The method includes applying a first fluid pressure to a control line of the microfluidic device, and closing the plurality of valves in response to the application of the first pressure. The method also includes closing the one-way valve in response to application of the first pressure, and applying a second fluid pressure to a second control line of the microfluidic device.

According to a specific embodiment of the present invention, a method of operating a microfluidic device having a plurality of input ports is provided. The method includes providing input fluid to one of the plurality of input ports, and actuating a set of valves to close a first portion of the input line connected to a subset of the plurality of input ports. The subset does not contain the one of the plurality of inlets. The method also includes flowing the input fluid through an input line connected to the one of the plurality of input ports, flowing the input fluid through the input line to a second portion of the input line and Close the second set of valves to separate multiple reaction chambers.

According to another specific embodiment of the present invention, there is provided a method of operating a programmable microfluidic device having an array of reaction sites in fluid communication with a first set of input lines and a second set of input lines. The method includes actuating a first set of valves operable to block the first set of input lines, and actuating a second set of valves operable to block a first portion of a subset of a second set of input lines. The method also includes loading a sample into the reaction site through a second portion of the second set of input lines and actuating a third set of valves operable to separate the reaction sites.

According to yet another embodiment of the present invention, a microfluidic device is provided. The microfluidic device comprises a predetermined number of input ports and a plurality of input fluid lines, each of the input ports is operable to receive one of a plurality of input fluids, each of the plurality of input fluid lines is in contact with the predetermined One of the number of input ports is in fluid communication. The microfluidic device also includes a set of valves each operable to close one of the plurality of input fluid lines. The number of the valve groups is less than the predetermined number. The microfluidic device also includes a manifold in fluid communication with each of the input fluid lines and a second set of valves each operable to close a portion of the manifold.

According to one embodiment of the present invention, there is provided a method of operating a programmable microfluidic device having an array of reaction sites in communication with a first set of input lines, a second set of input lines, and with the second set of input lines The connected manifolds are in fluid communication. The method includes actuating a first set of valves operable to close said first set of input lines, actuating a second set of valves operable to close a first portion of a subset of a second set of input lines, and actuating a second set of valves operable to close a first portion of a subset of a second set of input lines, and A third set of valves operates to deactivate the manifold. The method also includes deactuating the second set of valves, loading a plurality of samples into the reaction site via a second portion of the second set of input lines, and deactuating the second set of valves operable to separate The fourth valve group of the reaction site.

According to another embodiment of the present invention, a method of operating a programmable microfluidic device having an array of reaction sites in fluid communication with a first set of input lines and a second set of input lines is provided. The method includes actuating a first set of valves operable to block the first set of input lines, and actuating a second set of valves operable to isolate the reaction sites. The method also includes deactivating the second set of valves, loading a plurality of samples into the reaction site through a second set of input lines and actuating the second set of valves.

According to yet another embodiment of the present invention, a microfluidic device is provided. The microfluidic device includes a plurality of reaction sites and a first set of input lines providing fluid communication between a predetermined number of first input ports and the plurality of reaction sites. The number of the first group is the predetermined number. The microfluidic device also includes a second set of input lines providing fluid communication between a predetermined number of second input ports and the plurality of reaction chambers. Each of the second input line groups includes a trunk portion and a branch portion, and the number of the second groups is less than the predetermined number. The microfluidic device also includes a programmable input device operable to fill the reaction chamber with the first set of input lines or the second set of input lines.

According to a specific embodiment of the present invention, a method of providing a microfluidic device having a plurality of control lines is provided. The method includes actuating a first control line and placing a valve in a first state. The method also includes thereafter actuating a second control line operable to place the valve set in a second state. A valve in the first state prevents the set of valves from being placed in the second state.

According to another embodiment of the present invention, a method of providing a microfluidic device having a plurality of control lines is provided. The method includes establishing a first state of the microfluidic device by actuating a first control line and then actuating a second control line, and establishing the state of the microfluidic device by actuating the second control line and then actuating the first control line. The second state of the microfluidic device.

According to yet another embodiment of the present invention, a microfluidic device is provided. The microfluidic device includes a first valve and a second valve. The microfluidic device also includes a control line in fluid communication with the first valve and the second valve, and a pressure source in fluid communication with the control line. The microfluidic device also includes a one-way valve disposed in the control line between the pressure source and the second valve.

According to another alternative embodiment of the present invention, a microfluidic system is provided. The microfluidic system includes a carrier. The carrier includes a plurality of first input ports and a plurality of first input lines. Each of the first plurality of input lines is in fluid communication with one of the first plurality of input ports. The carrier also includes a plurality of second input ports and a plurality of second input lines. Each of the plurality of second input lines is in fluid communication with one of the plurality of second input ports. The carrier also includes a first pressure source and a second pressure source. The microfluidic system also includes a microfluidic device mounted on the carrier. The microfluidic device includes a plurality of third input lines and a plurality of fourth input lines. Each of the third plurality of input lines is in fluid communication with one of the first plurality of input lines, and each of the fourth input lines is in fluid communication with one of the second plurality of input lines. The microfluidic device also includes a first control line in fluid communication with the first pressure source, a one-way valve operable to block at least a portion of the first control line, and a fluid connection with the second pressure source. Connected second control line.

The present invention has many advantages over conventional techniques. For example, the present technology allows customization of microfluidic devices after fabrication, enabling users to provide multiple programmable panel setups. These and other embodiments of the invention, along with its many advantages and features, are described in more detail below and in connection with the accompanying drawings.

Description of drawings

Figure 1 is a simplified schematic diagram of a microfluidic device according to one embodiment of the present invention.

Figure 2A is a simplified schematic diagram of a one-way valve according to one embodiment of the present invention.

Figure 2B is a simplified top view showing the one-way valve shown in Figure 2A.

Figure 3 is a simplified flowchart of a method of operating a microfluidic device according to one embodiment of the present invention.

Figure 4 is a simplified flowchart of a method of operating a microfluidic device according to another embodiment of the present invention.

Figure 5 is a simplified flowchart of a method of operating a microfluidic device according to yet another embodiment of the present invention.

Figure 6A is a schematic diagram of a reaction chamber in an array according to one embodiment of the invention.

Figure 6B is a simplified perspective view of control lines, fluid input lines, and reaction chambers in an array according to one embodiment of the invention.

Figure 7 shows a simplified method of programming a programmable microfluidic device according to one embodiment of the present invention.

Figure 8 shows a simplified method of programming a programmable microfluidic device according to another embodiment of the present invention.

Figure 9 shows a simplified method of programming a programmable microfluidic device according to yet another embodiment of the present invention.

Figure 10 is a simplified schematic diagram of a programmable microfluidic device according to one embodiment of the present invention.

Figure 11 is a simplified schematic diagram of a microfluidic system according to one embodiment of the invention.

detailed description

Figure 1 is a simplified schematic diagram of a microfluidic device according to one embodiment of the present invention. In a specific embodiment, the microfluidic device shown in Figure 1 comprises a programmable high density digital array. The microfluidic device 100 includes a first pressure source 102 and a second pressure source 104 . In FIG. 1 , a first pressure source 102 is referred to as a containment accumulator, which, as described in more detail below, is in fluid communication with a control line operable to close the connection with the array. Valves associated with reaction chambers (not shown) in 106 . Since actuation using the pressure source 102 closes the reaction chamber valves and contains the sample and/or reagents in the reaction chamber, the pressure source 102 is referred to in some embodiments as a containment accumulator.

In operation, a liquid is placed in a pressure accumulator or source of pressure which is then connected to an external positive pressure supply such as a container containing compressed air. Under pressure, the compressed air or other fluid pushes the liquid in the pressure accumulator into the control line, thereby actuating the valve. Thus, the pressure source typically does not contain a pressurized fluid when manufactured, but rather provides a container in which the pressurized fluid can be accumulated and stored during operation of the microfluidic device to apply pressure to the control line upon activation. As described more fully throughout this application, the pressure accumulator is operable to maintain pressure in the control line after activation. Additional description of digital arrays suitable for practicing embodiments of the present invention is provided in co-pending and commonly assigned US Provisional Patent Application No. 61/044,417, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes.

Figure 6A is a schematic diagram of a reaction chamber in an array according to one embodiment of the invention. As an example, some embodiments of the invention employ a unit cell having a reaction chamber with lateral dimensions of 100 μm x 60 μm and a height of 125 μm. In this exemplary embodiment, the chamber has a volume of about 0.75 nl. Such small chamber volumes enable the use of smaller sample volumes and reduce operating costs. A 50 μm diameter via connects the reaction chamber and the assay/sample input line. The reaction chambers were placed with a first lateral chamber spacing of 110 μm and a second lateral chamber spacing of 200 μm. A 50 μm x 50 μm valve operable to prevent flow through the input line is provided in a layer different from the layer containing the assay/sample input line. The particular geometry of the cell is not intended to limit embodiments of the invention, but merely provides an example of a specific embodiment. In another embodiment, other device geometries suitable for a particular application are employed.

According to some embodiments of the invention, fluid flow through the microfluidic device is provided using input lines having a width ranging from about 5 μm to about 400 μm and a depth ranging from about 5 μm to about 75 μm. A control line having a width ranging from about 5 μm to about 400 μm and a depth ranging from about 5 μm to about 75 μm is used to valve off fluid flow through the input line. Reaction chambers having a width in the range of about 10 μm to about 500 μm, a length in the range of about 10 μm to about 500 μm, and a height in the range of about 5 μm to about 500 μm are employed in some embodiments. The geometries of these devices are provided by way of example and are not intended to limit the embodiments described herein.

As shown in Figure 6A, a plurality of input lines 620 are provided through which fluid can flow in a horizontal direction. Three input lines are shown, but some embodiments of the invention employ more than three, eg, 11 input lines. With multiple input lines a single sample can be distributed across multiple input lines, which provides multiple copies of a given sample. As described in the more complete description of FIG. 6B, the input lines or channels are at least partially contained in the first layer of the microfluidic device. Referring to FIG. 1 , the input lines are in fluid communication with an assay input line 132 on the left side of the array 106 and in fluid communication with a sample input line 142 on the right side of the array 106 . Thus, fluid from either the assay input 130 or the sample input 140 can be provided to the input line and thus to the reaction chamber.

Multiple control lines 610 are provided in a vertical direction, enabling control of fluid flow through the input lines. Two input lines are shown, but some embodiments of the invention employ more than two control lines, eg, 70 control lines. In another embodiment, there are 71 control lines. The plurality of control lines form separate reaction chambers along the length of the input line, providing multiple reaction chambers containing the same sample. As described in the more complete description of FIG. 6B, the control lines or channels are at least partially contained in the second layer of the microfluidic device. Referring to FIG. 1 , the control line is in fluid communication with a portion 114 of a lockout control line 110 which is in fluid communication with the containment accumulator 102 . At the intersection of the control line and the input line is formed a valve 615 that is actuated in response to fluid pressure in the control line and is operable to prevent fluid flow through the input line. Typically, the multilayer microfluidic device comprises multiple elastomeric layers and the valve 615 comprises a deflectable membrane. In the embodiment shown in Figures 6A and 6B, the deflectable membrane of the valve is deflected into the fluid channel located above the intersection with the control channel. Thus, the embodiment shown employs a "push up" valve, where the deflectable membrane deflects upward into the fluid channel to close the fluid channel where the valve is located. For the valve shown in Figures 6A and 6B, releasing the fluid pressure present in the control passage causes the deflectable membrane to return to the undeflected position thereby opening the closed valve.

Fluid flowing through input line 620 passes through hole 625 in a direction perpendicular to the plane of FIG. 6A and flows upward into reaction chamber 630, which is at least partially contained in the microfluidic device as described in the more complete description of FIG. 6B. in the third layer. Thus, said through hole is at least partially contained in at least said second or third layer of the microfluidic device. Typically, the vias are formed by removing a portion of the second or third layer using a laser ablation process. Since the microfluidic device is air permeable, the blind fill technique can be used to fill the reaction chamber and perform various chemical, biological or other experiments. Those skilled in the art understand that after fluid is present in the reaction chamber, actuation of the control line will cause the valve to close and retain the fluid in the reaction chamber for a predetermined period of time.

Figure 6B is a simplified perspective view of control lines, fluid input lines, and reaction chambers in an array according to one embodiment of the invention. An array (eg, array 106 shown in FIG. 1 ) is part of a multilayer microfluidic device. Each layer typically comprises an elastomeric structure with one or more grooves, channels, chambers, and the like. As shown herein, the first layer 601 includes a plurality of control channels 610 and further control channels 611 arranged in an array of parallel channels. The control passage 610 and the further control passage 611 are in fluid communication with one or more pressure sources or pressure accumulators. Thus in one embodiment, control passage 610 is in fluid communication with pressure accumulator 102 and control passage 611 is in fluid communication with pressure accumulator 104 . Although a single control channel 611 is shown in FIG. 6B , those of ordinary skill in the art will understand that the single channel shown is representative of one or more control channels associated with layer 601 .

Furthermore, the control channels are not limited to locations relative to layer 601 . The control channels can be placed in other layers as appropriate for specific applications. For example, in a multiple control (control-on-control) implementation, in order to make the first control line (such as control channel 611) control the second control line (such as control channel 610), the second control line Placed in both the first layer 601 and the second layer 602 according to the length of the control channel. Vias similar to vias 625 are used to transition the second control lines from the first layer 601 to the second layer 602 . By passing the second control line over the first control line, a valve is formed at the intersection of the two control lines. Once the first control line is actuated, the flexible membrane at the valve position between the first control line and the second control line pushes upwards to block the second control line located in layer 602 at the valve position. Thus, as described more fully hereinafter, "push-up" valves may be formed between different control lines or between a control line and a fluid input line. Referring to FIG. 1 , the control line connected to the valve 128 transitions from the lower layer to the upper layer of the structure through the through hole, and then passes through the control line 122 so that the control line 122 can actuate the partition valve 124 . The control line associated with valve 128 is then threaded back into the first layer through another through hole to pass and actuate the valve of fluid input line 132 . Once control line 122 is actuated, divider valve 124 blocks flow through the control line connected to valve 128 , preventing valve 128 from closing normally in response to control line 110 actuation.

The second layer 602 contains a plurality of fluid input channels 620 also arranged in an array of parallel channels. In the embodiment shown in Figures 6A and 6B, the control channel 610 and the input channel 620 are arranged perpendicular to each other. Actuation of valves present in the microfluidic device is achieved by applying pressure to a liquid (typically a substantially incompressible fluid) present in the control channel 610 . Typically, the liquid is placed in an accumulator or pressure source and a pressurized fluid (eg, air, nitrogen, etc.) is provided to the accumulator. The increase in pressure in the accumulator brings fluid under pressure into the control line. The deflectable membrane forming the top of the layer 601 deflects upwards into the fluid input channel 620 in response to the applied pressure. Thus, a "push up" valve is created at the intersection of the control channel and the fluid input channel. Some other embodiments may employ "push down", "pull down" or "pull up" valves by repositioning various control and fluid layers.

In some embodiments of the invention that provide multiple controls, one or more additional control layers (eg, lower layer 601 ) may be provided, or by routing control lines into 602 layers. The one or more additional control layers may contain additional control lines (not shown) which, once actuated, will pass through the control lines present in the valve closure layer 601 . Thus, by applying fluid pressure to the control fluid (eg, liquid) present in one or more additional control lines, the flexible membrane is deflected to the control line 610, preventing fluid from flowing through the control line. Accordingly, some embodiments of the invention provide a layer of control, also referred to as multiple control. Many variations, improvements, and substitutions will be apparent to those of ordinary skill in the art.

The second layer also includes a plurality of through holes 625 creating fluid channels that provide fluid flow from the input channels to the third layer 603 . The third layer 603 comprises a plurality of reaction chambers 630 which are in fluid communication with the fluid input channels through through holes. In the embodiment shown, the reaction chamber 630 is formed in communication with the bottom of the layer 603, in other words, the reaction chamber is open from the bottom. Therefore, the vias are completely contained in the 602 layer. In other embodiments, the vias may be included in both layers 602 and 603 to suit a particular application.

In some embodiments, a microfluidic device may comprise one or more layers prepared according to spin-on or cast fabrication protocols. For example, a spinning protocol may involve placing a polymeric material on a patterned disk or mold, and spinning the disk to create a layer of polymer on the disk. Exemplary polymers include polymethacrylate, polystyrene, polypropylene, polyester, fluoropolymers, polytetrafluoroethylene, polycarbonate, polysilicon, and polydimethylsiloxane (PDMS). Casting protocols can involve, for example, casting the PDMS material onto a patterned template or mold, which can result in a PMDS layer that can be peeled or pulled intact from the mold. Typically, layers produced by casting fabrication techniques are thicker than those produced by spin fabrication techniques. A resilient block may contain one or more cast or rotated layers in any desired combination.

In some embodiments, first layer 603 may be fabricated according to a rotational scheme. For example, PDMS can be placed on a mold with raised portions corresponding to various desired control channels 610 . The mold can be rotated to create a thin layer of PDMS across the mold. After curing, the first layer 601 can be peeled from the mold and attached to a suitable rigid substrate (eg glass, silicon) or plastic (polystyrene). Alternatively, the first layer 601 may remain attached to the mold. The first layer 601 may contain openings, grooves, or other voids that at least partially form or define the control channel 610 .

To create the second layer 602, a rotational scheme may be used, placing the PDMS on a second mold with raised portions corresponding to the various desired fluid input channels. The second mold may also include, for example, form raised or undulated portions corresponding to the indicia aligned in the second layer 602 . These alignment marks can be used in the laser ablation used to form the vias 625 so that the laser ablation is directed to the alignment marks during the ablation process. The second mold can be rotated to provide a thin layer of PDMS over the entire second mold. The second layer 602 may include openings, grooves, or other voids that at least partially form or define the input channel 620 . In some cases, second layer 602 may be exposed to one or more of the laser ablations described above. The ablative laser beam directed at the second layer 602 may form the via 625 . After the second layer 602 is sufficiently cured, the first layer 601 can be peeled from the second mold and brought into alignment and contact with the first layer. The second layer can be glued to the first layer so that the two layers adhere to the rigid substrate.

To create the third layer 603 using the casting scheme, PDMS can be cast on a third mold with raised portions corresponding to the various desired reaction chambers 630 . After curing, the third layer 603 may be peeled from the third mold and aligned and contacted with the second layer 602 . A third layer can be glued over the second layer so that all three layers adhere to the rigid substrate. Materials from which microfluidic devices can be fabricated include, but are not limited to, elastomers, silicon, glass, metals, polymers, ceramics, inorganic materials, and/or combinations of these materials.

Referring again to FIG. 1 , the pressure source 102 is in fluid communication with the closed control line 110 . The lockout control line includes several sections described more fully below and a one-way valve 112 (also known as a check valve). Additional information on check valves is provided in co-pending and commonly assigned International Patent Application No. PCT/US07/080489 (published as International Publication No. WO 2008/043046 A2, the entire disclosure of which is incorporated herein by reference in its entirety). describe. The first section 114 receives an actuation pressure from the pressure source 102 and is operable to close containment valves present in the array 106 . Array 106 is suitable for performing a variety of microfluidic experiments, as will be appreciated by those skilled in the art. Samples, reagents, etc. present in the reaction chambers in the array can thus be retained in the reaction chambers by closing the containment valves in response to actuation of the lockout control line 110 . Section 114 of lockout control line 110 has no check valve, which allows the containment valve to reopen after removal of the actuating pressure from pressure source 102 .

The second section 116 of the lockout control line 110 is downstream of the one-way valve 112 . Since check valve 112 is operable to prevent fluid flow from section 116 to section 114, actuation of pressure source 102 will cause control fluid (typically a liquid) to pass through check valve 112 and close valves 118a-118e. The thin lines associated with the lockout control line 110 shown in FIG. 1 represent the "flyover" section of the control line, where the design of the control line prevents the assay input line 132 (described more fully below) from being in the lockout control line. Line 110 is activated and then closed. The bold lines associated with valves 118a-118e represent valves operable to close or deactivate manifold 135 (providing fluid flow between assay input lines 132). Closure of valves 118a-118e prevents assay flow from one assay input line to the other through manifold 135, referred to as deactivation of the manifold.

Upon actuation of the latch control line 110 by the pressure source 102, the valves 118a-118e close and the one-way valve 112 keeps the valves 118a-118e closed after the latch control line actuation pressure is removed. Contrary to valves 118a-118e, containment valves actuated by section 114 will open upon removal of the actuating pressure. Thus, the spatial position of the check valve in the latching control line provides one or more segments characterized by latching or non-latching behavior. As is clear to those skilled in the art, the use of additional check valves in the control line provides an additional section with latching behaviour.

Lockout control line 110 also includes a third section 128 having a valve operable to close assay input line 132 from assay input 130 to array 106 . Assay input 130 may also be referred to as an input port. Since the third section 128 is also downstream of the one-way valve 112, actuation of the lockout control line 110 by the pressure source 102 closes the valve in the third section 128 and blocks the assay input line 132 and the array 106 and the Flow between reaction chambers. The valve of section 128 remains closed after the actuation pressure of the latch control line 110 is removed.

A second pressure source 104, referred to as an interface accumulator, is provided in fluid communication with a second control line 120. The second control line also comprises several segments 121 , 123 and 125 . Section 121 contains elevated region 122 to enable actuation of independent valve 124 without closing assay input line 132 . Additional description of valve 124 (referred to herein as an independent or decoupling valve) is provided throughout this specification and described in more detail below. Although the particular independent or partition valve 124 described in FIG. 1 is actuated using the second pressure source 104, this is not required in the present invention. In other embodiments, the individual or partition valves 124 may be mechanical, electrostatic, fluidic, electromechanical, thermodynamic, piezoelectric, or the like. Thus, although the isolation valve 124 is actuated with the second pressure source 104 shown in FIG. 1 , this is not required in some embodiments. Furthermore, while a single second pressure source 104 is used to control multiple valve groups including separate valves, other embodiments may employ a combination of fluid actuation of multiple pressure sources, electrostatic actuation, and the like. Numerous variations, improvements, and substitutions will be apparent to those of ordinary skill in the art.

Section 123 contains valves 134 operable to close five of the six illustrated assay input lines 132 . Since only five valves 134 are provided in the embodiment shown in FIG. 1, actuation of valves 134 in section 123 by pressure source 104 will still leave the rightmost assay input line open. Section 125 includes a one-way valve 127 and a valve 126 operable to close a sample input line 142 in fluid communication with a sample input (ie, input port) 140 and array 106 . It should be noted that although assay and sample inputs are shown in Figure 1, the invention is not limited to assay and sample inputs only, other inputs are included within the scope of the invention.

Placing a one-way or check valve 127 in section 125 of the lockout control line 120 allows 126 the valve to close and remain closed after pressure is applied to the section, preventing fluid flow to and from the array 106 through the sample input line. Since sections 121 and 123 do not contain check valves, deactuation of these sections will cause valves 134 and 124 to reopen.

Some embodiments of the invention provide asynchronous logic functions in the microfluidic device 100 . For example, some embodiments of the present invention provide multiple controls due to the use of a separate or partitioned valve 124 shown in FIG. 1 actuated by a control line 120 that is separate and independent from the other control lines 110 shown. In FIG. 1 , applying pressure to the latch control line 110 prior to actuating the isolation valve 124 latches the containment valve 128 . On the other hand, actuating the latch control line 120 through the interface accumulator 104 to close the partition valve 124 will prevent the containment valve 128 from closing until the latch control line 110 is pressurized by the containment accumulator 102 . In other words, if the isolation valve 124 is closed when pressure is initially applied to the valve 128 , the closed state of the isolation valve prevents the applied pressure from reaching the valve 128 . Thus, the sequence of actuation of control lines 110 and 120 results in different valves being operated, which provides asynchronous logic.

Multiple controls provided by embodiments of the present invention allow array 106 to be "programmed." For example, array 106 may be used in a variety of configurations because array 106 is programmed in a sequence in which multiple valves close, latch, or reopen. In some embodiments described herein, using the 2 pressure sources shown provides three different layouts. These multiple arrangements are provided using check valves as well as divider valves (which can be considered as one in a stacked control valve bank). The split valve, which may limit the control of valve 128 that may be achieved in response to actuation of the latch control line 110, is only one example of a stacked control valve and other arrangements are within the scope of the invention.

The use of check valves in predetermined sections of the control line provides partial lockout of the control line. For example, valves 118 and 128 may be latched by initiating actuation of control line 110 . However, actuation of the initial control line 120 results in closure of the partition valve. Subsequent actuation of control line 110 results in latching of valve 118 but has no effect on valve 128 . Subsequent de-actuation of control line 120 (eg, by releasing pressure applied to the interface accumulator) while maintaining control line 110 actuated results in latching of valve 128 after reopening of the partition valve.

In the microfluidic device 100 shown in FIG. 1 , the six assay input lines 132 are each divided into four input lines, providing a total of 24 assay input lines through the containment valve 128 . Additionally, 24 sample input lines 142 are shown. These specific numbers of input lines are examples only, and other embodiments provide other specific numbers of input lines. For example, in one specific embodiment, the schematic diagram shown in Figure 1 shows only one half of the microfluidic device (eg, the right side of the device), with a matching set of inputs on the left side of the device. Many changes, improvements, and substitutions will be apparent to those of ordinary skill in the art.

Figure 2 is a simplified cross-sectional view of a one-way valve according to one embodiment of the present invention. Figure 2B is a simplified top view of the one-way valve shown in Figure 2A. Referring to FIG. 2A , actuating fluid (typically a liquid) flows into check valves 112 / 127 through inlet 210 and through chamber 220 through through hole 215 . The membrane 230 is lifted or held in a substantially horizontal position by the flow of the actuating fluid so that the actuating fluid passes through the inlet 210 from left to right. Actuating fluid flows through the chamber 220 through the through holes 240 a - 240 d and the through hole 242 and out the outlet 250 . Thus, in the embodiment shown, there is one input through-hole and five output through-holes for a total of six through-holes per check valve. As shown in the top view of Figure 2B, additional structural elements and components are provided depending on the particular design.

When the actuating fluid pressure is removed, membrane 230 collapses over layer 260 , preventing backflow through inlet 210 . Thus, one-way valves 124/127 provide flow from inlet 210 to outlet 250, but prevent flow in the reverse direction. Additional descriptions of one-way valves are provided in the co-pending and commonly-assigned applications cited above.

Some embodiments of the invention provide microfluidic devices incorporating one-way or check valves. In one embodiment, the microfluidic device comprises a first valve and a second valve. Typically, the first valve is one of a plurality of valves forming a first valve group and the second valve is one of a plurality of valves forming a second valve group. A control line in fluid communication with the first valve and the second valve is provided as part of the microfluidic device along with a pressure source in fluid communication with the control line. Referring to FIG. 1 , manifold 135 is deactivated by actuating control line 110 by applying pressure to containment accumulator 102 , causing valve 615 in array 106 and valves 118a - 118e to actuate. The microfluidic device also includes a one-way valve disposed in a control line between the pressure source and the second valve. For example, initially open valve 112 is located on control line 110 between housing accumulator 102 and valves 118a-118e. Check valve 112 provides latching of valves 118a-118e after reducing or removing pressure on control line 110, while the valves present in array 106 (non-latching valves) can reopen to allow sample to flow into the reaction chamber. Thus, by employing check valves to lock a predetermined number of valves in a microfluidic device, control of fluid flow and separation of samples in a microfluidic device is provided that is not possible with conventional designs.

In the embodiment shown in FIGS. 1 and 6A , valve 615 is configured to separate the reaction chambers disposed on the microfluidic device, while valves 118 a - 118 e are configured to connect six lines to assay input port 130 (coupled). The first fluid input line is separated from the second fluid input line of the six lines connected to the assay input port. As described more fully in this specification, the ability to close and latch valves 118a-118e, thereby disabling manifold 135, enables microfluidic devices to be programmed in a variety of ways, which enables fluid flow in input line 132 to be tailored to specific applications. be separated or merged.

In addition to integrating check valves into microfluidic devices with array layouts, some designs provide independent valves, also known as partition valves. For example, a partition valve that is controlled independently of other valves in the device can be operated to prevent the actuation of one or more valves that control Fluid flow in fluid input lines. For example, isolation valve 124 may be closed prior to actuation of control line 110 , thereby preventing closure of valve 128 in response to actuation of control line 110 .

Figure 10 is a simplified schematic diagram of a programmable microfluidic device according to one embodiment of the present invention. The elements shown in FIG. 10 may be provided in a carrier or as shown in FIG. 11 in a microfluidic device attached to a carrier. As shown in FIG. 10 , the outline of a microfluidic device mounted on a carrier is indicated by dashed line 1050 . Referring to Figure 10, two vent sources are provided on opposite sides of the microfluidic device. Discharge sources typically provided as part of the carrier may not be employed in all embodiments. 10 shows 48 sample input lines 1010 at the top, wherein 24 sample input lines are arranged on the left side of the microfluidic device and provide samples to the left side of the array 106, and 24 sample inputs are arranged on the right side of the microfluidic device. Lines and samples are provided to the right of the array 106 . The 48 sample lines are typically provided in a carrier and are in fluid communication with the array 106 through vias 1030 formed in the microfluidic device and aligned with the ends of the sample lines. Sample input lines 142 are provided in the microfluidic device from the through-holes 1030 to the array 106. The 48 sample lines are pressurized by a common pressure source (not shown) that is compatible with where the microfluidic device is placed. The sample portion of the carrier is ligated thereon. Thus, a sample from the 48 sample ports can be loaded into the microfluidic device and pushed into the array 106 via the sample input line. Referring to FIG. 1 , the right portion of array 106 is shown for clarity, wherein the 24 sample input lines of the right portion of FIG. 10 are shown as sample input lines 142 . Accordingly, some embodiments of the invention provide 48 sample input lines with up to 48 different samples. Other implementations provide different numbers depending on the specific application.

In FIG. 10 is shown a control line 1004 which is connected to the interface reservoir 104 shown in FIG. 1 . Additionally, several check valves (CVs) are shown in Figure 10, which provide unidirectional fluid flow through control lines in the microfluidic device. Referring to the lower portion of the array 106, additional CVs are provided for the control lines connected to this portion of the array. A hydration line is provided on the exterior of the array 106 . These hydration lines provide a source of hydration fluid that acts to reduce or prevent evaporation of the fluid in the array.

The 48 input lines shown in the lower part of Figure 10 comprise 12 assay input lines (6 to the left of array 106 and 6 to the right of array 106). The 12 assay lines are pressurized by a common pressure source (not shown) that can be connected to the assay portion of the carrier on which the microfluidic device is placed. Thus, 12 assay port assays can be loaded into the microfluidic device and pushed into 2 arrays 106 via assay input lines. Referring to FIG. 1 , the right array 106 is shown for clarity with the 6 sample input lines shown as assay input lines 132 in the right portion of FIG. 10 . Accordingly, some embodiments of the invention provide 12 assay input lines with up to 12 different assays. Assay input lines pass between the central two arrays 106 of the microfluidic device, 6 lines branch to the right array 106 and 6 lines branch to the left array 106 . Use of the illustrated manifold can provide fewer than 12 assays, eg, as few as a single assay. Other implementations provide different numbers depending on the specific application.

Of the other 36 input lines shown in the lower part of Figure 10, 2 lines are used for hydration and 34 are unused in this implementation. Assay input line 1020 provides the input of a single input line into array 106 , while assay input line 1022 branches into multiple input lines (eg, 4 lines) into assay 106 .

Figure 6A shows an example of 3 input lines per sample, but in the embodiment shown in Figure 10, there are 11 input lines. Also, in Figure 6A, 2 control lines are shown in Figure 6A, but in the embodiment shown in Figure 10, there are 71 control lines that can be manipulated to form 70 reaction chambers/input lines. Thus, in this embodiment there are 770 reaction chambers (11 input lines x 70 reaction chambers) for each sample. With 48 samples distributed into 770 reaction chambers, a high density integrated fluidic circuit (IFC) is provided. In one embodiment, the microfluidic device is capable of testing up to 48 individual samples at one time. Each of the up to 48 samples was distributed into independent groups of 770 reaction chambers, delivering a total of 36,960 simultaneous digital PCR reactions. In another embodiment, removing some of the programmability of the microfluidic device increases the number of reaction chambers/sample for the same device area and component size. For example, one embodiment uses up to 48 samples with 814 reaction chambers/sample.

Using products available from the assignee of the present invention, the entire digital PCR process can be completed in less than 4 hours. Furthermore, the microfluidic devices described herein are components of complete genetic analysis systems. The system may comprise a microfluidic device, a controller for the microfluidic device, a BioMark ^™ system or an integrated thermal cycler, an EP1 reader and associated software. The microfluidic devices described herein are compatible with off-the-shelf reagents and dispensing designs in microwell formats. Many changes, improvements, and substitutions will be apparent to those of ordinary skill in the art.

Figure 11 is a simplified schematic diagram of a microfluidic device according to one embodiment of the present invention. The microfluidic system includes a carrier 1100 and a microfluidic device 1108 mounted on the carrier. Microfluidic device 1108 incorporates the elements described in FIG. 1 . The carrier comprises a plurality of first input ports or holes 1105 arranged in a bank 1106a. The carrier also has a plurality of first input lines 1115 (eg, 48 input lines) in fluid communication with the plurality of first input ports. There is an outer perimeter around the first plurality of inlets to enable pressurization of the first inlets with a common pressure source. In one exemplary application, 48 samples are loaded into a first input port, and applying pressure to the port from the top of the carrier causes the 48 samples to be pushed through a plurality of first input lines and ultimately into fluid on a microfluidic device. Wire.

The carrier also includes a plurality of second input ports 1105 and a plurality of corresponding second input lines 1115 arranged on the stage 1106b. Similar to the first plurality of input ports, the second plurality of input ports is surrounded by an outer perimeter such that fluid dispensed into the second input ports can be pushed through the second input line and into the fluid line in the microfluidic device.

In order to provide fluid communication between the carrier and the microfluidic device placed on the carrier, the microfluidic device has a through hole 1114 formed in the lower part of the microfluidic device, which is connected to the first input line and the second The ends of the input lines are partially aligned. Fluid flowing through the input line passes through the through hole up into the fluid line provided by the microfluidic device. As shown in FIG. 10 , the fluid line connected to the sample input port enters the opposite side of the microfluidic device through through holes 1030 arranged vertically along the left and right sides of the microfluidic device in the figure, and flows to the center of the microfluidic device. In FIG. 6A, these sample input lines may correspond to the horizontally flowing input lines 620 in FIG. 6A. Using these 48 sample input lines, the reaction chambers in each row of the microfluidic device can be filled with different samples.

Through holes 1040 are also provided in the lower part of the microfluidic device and are aligned with the second plurality of input lines 1020 . As shown in FIG. 10 , fluid loaded into the second input port (as measured) flows through the second input line 1020 , through the through hole 1040 and into the manifold 1060 . The microfluidic device contains valves operable with the manifold to cause 1 fluid (sample or assay) or 12 different fluids to be provided into reaction chambers in two arrays. If the application requires a single input fluid, the manifold is opened and the input fluid flows to all fluid lines passing vertically through the center of the array in FIG. 10 . The fluid lines branch off as they pass through the center of the array, eventually flowing through the reaction chamber as they flow through through holes 1030 on the sides of the array. Referring to Figure 6A, these assay input lines may correspond to input lines 620 that pass under the reaction chamber. In this example, the flow on the left array side is from center to left and the flow on the right array is from center to right.

Alternatively, manifold 135 can be closed to introduce 12 different fluids (eg, assays) to be introduced into the reaction chamber (ie, 6 different assays per array). As shown in FIG. 10 , the fluids flow vertically between the arrays and then branch out, with each of the 12 fluids flowing into the rows of the array, passing horizontally from the center to vias 1030 adjacent the edges.

Pressure accumulators 1106c and 1106d are provided on the carrier to enable actuation of control lines and check valves present on the microfluidic device. In a similar manner to the fluidic lines, the control lines 1002 and 1004 in the carrier are in fluid communication with the control lines 110 and 120 on the microfluidic device through through holes formed in the microfluidic device. Thus, while Figure 1 shows the interface of the carrier and housing the reservoirs and control lines in the microfluidic device, it should be understood that this schematic is a simplified one for clarity and convenience. Additional details regarding supports upon which microfluidic devices may be placed are provided in US Patent Application Publication No. 2005/0214173, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

The carrier 1100 has integrated pressure accumulator holes 1101 and 1102 with dry holes 1103, 1104 in each of them for accommodating a valve, preferably a check valve attached to the cover. Carrier 1100 also includes one or more well stations 1106a, b, c, and d, each having one or more wells 1105 (also referred to as input ports) therein. The wells 1105 of each carrier 1100 have channels leading from the wells 1105 to the microfluidic devices 1108 placed in positions 1107 of the carrier. Orifice stations 1106c and 1106d are typically used to provide the pressure used to actuate the control lines present in the microfluidic device 1108 . Fluidic lines for the control fluids are provided, connecting the wells in the well station with valves or other control devices present in the microfluidic device. The microfluidic device is preferably an elastomeric block formed from two or more layers of elastomeric material with microfabricated grooves or channels formed therein.

There are one or more channels in the microfluidic device in fluid communication with one or more through-holes 1114, which in turn provide fluid communication between the channels in the microfluidic device and the channels in the carrier, which then lead to the array of holes 1106a - the holes 1105 in d, thereby providing fluid communication between the holes 1105 in the carrier 1100 and the channels in the microfluidic device 1108. Accumulator hole crowns 1109 and 1110 connect to accumulator holes 1101 and 1102 to form accumulator chambers 1115 and 1116 . Accumulator orifice tops 1109 and 1110 contain valves 1112 and 1111 respectively, which are preferably check valves that introduce gas under pressure into accumulator chambers 1115 and 1116 and retain the gas. When present in accumulator chambers 1115 and 1116 , valves 1111 and 1112 are located inside dry bores 1102 and 1104 so that liquid does not contact valves 111 and 1112 . Valves 1111 and 1112 are preferably opened mechanically by depressing shaves, needles, etc. in the preferred check valves, which overcome the self-closing force of the check valves to release pressure from the accumulator chamber, thereby reducing the pressure in the accumulator chamber. contained fluid pressure.

Carrier 1100 and its associated components may be fabricated from polymers such as polypropylene, polyethylene, polycarbonate, high density polyethylene, polytetrafluoroethylene PTFE or Teflon(R), glass, quartz, metal (such as aluminum), transparent materials, polysilicon, etc. Accumulator orifice tops 1109 and 1110 also contain adjustment screws that are removable to introduce or remove gas or liquid from accumulator chambers 1115 and 1116 . Preferably, valves 1112 and 1111 are actuated to relieve fluid pressure in accumulator chambers 1115 and 1116 that would otherwise remain therein. Use of notch 1117 facilitates proper placement of the microfluidic device in other instruments, eg, instruments used to manipulate or analyze the microfluidic device or reactions performed therein.

Figure 3 is a simplified flowchart showing a method of operating a microfluidic device according to one embodiment of the present invention. In the embodiment shown in Figure 3, a 48-panel layout of the digital array is provided. A first pressure is applied to pressure source 102 (containing the accumulator), which results in actuation of latch control line 110 (310). In a specific embodiment, said first pressure is 30 psi. In other embodiments, other pressures are used depending on the particular application. Referring to FIG. 1 , all valves (not shown) in array 106 will be closed due to the pressure in section 114 . Pressure is applied to section 116 through one-way valve 112, which causes closing and latching of valves 118a-118e. The elevated portion of section 116 prevents the assay input line from closing due to actuation of section 116 . Closure of valve 118 closes the flow of input fluid through manifold 135, thereby isolating the six assay input lines connected to assay input 130 from each other.

It should be noted that in the control flow shown in Figure 3, the pressure source 104 is not used. Therefore, there is no actuation of control line 120 or closure of partition valve 124 . Thus, in response to actuation of the latch control line 110, the containment valve 128 is closed and latched. Thus, upon application of a first pressure (eg, 30 psi) by the containment accumulator, the array valves close and valves 118 and 128 close and latch.

Decreasing the first pressure (312) of the pressure source 102 reopens the array valve (not shown). In some embodiments, the pressure is removed during process (312) to bring the applied pressure to zero. As noted above, valves 118 and 128 will remain latched in the closed state. Sample supplied from sample input or port 140 is loaded ( 314 ) into the reaction chambers in the array through sample input line 142 . The design of the chip provides for loading through sample input line 142, assay input line 132, or both. Since in this embodiment the assay input line is closed by the lock valve 128, loading takes place via the sample input line. After sample loading is complete, a second pressure is applied to pressure source 102 (316), thereby closing the array valves and isolating the samples in the reaction chambers. In some embodiments, the second pressure is equal to the first pressure (eg, 30 psi), although this is not required in the present invention. Other suitable pressures for closing the array valves are within the scope of the present invention. Furthermore, although the first pressure is removed in some embodiments, this is not required in the present invention, as some embodiments can reduce the pressure to a non-zero value and still provide sufficient fluid flow to load the sample.

Thus, the embodiment shown in Figure 3 provides for a loading of 48 samples (ie, 24 indicated sample input lines x two sides of the device). Other sample layouts are also provided by the programmable digital arrays described herein, as discussed below.

It should be understood that the specific steps shown in Figure 3 provide one specific method of operating a microfluidic device according to one embodiment of the present invention. Other sequences of steps may also be implemented according to some alternative embodiments. For example, some alternative embodiments of the invention may perform the steps listed above in a different order. Furthermore, a single step shown in FIG. 3 may contain multiple sub-steps, which may be performed in various orders depending on the requirements of the single step. Furthermore, additional steps may be added or removed depending on the particular application. Many changes, improvements, and substitutions will be apparent to those of ordinary skill in the art.

Figure 4 is a simplified flowchart showing a method of operating a microfluidic device according to another embodiment of the present invention. Unlike the 48-panel layout depicted in FIG. 3, a single-panel layout is provided in the embodiment shown in FIG. A first pressure is applied to pressure source 104 ( 410 ), which causes actuation of latch control line 120 . Pressure will be applied through one-way valve 127 to close and latch valve 126 in section 125 . Therefore, there is no flow through the sample input line 142 in this layout. Elevated section 122 delivers control pressure to close partition valve 124 .

Section 123 will communicate the control pressure in lockout control line 120 to valves 134 associated with 5 of 6 assay input lines 132 . Therefore, valve 134 will be closed so that flow is only through the rightmost assay input line.

A single sample is loaded via the rightmost assay input line (412). Since section 116 of latch control line 110 is not actuated in this arrangement, manifold 135 is open so that a single sample contained in the rightmost sample input is provided to all sample input lines 132 . Thus, the array is loaded with the assay input using a single sample from the rightmost sample assay input. Applying the second pressure to the pressure accumulator 102 actuates the latch control line 110 and closes the array valves connected to the section 114 . Thus, multiple layouts using a single programmable digital array are possible, for example, single-panel layouts, using different control flows.

It should be understood that the specific steps shown in FIG. 4 provide a specific method of operation of the microfluidic device according to another embodiment of the present invention. Other sequences of steps may also be implemented according to alternative embodiments. For example, some alternative embodiments of the invention may perform the steps listed above in a different order. Furthermore, a single step shown in FIG. 4 may contain multiple sub-steps, which may be performed in various orders depending on the needs of the single step. Furthermore, additional steps may be added or removed depending on the particular application. Many changes, improvements, and substitutions will be apparent to those of ordinary skill in the art.

Figure 5 is a simplified flowchart showing a method of operating a microfluidic device according to yet another embodiment of the present invention. In the embodiment shown in Figure 5, a 12-panel layout of the digital array is provided. A first pressure is applied to pressure source 104 (510). In a specific embodiment, the first pressure is 45 psi, although this is not required in the present invention and other suitable pressures may be used. As discussed with respect to process (410) of FIG. 4, the latch control line 120 is actuated. Pressure is applied through one-way valve 127 to close and latch valve 126 in section 125 . Therefore, there is no flow through the sample input line 142 in this layout. Elevated section 122 will deliver the control pressure to close partition valve 124 . As discussed below, the temporary closure of the partition valve prevents containment valve 128 from closing upon subsequent actuation of control line 110 . Section 123 will communicate the control pressure of lockout control line 120 to valves 134 associated with 5 of 6 assay input lines 132 . Therefore, the valve 134 is temporarily closed.

Applying a second pressure to the pressure source 102 (512) actuates the latch control line 110. In a specific embodiment, the second pressure is 30 psi, which is less than the first pressure (eg, 45 psi). As discussed below, the second pressure is sufficient to close the intended valve without opening the partition valve 124 that was closed in process (510). Other pressures may be used depending on the application. Actuation of section 114 closes the valves in array 106 . Pressure is applied to section 116 via one-way valve 112, which causes closing and latching of valves 118a-118e. The elevated portion of section 116 prevents the assay input line from closing due to actuation of section 116 . Closure of valve 118 closes the flow of input fluid through manifold 135 , thereby isolating each of the six assay input lines connected to assay input 130 .

Unlike initial actuation of control line 102 , initial actuation of control line 104 closes the partition valve, thereby providing a programmable sequence in which actuation of control line 102 does not cause valve 128 associated with assay input line 132 to latch. This multiple control feature provides asynchronous logic functionality employing some embodiments of the present invention, which is not available using conventional microfluidic devices.

The pressure applied to pressure source 102 is reduced, eg, to zero (514). Because section 116 is downstream of one-way valve 112, valve 118 continues to latch in the closed position after section 116 is deactuated, which prevents fluid flow between the six assay input lines. Deactivation of section 114 opens the valves in the array, allowing the sample to be loaded during subsequent loads. As discussed above, valve 128 is not latched during process (512).

The pressure applied to the pressure source 104 is reduced, eg, to zero (516). Valve 134 is reopened by reducing the pressure in section 123 . As discussed above, assay input 130 is used to provide input to the reaction chambers in the array. Partition valve 124 is reopened by reducing the pressure in elevated section 122 in section 121 . Since no pressure is applied to control line 110 at this stage of the programming process, valve 128 remains open. Although shown as reducing the pressure to zero, these specific pressures are not required for the invention and other suitable pressures may be used.

Samples are loaded (518) from the 6 assay inputs. Separation between assay inputs is maintained by de-functionalization of manifold 135 due to previous latching of valve 118 . Thus, in this layout, a 12-panel layout (6 assay inputs x 2 sides of the device) is provided. A third pressure is applied to pressure source 102 ( 520 ) to close the array valve associated with section 114 . Furthermore, since the partition valve 124 is reopened, the valve 128 is latched closed.

It should be understood that the specific steps shown in Figure 5 provide a specific method of operating a microfluidic device according to yet another embodiment of the present invention. Other sequences of steps may also be implemented according to alternative embodiments. For example, alternative embodiments of the invention may perform the steps listed above in a different order. Furthermore, a single step shown in FIG. 5 may contain multiple sub-steps, which may be performed in various orders based on the single step. Furthermore, additional steps may be added or removed depending on the particular application. Many changes, improvements, and substitutions will be apparent to those of ordinary skill in the art.

Table 1 provides the actuation pressure and loading process for one of several possible panel layouts for the programmable digital array microfluidic devices described herein. The pressures applied to pressure sources 102 and 104 are described as exemplary pressures, although other suitable pressures are within the scope of embodiments of the present invention.

Table 1

The integration of check valves and divider valves with the digital array provides a multi-user programming layout using a single high-density digital array chip. Accordingly, some embodiments of the invention provide cost savings and increased experimental throughput. The serial or asynchronous logic described herein provides enhanced device functionality since the state of a particular valve is not only dependent on the actuation of a control line in fluid communication with the particular valve, but also on the state of a separate valve actuated by an independent control line . Therefore, the sequence of pressurization determines the logic output of the panel layout. Accordingly, some embodiments of the present invention provide device layouts and functions not achievable with conventional microfluidic devices.

According to one embodiment of the present invention, a method of setting up a microfluidic device having a plurality of control lines is provided. Serial or asynchronous logic is implemented according to the method. The method includes actuating a first control line and placing a valve in a first state. A valve (which may be one of a plurality of valves) may be placed in a closed state in response to actuation of said first control line. For example, isolation valve 124 may close in response to applied pressure to interface accumulator 104 and pressurization of control line 120 . As described more fully throughout this specification and described in more detail below, the separation valve may provide multiple control functions due to its placement between valve 128 and containment accumulator 102 , which provides serial logic operation.

The method also includes, after placing the valve in the first state, actuating a second control line operable to place the valve set in the second state. The fact that the valve is in the first state (ie, closed) prevents the valve set from being placed in the second state (eg, closed state). As shown in FIG. 1 , closing isolation valve 124 prior to actuation of control line 110 prevents valve 128 from closing (and latching) in response to actuation of control line 110 . Accordingly, some embodiments of the present invention provide serial logic functions in which the sequence of valve actuations produces different outcomes selected from several final states. In this example, initial actuation of control line 110 results in closing and latching of valve 128 . On the other hand, initial actuation of the control line 120 results in the closing of the partition valve 124 which, while held in the closed state, prevents the closing and latching of the valve 128 . Although FIG. 1 shows control lines 110 and 120 actuated by applying pressure to separate pressure sources 102 and 104, other methods of actuation are within the scope of the present invention.

According to another embodiment of the present invention, another method of implementing serial logic using a microfluidic device is provided. A method of setting up a microfluidic device having a plurality of control lines includes establishing a first state of the microfluidic device by actuating a first control line and then actuating a second control line. The first state of the microfluidic device may include setting the valve to an open or closed state in response to actuation of the plurality of control lines. For example, actuating the control line 120 prior to actuating the control line 110 results in closure of the isolation valve 124, as described above. Thus, valve 128 does not latch closed in response to actuation of control line 110 .

The method also includes establishing a second state of the microfluidic device by actuating a second control line prior to actuating the first control line. The programmable nature of the microfluidic devices described herein provides for different final states of the microfluidic device depending on the sequence of valve actuation. Thus, in this example, actuating the control line 110 prior to actuating the control line 120 results in the closing and latching of the valve 128 followed by the closing of the partition valve 124 . Closing the partition valve after latching valve 128 has no effect because check valve 112 maintains a constant pressure on the control line downstream of the check valve, preventing closure of the partition valve from affecting the state of the microfluidic device.

As an example of a microfluidic device implementing serial logic, a first state of a microfluidic device may include a closed or blocked state of a first set of input lines (eg, input line 142 blocked by valve 126 ) and communication with multiple input lines in the microfluidic device. A second set of input lines (eg, input line 132) in fluid communication with the reaction chambers. The second state may comprise the first set of input lines (e.g., input lines 142) in fluid communication with a plurality of reaction chambers in the microfluidic device, and a second set of input lines (e.g., valve 128) in a closed or blocked state. blocked input line 132). The second state may also include a set of valves (eg, valves 118a-118e) in a closed state, preventing fluid flow through the manifold connected to the second set of input lines.

It should be noted that although the separation valve is actuated by control line 120, other embodiments may employ another separation valve actuated by control line 110 in place of or in addition to the separation valve shown. Thus, for example, an overhead line may lead from containment accumulator 102 to an additional divider valve located between interface accumulator 104 and check valve 127 . Closing this additional isolation valve prior to applying pressure to section 125 of the control line connected to interface accumulator 104 prevents valve 126 from closing and latching in response to actuation of control line 120 . Thus, although Figure 1 shows a specific implementation, the present invention is not limited to this specific implementation and other microfluidic device designs are included within the scope of the present invention.

Furthermore, while the embodiment shown in FIG. 1 and the alternative geometries discussed above provide two levels of multiple control by employing a separate valve that is actuated by applying pressure to one of the two control lines, the present invention The implementation provides additional levels of multiple control. For example, additional independent valves (eg, electrostatically actuated) can be integrated into the design of the microfluidic device to provide a third level of control. In addition to actuation of the 2 control lines shown, initial actuation of this additional independent valve will drive a logic output of device status.

Figure 7 shows a method of programming a programmable microfluidic device according to one embodiment of the present invention. In the embodiment shown in Figure 7, the microfluidic device is programmed to partition the reaction chambers of the microfluidic device into 48 individual panels. The specific number of panels depends on the actual design of the microfluidic device, and in alternative embodiments may be a different number, such as 12 or 192 panels. The reaction chambers of each individual panel can be filled with different samples, providing in this embodiment the simultaneous detection of up to 48 different samples.

As shown in FIG. 1 , the microfluidic device has an array of reaction sites or chambers arranged in an array 106 . The reaction sites are in fluid communication with the first set of input lines 132 and the second set of input lines 142 . The method includes actuating (710) a first set of valves operable to block (712) a first set of input lines. For example, the first set of valves is valve 128 , also referred to as a containment valve operable to block or close input fluid line 132 . Fluid flow through input line 132 is prevented, such as by closing valve 128 by applying pressure to containment accumulator 102 . The control line 110 is a latching control line due to the presence of the one-way or check valve 112 which remains closed after the containment valve 128 is closed.

The method also includes actuating (714) a second set of valves operable to separate (716) the reaction sites. As shown in Figure 6B, a reaction chamber 630 is defined in the elastomeric layer 603 of the microfluidic device. The second set of valves is arranged in an array and is shown as valve 615 in FIG. 6A . In addition to the first valve group 128 and the second valve group 615, a third valve group 118a-118e is actuated simultaneously with said first valve group. The presence of check valve 112 also enables valves 118a-118e to latch when closed. In the embodiment shown in FIG. 1 , the first set of valves and the second set of valves are simultaneously actuated or closed in response to pressure applied to control line 110 .

After the first, second, and third sets of valves are closed, the second set of valves is deactuated ( 718 ), for example, by reducing or eliminating pressure applied by pressure source 102 to control line 110 . Deactuation of the valve results in opening of the valve, which allows fluid to flow through an input line connected to the valve. A plurality of samples is loaded into the reaction site via the second set of input lines 142 (720). In FIG. 1 , the sample is contained in sample input or port 140 . Each side of the microfluidic device contained 24 different input ports, providing a total of 48 input ports, although this specific number may vary depending on the specific design of the microfluidic device. The sample port can contain 48 different samples or fewer total samples when a single sample is provided at multiple input ports. Thus, although Table 1 refers to a 48-panel layout, the programmable microfluidic device actually provides up to 48 individual panels depending on the samples provided at the 48 sample ports. After loading the sample into the reaction site, the second set of valves is actuated a second time to separate the reaction chambers from each other (722).

It should be understood that the specific steps shown in Figure 7 provide a specific method for programming a microfluidic device according to one embodiment of the present invention. Other sequences of steps may also be performed according to alternative embodiments. For example, alternative embodiments of the invention may perform the steps listed above in a different order. Furthermore, a single step shown in FIG. 7 may contain multiple sub-steps, which may be performed in various orders from a single step. Furthermore, additional steps may be added or removed depending on the particular application. Many changes, improvements, and substitutions will be apparent to those of ordinary skill in the art.

Figure 8 shows a method of programming a programmable microfluidic device according to another embodiment of the present invention. The method shown in Figure 8 provides a method for operating a programmable microfluidic device with a single panel and all reaction chambers containing the same sample. As shown in Figure 1, the programmable microfluidic device has an array of reaction sites in fluid communication with a first set of input lines and a second set of input lines. In the exemplary embodiment shown in FIG. 8 , the first set of input lines are input fluid lines 142 connected to sample input 140 and the second set of input lines are input fluid lines 132 connected to assay input 130 . The first input line set is actuated to a closed position (810), flow through the first input line set is blocked (ie, closed) (812). Thus, in one example, pressurizing the interface accumulator 104 causes the valve 126 to close, preventing fluid flow through the input line 142 . The presence of the check valve 127 causes the first valve group to be latched in the closed position.

The method also includes actuating (814) a second set of valves operable to block (816) the first portion of the second sub-set of input lines. Referring to FIG. 1 , the first portion of the second set of input lines is the portion of input line 132 below valve 134 , the second set of valves being valves 134 operable to block or shut off the associated six assay inputs 130 5 of the 6 input lines 132. Thus, in this embodiment, the subgroup of input lines is 5 of the 6 input lines, using the right-most assay input port (connected to the right-most input line) shown in Figure 1 to provide the loading reaction chamber or site samples. Input lines not included in the subgroup (ie, the rightmost input line) are open and used to load the sample into the reaction chamber as described more fully below. No other assay inputs were used in this implementation. In the embodiment shown in FIG. 1 , the pressure applied by interface accumulator 104 actuates valve 126 and valve 134 simultaneously.

Referring to FIG. 1 , actuation of the valves through the use of control line 110 is not implemented. Thus, loading a sample into the rightmost assay input will cause the sample to flow through section 116 and through manifold 135 to the second portion of the second set of input lines (i.e., the end of input line 132 above valve 134). part). Samples are loaded into reaction chambers or sites in array 106 through the second set of input lines (818). It should be noted that the manifold is deactivated (ie, flow through the manifold is prevented) by actuation via section 116 of control line 110 which in this embodiment is not pressurized. Thus, the manifold opens and connects all 6 input fluid lines at the beginning of the second section of input lines 132 . Through manifold 135 , the sample is distributed to a branched portion of input line 132 , eventually flowing through 24 input lines, through open valve 128 . Thus, a single sample is provided to all reaction chambers in array 106 . To partition the sample in the reaction chamber (822), a third valve set (820), such as containment valve 615 shown in FIG. 6A, is actuated. Applying pressure to the control line 110 by using the containment accumulator 102 provides the pressure to close the valves 615 in the array 106 .

It should be understood that the specific steps shown in FIG. 8 provide a specific method for programming a microfluidic device according to another embodiment of the present invention. Other sequences of steps may also be implemented according to alternative embodiments. For example, alternative embodiments of the invention may perform the steps listed above in a different order. Furthermore, a single step shown in FIG. 8 may contain multiple sub-steps, which may be performed in various orders from a single step. Furthermore, additional steps may be added or removed depending on the particular application. Many changes, improvements, and substitutions will be apparent to those of ordinary skill in the art.

Figure 9 shows a simplified method of programming a microfluidic device according to yet another embodiment of the present invention. The programmable microfluidic device has an array of reaction sites in fluid communication with a first set of input lines, a second set of input lines, and a manifold connecting the second set of input lines. The method includes actuating (910) a first set of valves operable to block (912) a first set of input lines. Referring to FIG. 1 , the first set of valves may be valve 126 downstream of check valve 127 . The presence of check valve 127 enables valve 126 to latch in the closed position after actuation. The second valve set (914) is actuated to block the first portion of the second input line set sub-set (916). Application of pressure from interface accumulator 104 to control line 120 results in actuation of valve 126 and valve 134 .

Referring to FIG. 1 , the first part of the second input line group is the input line 132 part under the valve 134, and the second valve group is the valve 134, which can be operated to block or close the 6 input lines connected to the 6 measuring inputs 130. 5 of the input lines. Thus, in this embodiment, the subset of input lines is 5 out of 6 input lines. Because the valves 134 are not blocked by the one-way valves, fluid can flow through the first portion of all six input lines 132 to the manifold 135 when these valves are reopened. In the embodiment shown in FIG. 1, application of pressure from interface accumulator 104 actuates valve 126 and valve 132 simultaneously.

The method also includes actuating (918) a third set of valves operable to deactivate (920) the manifold. In an exemplary embodiment, actuation of the third set of valves includes applying a predetermined pressure to a pressure source (eg, housing accumulator 102 ) in fluid communication with the second set of valves. Referring to FIG. 1 , actuating valves 118 a - 118 e by applying pressure to section 116 of control line 110 blocks flow through the manifold to provide separation of fluid flowing through input line 132 . The presence of the check valve 112 causes the valves 118a-118e to latch in the closed position. As described more fully below, deactivation of manifold 135 allows 12 samples (6 samples per side of the microfluidic device) to be loaded into the reaction chamber from 6 assay inputs 130 . As discussed in the embodiment of Figure 7, fewer than 12 samples can be loaded by providing the same sample to more than one assay input. Many changes, improvements and substitutions will be apparent to those skilled in the art.

Referring again to FIG. 1 , it should be noted that initial actuation of control line 120 prior to actuation of control line 110 results in not only closing and latching of valves 126 and 134 , but also closure of divider valve 124 . Since the separation valve is disposed between containment accumulator 102 and valve 128 , closure of the separation valve prevents valve 128 from closing in response to actuation of control line 110 . In the embodiment shown in FIG. 9, the pressure used to actuate control line 120 is a predetermined pressure sufficient to prevent actuation of control line 110 causing valve 128 to close. As shown in Table 1, a pressure of 45 psi is sufficient to hold valve 128 in the open position while 30 psi is applied to control line 110 . The specific values of 45 psi and 30 psi are not required by the invention and other pressures may be used to perform this multiple control function.

The method also includes deactuating the second set of valves (922). Typically, the second set of valves is deactuated by reducing the first pressure applied to the first pressure source, for example to 0 psi, so that it assumes the open position. The reduction in pressure applied to the control line returns the flexible membrane deflected into the input fluid line to a substantially undeflected position, restoring fluid flow through the input fluid line. After reopening valve 134, the sample provided in assay input 130 is loaded into the reaction site (924). As shown in FIG. 1 , a first portion of each of the second set of input lines (e.g., the portion of input line 134 below valve 134) is in fluid communication with an assay input configured to receive one of a plurality of samples. one. The sample flows through the first and second portions of the second set of input lines to the reaction site. As shown in Figures 6A and 6B, the reaction sites may be formed in the form of a plurality of reaction chambers, wherein each reaction chamber is in fluid communication with the second portion of the input fluid line 132 through a through hole through which the input fluid The wires reach the reaction chamber, which may be arranged in an elastomeric microfluidic device layer above the layer containing the fluidic wires.

As shown in FIG. 1 , the portion of input line 132 located above valve 134 branches into four sets of input lines that are in fluid communication with reaction sites or chambers in array 106 . By providing 6 different samples in each assay input port, a total of 12 panels can be defined in the array (since Figure 1 only shows the first side of the microfluidic device). As will be apparent to those skilled in the art, using one sample in more than one input port results in fewer than 12 panels being produced. To isolate the reaction sites, a fourth set of valves (926), such as valve 615, is actuated.

In some implementations, the method further includes actuating a valve to prevent closure of a fifth set of valves, wherein the fifth set of valves is operable to close the second portion of the second set of input lines. As shown in FIG. 1 , closure of the isolation valve 124 prevents the valve 128 from closing in response to actuation of the control line 110 . In the embodiment shown in FIG. 9, an initial pressure of 45 psi applied to control line 120 causes closure of the divider valve, leaving the second portion of input line 132 open while the manifold is stopped by closing valves 118a-118e. use. The separation valve may be actuated by applying pressure to a control line or may be actuated by other means including, but not limited to, mechanical, electrostatic, and the like. In applications using pressure-based control lines, multiple valves and/or valve banks can be actuated simultaneously using a single control line. Thus, when interface accumulator 104 is pressurized, isolation valve 124 and valves 126 and 134 are simultaneously actuated in response to actuation of control line 120 .

The programmable nature of the microfluidic devices provided herein is evidenced by the fact that changing the sequence of valve actuation steps results in different states of the valves in the array, and thus of the array. For example, in the embodiment shown in Figure 9, 12 samples can be loaded into the array by closing the isolation valve 124 and then applying pressure to the control line 110, which deactivates the manifold 135, The pressure to accumulator 102 is then reduced, and then the pressure to accumulator 104 is reduced. In this example, pressure is maintained on accumulator 104 while pressure is applied through accumulator 102 to prevent valve 128 from closing in response to actuation of control line 110 by applying pressure using accumulator 102 . On the other hand, releasing the pressure applied to the accumulator 104 while applying pressure through the accumulator 102 results in the closing of the valve 128 . Thus, two different states of the microfluidic device, the ability to load a sample through the input line 132 or to block the input line 132, are achieved by changing the sequence of valve actuation. This programmable feature provides benefits not available with conventional designs.

It should be appreciated that the specific steps shown in Figure 9 provide a specific method of programming a microfluidic device according to yet another embodiment of the present invention. Other sequences of steps may also be implemented according to some alternative embodiments. For example, some alternative embodiments of the invention may perform the steps listed above in a different order. Furthermore, a single step shown in FIG. 9 may contain multiple sub-steps, which may be performed in various orders from a single step. Furthermore, additional steps may be added or removed depending on the particular application. Many changes, improvements, and substitutions will be apparent to those of ordinary skill in the art.

It is to be understood that this invention is not limited to the particular methodology, protocols, reagents, etc. described herein, as variations in these will be appreciated by those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. It should also be noted that as used herein and in the appended claims, unless the context clearly dictates otherwise, an unspecified number includes the plural. Thus, for example, reference to "a cell" refers to one or more cells, equivalents of which are known to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Embodiments of the invention and details of its various features and advantages are explained more fully with reference to the non-limiting embodiments and examples which are described and/or illustrated in detail in the accompanying drawings and the following description. It should be noted that features shown in the drawings are not necessarily drawn to scale, and features of one embodiment may be used in other embodiments, even if not explicitly stated herein, as will be understood by those skilled in the art. Descriptions of well-known components and processing techniques may be omitted so as not to unnecessarily obscure the embodiments of the invention. The examples used herein are provided merely to facilitate understanding of the manner in which the invention may be practiced and to further enable those skilled in the art to practice the embodiments of the invention. Accordingly, the examples and embodiments herein should not be construed as limiting the scope of the invention, which is defined only by the appended claims and applicable law. Furthermore, it should be noted that like reference numerals indicate like parts throughout the several views of the drawings.

Therefore, the "Definitions" section is given below, in which some terms related to the present invention are specifically defined for clarity, but all definitions are consistent with the understanding of these terms by those skilled in the art. Specific methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All references referred to herein are incorporated by reference in their entirety.

definition

PNA is peptide nucleic acid (peptide nucleic acid)

LNA is locked nucleic acid (locked nucleic acid)

DA is a dynamic array (dynamic array)

PCR is polymerase chain reaction

BSA is bovine serum albumin (bovine serum albumin)

FRET is Fluorescence resonance energy transfer

GT for genotyping

PEG is polyethylene glycol

PLP is a padlock probe

The term "proximate" as used herein generally refers to the position of a primer relative to a probe on the complementary strand of a target nucleic acid analyte. Primers and probes may be separated by about 1 to about 20 nucleotides, more specifically, by about 1 to about 10 nucleotides, or may be directly adjacent to each other.

The term "analyte" as used herein generally refers to a nucleic acid molecule or mixture of nucleic acid molecules as defined below to be detected or quantified using the methods of the invention. The terms "target nucleic acid analyte" and "nucleic acid analyte" are used interchangeably with the term "analyte" in the present invention.

The terms "complementarity" or "complementarity" as used herein may encompass the natural association of polynucleotides by base pairing under permissive salt and temperature conditions. For example, the sequence "A-G-T" binds its complement "T-C-A". Complementarity between two single-stranded molecules can be "partial", in which only some of the nucleic acids bind, or complete when there is overall complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands plays an important role in the efficiency and strength of hybridization between nucleic acid strands. This is especially important in amplification reactions that depend on binding between nucleic acid strands and the design and use of molecules.

As used herein, the term "covalently linked" generally refers to the attachment of one molecular structure to another molecular structure by a covalent chemical bond.

The term "dye" as used herein generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation at wavelengths greater than or equal to 340 nm.

The term "fluorochrome" as used herein generally refers to any dye that emits longer wavelength electromagnetic radiation by a fluorescence mechanism upon illumination by a source of electromagnetic radiation (eg, a lamp, photodiode, or laser).

The term "GT sample buffer" as used herein generally refers to a buffer capable of blocking the binding sites of reaction channels and chamber surfaces in a DA chip. The buffer prevents the loss of reaction components during the chip loading process or reaction. It also reduces the use of additional Taq-Gold polymerase by less than about 80% in terms of reagent costs. 20×GT buffer may include the following combinations: Betaine (FW 117.15), BSA, T20 (in PBS) (Thermo Scientific, Rockford, IL), (in PBS) (Thermo Scientific, Rockford, IL), (in TBS) (Thermo Scientific, Rockford, IL), T20 (in TBS) (Thermo Scientific, Rockford, IL), glycerol, PEG 20,000, PEG MME550, PEG MME5000 and Tween 20.

The term "homogenous assay" as used herein generally refers to a method of detecting or quantifying a nucleic acid analyte that does not require post-assay processing to record assay results. Homogeneous assays can be performed in closed tubes or microfluidic arrays without the need for further addition of reagents or supplemental chemicals after assay initiation to document results. Homogeneous assays enable real-time recording of assay results, which means that assay results can be continuously recorded as the assay progresses over time.

The term "hydrolysis probe" as used herein is generally described in US Patent No. 5,210,015, which is incorporated herein by reference in its entirety. The hydrolysis probe utilizes the 5'-nuclease activity present in the thermostable Taq polymerase used in the PCR reaction ( Probe Technology, Applied Biosystems, Foster City CA). The hydrolysis probe is labeled with a fluorescent detection dye (such as fluorescein) and a receptive dye or quencher. Typically, a fluorescent dye is covalently attached to the 5' end of the probe and a quencher is attached to the 3' group of the probe. When the probe is intact, the fluorescence of the detection dye is quenched due to fluorescence resonance energy transfer (FRET) . The probe can be annealed downstream of a primer that defines one end of the PCR reaction nucleic acid target analyte amplification target. Utilizing the polymerase activity of the Taq enzyme, amplification of the target nucleic acid analyte is directed by one primer upstream of the probe and a second primer downstream of the probe but annealing to the opposite strand of the target nucleic acid. As the upstream primer is extended, Taq polymerase reaches the region where the labeled probe anneals, recognizes the probe-template hybrid as a substrate, and hydrolyzes the phosphodiester bond of the probe. The hydrolysis reaction irreversibly releases the quencher dye from quenching of the reporter dye, thus resulting in an increase in detector fluorescence with each successive cycle of PCR. Specifically, the hydrolysis probes of the present invention are capable of common 8-mer or 9-mer motifs in human and other transcriptomes and can have a high _Tm of about 70°C by employing LNA analogs.

The term "label" as used herein refers to any atom or molecule that can be used to provide a detectable and/or quantifiable signal. In particular, the label can be attached to a nucleic acid or protein. A label can provide a signal detectable by fluorescence, radioactivity, colorimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.

The term "nucleic acid" as used herein generally refers to cDNA, DNA, RNA, single- or double-stranded and any chemical modifications thereof, such as PNA and LNA. LNAs are described in US Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, which are hereby incorporated by reference in their entirety. Nucleic acids can be of any size. Nucleic acid modifications can include the addition of chemical groups that add additional charge, polarity, hydrogen bonding, electrostatic interactions, and functionality to individual nucleic acid bases or to the nucleic acid as a whole. This modification can include modified bases such as 2'-position sugar modification, 5-position pyrimidine modification, 8-position purine modification, modification of cytosine exocyclic amine, substitution of 5-bromouracil, backbone modification, methyl Chemicalization, abnormal base pairing combinations such as isobase isocytidine and isoguanidine, etc. Nucleic acids can be derived from entirely chemical synthetic processes (e.g., solid-phase-mediated chemical synthesis) or from biological sources (e.g., by isolation from virtually any species from which nucleic acids can be provided) or from processes involving the manipulation of nucleic acids by molecular biological tools (e.g. DNA replication, PCR amplification, reverse transcription) or from a combination of these processes.

The term "nucleic acid probe" as used herein is a nucleic acid that carries at least one covalently attached dye, such as a fluorescent dye. In particular, the probes do not contain sequences complementary to the sequences used to prime the PCR reaction.

The term "padlock probe" or "PLP" as used herein generally refers to a linear oligonucleotide having a length of about 100 base pairs. The sequences at the 3' and 5' ends of the PLP are complementary to adjacent sequences in the target nucleic acid analyte. In the central non-complementary region of the PLP there is a "tag sequence" that can be used to identify a particular PLP. The tag sequence may be flanked by universal primer sites or unique and/or specific primer sites that allow PCR amplification of the tag sequence. After hybridization to the target, the 5' and 3' ends of the PLP are in close proximity and can then be ligated. The resulting product is a circular probe molecule attached to the target nucleic acid analyte. For example you can use Real-time PCR amplifies and quantifies and/or detects the tag region of circularized PLP. The presence and amount of the amplicon can be correlated to the presence and amount of the target sequence in the sample. For a description of PLP see, for example, Landegren et al., 2003, Padlock and proximity probes for in situ and array-based analyses: tools for the post-genomic era, Comparative and Functional Genomics 4:525-30; Nilsson et al., 2006, Analyzing genes using closing and replicating circles Trends Biotechnol. 24: 83-8; Nilsson et al., 1994, Padlock probes: circularizing oligonucleotides for localized DNA detection, Science 265: 2085-8. The above references are incorporated herein by reference in their entirety.

The term "PCR" as used herein generally refers to a method for amplifying, detecting or quantifying a specific region of an analyte. Those skilled in the art will appreciate that there are several variations on the basic PCR technique, such as allele-specific PCR, assembly PCR (assembly PCR) or polymerase cycling assembly (PCA), colony PCR, helicase-dependent Sexual amplification, hot-start PCR, intersequence-specific (ISSR) PCR, inverse PCR, ligation-mediated PCR, methylation-specific PCR, multi-site ligation-dependent probe amplification, multi-site PCR, nested PCR, overlap extension PCR, quantitative PCR, quantitative real-time PCR, RT-PCR, thermal asymmetric interlaces (TAIL) PCR, touchdown PCR, and PAN-AC. Furthermore, those skilled in the art will understand how to apply these changes to the underlying PCR technology.

The term "purification" as used herein generally refers to any process by which a protein, polypeptide or nucleic acid is separated from other components or compounds on the basis of charge, molecular size or binding affinity.

The term "quencher" as used herein generally refers to a dye that reduces the fluorescence emission of another dye.

The term "querying" as used herein generally refers to determining whether a target-specific probe is associated with (eg, linked to or linked to) a nucleic acid analyte, and optionally quantifying the amount of the target-specific probe in a sample.

"Sample" as used herein generally refers to tissue or fluid samples from humans or animals, including but not limited to plasma, serum, spinal fluid, lymph fluid, external sections of skin, respiratory system, intestine and genitourinary tract, tears, saliva, Samples of blood cells, tumors, organs, tissues, and in vitro cell culture components. Specifically, samples can be single cells, paraffin-embedded tissue samples, and needle biopsies. Additionally, samples may include environmental samples such as lake water and food samples.

As used herein, the words "substantially purified" or "substantially isolated" generally include nucleic acid or amino acid sequences taken, isolated or isolated from their natural environment, which are at least about 60%, especially at least about 75%, most especially At least about 90% other components associated therewith, and include recombinant or cloned nucleic acid isolates and chemically synthesized analogs or systemically biologically synthesized analogs.

Considering the great diversity of polymer chemistries, precursors, synthesis methods, reaction conditions, and available additives, there is a large number of available elastomeric systems that can be used to fabricate elastic modules, layers, membranes, microvalves, pumps, etc. Variations in the materials used may in some cases be dictated by the need for specific material properties, namely solvent resistance, stiffness, gas permeability or temperature stability. There are many types of elastic polymers. A brief description of the most common elastomer types is given here in order to show that even with relatively "standard" polymers there are many possibilities for bonding. Commonly used elastomeric polymers include polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), polyurethane, and silicone or polysiloxane.

Polyisoprene, polybutadiene, and polychloroprene are all polymerized from diene monomers and therefore have one double bond per monomer when polymerized. This double bond converts the polymer into an elastomer by vulcanization (generally, sulfur is used to form crosslinks between the double bonds by heating). This enables easy homogeneous multilayer softlithography by incomplete vulcanization of the layers to be bonded; photoresistencapsulation is enabled by a similar mechanism.

Pure polyisobutylene has no double bonds, but is used as an elastomer by crosslinking by introducing a small amount (about 1%) of isoprene during polymerization. The isoprene monomer gives pendant double bonds on the polyisobutylene backbone, which can then be vulcanized as described above.

Poly(styrene-butadiene-styrene) is produced by living anionic polymerization (ie, there is no natural chain termination step in the reaction), so "living" polymer ends can exist in the cured polymer species. This is a natural candidate for photoresist encapsulation systems (where there will be a lot of unreacted monomer in the liquid layer cast on top of the cured layer). Incomplete curing allows for homogeneous multi-layer soft printing (A combined with A). This chemistry also facilitates having one layer with extra butadiene ("A") and coupling agent and another layer ("B") lacking butadiene (for homogeneous multilayer soft printing). SBS is a "thermoset elastomer", meaning that above a certain temperature it melts and becomes plastic (as opposed to elastic); lowering the temperature regenerates the elastomer. Thus, the layers can be bonded together by heating.

Polyurethanes are made from diisocyanates (A--A) and diols or diamines (B--B); due to the variety of diisocyanates and diols/amines, the number of different types of polyurethanes is huge . However, the A-versus-B properties of the polymers make them useful for heterogeneous multilayer soft printing like RTV 615: by using an excess of A-A in one layer and an excess of B-B in another layer.

Silicone polymers have a wide range of structural variations and are available in a variety of commercially available formulations. Vinyl-to-(Si--H) crosslinking of RTV 615 has been discussed (enables heterogeneous multilayer soft printing and photoresist encapsulation), but these are just a few for siloxane polymer chemistry One of the methods.

In addition to using the simple "neat" polymers described above, crosslinkers can be added. Some reagents, such as monomers with pendant double bonds for vulcanization, are suitable for homogeneous (A to A) multilayer soft printing or photoresist encapsulation; in this method the same reagents. Complementary reagents (ie, one monomer with pendant double bonds and the other with pendant Si-H groups) are suitable for heterogeneous (A to B) multilayer soft printing. In this method a complement is added to an adjacent layer.

The following is a non-exhaustive list of elastomers that may be used in conjunction with the present invention: polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), polyurethane and silicone polymers; or poly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F), poly(carborane-siloxane) (Dexsil), poly(acrylonitrile-butadiene ene) (nitrile rubber), poly(1-butene), poly(chlorotrifluoroethylene-difluoroethylene) copolymer (KeI-F), poly(ethyl vinyl ether), poly(vinylidene fluoride) ), poly(vinylidene fluoride-hexafluoropropylene) copolymer (Viton), polyvinyl chloride elastomeric composition (PVC), polysulfone, polycarbonate, polymethyl methacrylate (PMMA) and polytetrafluoroethylene ( Teflon).

Allcock et al., Contemporary Polymer Chemistry, 2nd Edition describe elastomers as polymers that exist at temperatures between the glass transition temperature and the liquefaction temperature. Elastomers exhibit elastic properties due to the fact that the polymer chains are prone to torsional motion to untie the backbone chains under the action of a force, and the backbone chains curl back to their original shape when the force is not applied. Typically, elastomers deform when a force is applied and return to their original shape when the force is removed. The elasticity exhibited by an elastomer can be characterized by the modulus of elasticity. Some embodiments of the present invention use materials with an elastic modulus of about 1 Pa to about 1 TPa, or about 10 Pa to about 100 GPa, or about 20 Pa to about 1 GPa, or about 50 Pa to about 10 MPa, or about 100 Pa to about 1 MPa, although according to Moduli of elasticity outside these ranges may also be used as required by the particular application. In some cases, the modulus of elasticity of the material may be about 100 MPa (megapascals) or less. In other embodiments, the modulus of elasticity of the material is about 75 MPA or less, about 50 MPA or less, about 25 MPA or less, about 10 MPA or less, about 8 MPA or less, about 5 MPA or less or about 2MPA or less.

Embodiments of the present invention provide microfluidic devices comprising components, such as channels, valves, and chambers, that are at least partially contained, embedded, or otherwise formed by or within one or more layers or levels of an elastic modulus. Which formed. An exemplary microfluidic device has reagent flow channels or reagent lines formed in an elastomeric first layer. The reagent flow channel includes a containment valve and a chamber conduit. The microfluidic device may also have control channels or containment lines formed in the elastomeric second layer adjacent to the first layer. Additionally, the microfluidic device may comprise a sample flow channel or sample line formed in an elastomeric third layer adjacent to said second layer. The sample flow channel may include a containment valve and a chamber conduit. The control channel can be operatively connected to the reagent flow channel receiving valve and the sample flow channel receiving valve. The microfluidic device may comprise a reagent chamber in fluid communication with a reagent line, and a sample chamber in fluid communication with a sample line. The reagent and sample chambers may be in fluid communication with each other through reaction flow channels or lines formed in the elastomeric third layer. The reaction line may include an interface valve. The microfluidic device can also include interfacial channels formed in an elastomeric fourth layer adjacent to the third layer. The interface channel may be operably connected to the reactant flow channel interface valve.

Embodiments of the invention also encompass methods of making and using the microfluidic devices disclosed herein. For example, operation of the microfluidic device can include opening one or more individual valves, closing one or more interface valves, and flowing material through the individual valves and into one or more chambers, optionally under pressure. Techniques may also include changing the pressure in the seal line to close the separate valve to close the separate chamber, and changing the pressure in the interface line to open the interface valve. A first material from the first chamber can flow through the open interface valve into the second chamber, where the first material mixes or reacts with a second material contained therein.

It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes may be suggested by those skilled in the art, and that these are included within the spirit and purview of the application and the scope of the appended claims.

The following correspond to the original claims in the parent application, which are hereby incorporated as part of the specification:

1. A microfluidic device comprising

pressure source;

a control line in fluid communication with the pressure source;

a plurality of valves operated by said control line; and

A separate valve is located adjacent the control line and between the pressure source and the plurality of valves.

2. The microfluidic device of item 1, wherein said independent valve is constructed and arranged to block the flow of fluid through said control line.

3. The microfluidic device of item 1, wherein the independent valve is in fluid communication with a second pressure source.

4. The microfluidic device of item 1, wherein the control line comprises a latch control line comprising a one-way valve.

5. The microfluidic device of item 4, wherein said one-way valve is configured to prevent fluid flow from said valve to said pressure source.

6. The microfluidic device of item 5, further comprising a plurality of chamber valves.

7. The microfluidic device of item 6, wherein said control line provides actuation pressure to said plurality of valves and said plurality of chamber valves.

8. The microfluidic device of item 1, wherein said individual valve is operable to prevent said plurality of valves from closing.

9. The microfluidic device of item 1, further comprising a second plurality of valves operated by a second control line.

10. The microfluidic device of item 9, wherein said second control line comprises a second lockout control line comprising a second one-way valve.

11. A method of operating a microfluidic device having a valve and a control line having a valve set associated therewith, the method comprising:

closing the valve; and

Applying pressure to the control line; wherein the closed valve renders the set of valves associated with the control line inoperable.

12. The method of item 11, wherein closing the valve comprises:

applying a second pressure to the second control line; and

The valve is closed in response to the second pressure.

13. The method of item 11, wherein the first set of valves closes in response to applying a first pressure to said first control line.

14. The method of item 13, wherein said first valve group is blocked,

15. The method of item 11, further comprising:

maintaining said second pressure applied to said second control line; and

A second pressure less than the first pressure is applied to the first control line, wherein a valve set associated with the second control line closes in response to application of the second pressure.

16. The method of item 15, wherein the valve bank is blocked.

17. A microfluidic device comprising:

first valve;

second valve;

a control line in fluid communication with the first valve and the second valve;

a pressure accumulator in fluid communication with the control line; and

a one-way valve positioned adjacent the control line and between the pressure accumulator and the second valve.

18. The microfluidic device of item 17, wherein said control line between said pressure accumulator and said first valve has no one-way valve.

19. The microfluidic device of item 17, wherein said first valve is arranged to separate reaction chambers arranged in said microfluidic device.

20. The microfluidic device of item 17, wherein the second valve is arranged to separate the first fluid input line and the second fluid input line.

21. The microfluidic device of item 17, further comprising a third valve in fluid communication with said control line.

22. The microfluidic device of item 21, wherein the third valve is operable to prevent the flow of fluid through a fluid input line coupled to the reaction chamber.

23. The microfluidic device of item 21, further comprising a separation valve constructed and arranged to block fluid flow through said control line, wherein said separation valve is positioned between said pressure accumulator and said third pressure accumulator along said control line. between the valves.

24. The microfluidic device of item 23, wherein said isolation valve is configured to receive an actuation pressure from a second control line, said second control line being in fluid communication with a second pressure accumulator.

25. The microfluidic device of item 24, wherein said second control line is independent of said control line.

26. The microfluidic device of item 23, wherein said isolation valve is operable to prevent said third valve from closing in response to actuation of said control line.

27. A microfluidic device comprising:

a plurality of reaction chambers arranged in an array configuration, each of the plurality of reaction chambers having a first valve in fluid communication with one of the plurality of reaction chambers and a first valve in fluid communication with the one of the plurality of reaction chambers a connected second valve;

a first control line operable to actuate the first valve and the second valve;

an input line set in fluid communication with the plurality of reaction chambers;

a plurality of sample inlets in fluid communication with the set of input lines; and

a one-way valve disposed in the first control line.

28. The microfluidic device of item 27, further comprising a first set of valves in fluid communication with the first pressure accumulator and operable to block a portion of said set of input lines.

29. The microfluidic device of item 27, wherein said first valve set is downstream of said one-way valve.

30. The microfluidic device of item 27, further comprising:

a second set of input lines in fluid communication with the plurality of reaction chambers; and

A second plurality of sample inlets in fluid communication with the second set of input lines.

31. The microfluidic device of item 30, further comprising a second set of valves operable via a second control line.

32. The microfluidic device of item 31, wherein said second control line is in fluid communication with a second pressure accumulator and is operable to block a portion of said second set of input lines.

33. The microfluidic device of item 31, further comprising a second one-way valve arranged in said second control line.

34. The microfluidic device of item 31, further comprising a separation valve disposed in said second control line and operable to block fluid flow through said first control line.

35. A microfluidic device comprising:

Multiple reaction chambers;

a plurality of first input ports, wherein each of the plurality of first input ports is in fluid communication with one or more of the plurality of reaction chambers via one of the first plurality of input lines;

a plurality of second input ports, wherein each of the plurality of second input ports is in fluid communication with one or more of the plurality of reaction chambers via one of the second plurality of input lines;

a first pressure accumulator in fluid communication with a first control line, wherein the first control line is configured to close the first plurality of input lines;

a second pressure accumulator in fluid communication with a second control line, wherein the second control line is configured to close the second plurality of input lines;

a first one-way valve disposed in the first control line between the first pressure accumulator and the first plurality of input lines; and

a second one-way valve disposed in the second control line between the second pressure accumulator and the second plurality of input lines.

36. The microfluidic device of item 35, further comprising a valve set operable to separate each of said plurality of reaction chambers.

37. The microfluidic device of item 35, further comprising a valve operable to block said second control line and disposed between said second pressure accumulator and said second control line.

38. The microfluidic device of item 37, wherein said first control line is operable to actuate said valve.

39. The microfluidic device of item 35, further comprising a valve set operable to block a subset of said second plurality of input lines.

40. The microfluidic device of item 35, further comprising a valve set operable to block a manifold providing fluid communication between the second plurality of input lines.

41. A method of operating a microfluidic device having a plurality of valves and a one-way valve, the method comprising:

applying a first fluid pressure to a control line of the microfluidic device;

closing the plurality of valves in response to application of the first pressure;

closing the one-way valve in response to application of the first pressure; and

A second fluid pressure is applied to a second control line of the microfluidic device.

42. The method of item 41, further comprising reducing said first fluid pressure applied to said control line.

43. The method of item 42, wherein said plurality of valves remains closed after reducing said first fluid pressure applied to said control line.

44. The method of item 42, wherein reducing the first fluid pressure comprises applying no fluid pressure to the control line.

45. The method of item 41, wherein applying the second fluid pressure to the second control line includes closing a separation valve.

46. The method of item 45, wherein said isolation valve is operable to prevent fluid communication between said plurality of valves and said control line.

47. The method of item 41, wherein said microfluidic device has a plurality of reaction chambers and a valve set associated with each of said plurality of reaction chambers.

48. A method of operating a microfluidic device having a plurality of input ports, the method comprising:

providing input fluid to one of the plurality of input ports;

actuating a valve set to close a first portion of an input line connected to a subset of the plurality of input ports, wherein the subset does not include the one of the plurality of input ports;

flowing said input fluid through an input line connected to said one of said plurality of input ports;

flowing the input fluid through the input line to a second portion of the input line; and

Close the second set of valves to separate multiple reaction chambers.

49. The method of item 48, wherein closing the second set of valves comprises applying fluid pressure to a first pressure source.

50. The method of item 49, wherein closing the second set of valves comprises closing a manifold providing fluid communication between input lines.

51. The method of item 49, wherein actuating the set of valves comprises applying fluid pressure to a second pressure source.

52. The method of item 48, wherein flowing the input fluid to the second portion of the input line comprises flowing the input fluid through a manifold providing fluid communication between the input lines.

53. The method of item 48, wherein said valve set is actuated prior to closing said second set of valves.

54. The method of item 48, further comprising flowing the input fluid through a second set of input lines branching off into the second portion of each of the input lines.

55. A method of operating a programmable microfluidic device having an array of reaction sites in fluid communication with a first set of input lines and a second set of input lines, the method comprising:

actuating a first set of valves operable to block the first set of input lines;

actuating a second set of valves operable to block a first portion of a subset of the second set of input lines;

loading a sample into the reaction site via a second portion of the second set of input lines; and

A third set of valves operable to separate the reaction sites is actuated.

56. The method of item 55, wherein actuating the first set of valves comprises blocking the first set of valves.

57. The method of item 55, wherein actuating the first set of valves comprises applying a first pressure to a first pressure source in fluid communication with the first set of valves.

58. The method of item 55, wherein actuating said first set of valves and actuating said second set of valves are performed simultaneously.

59. The method of item 55, wherein actuating the third set of valves comprises applying a second pressure to a second pressure source in fluid communication with the third set of valves.

60. The method of item 59, wherein actuation of said third valve set is performed after loading a plurality of samples into said reaction site.

61. The method of item 55, wherein said sample flows from a first portion of said input line to a second portion of said second set of input lines through a manifold connecting a second portion of said second set of input lines, The first portion of the input lines is not included in the subgroup of the second set of input lines.

62. The method of item 55, wherein input lines not included in said subset of said second set of input lines are in fluid communication with an input port configured to receive said sample.

63. A microfluidic device comprising:

a predetermined number of input ports, each operable to receive one of a plurality of input fluids;

a plurality of input fluid lines, each of the plurality of input fluid lines is in fluid communication with one of the predetermined number of input ports;

a set of valves each operable to close one of the plurality of input fluid lines, wherein the number of sets of valves is less than the predetermined number;

a manifold in fluid communication with each of the plurality of input fluid lines; and

A second set of valves each operable to close a portion of the manifold.

64. The microfluidic device of item 63, wherein said predetermined number is twelve.

65. The microfluidic device of item 63, further comprising a first pressure source in fluid communication with said valve set.

66. The microfluidic device of item 65, further comprising a second pressure source in fluid communication with said valve set.

67. The microfluidic device of item 63, wherein said portion of said manifold comprises a fluid line connecting one of said plurality of input fluid lines to another of said plurality of input fluid lines.

68. The microfluidic device of item 63, wherein the number of said valve sets is a number less than said predetermined number.

69. The microfluidic device of item 63, wherein each of said plurality of input fluid lines branches into a plurality of input fluid lines.

70. A method of operating a programmable microfluidic device having an array of reaction sites in fluid communication with a first set of input lines, a second set of input lines, and a manifold connecting the second set of input lines, The methods include:

actuating a first set of valves operable to close the first set of input lines;

actuating a second set of valves operable to close a first portion of a subset of the second set of input lines;

actuating a third set of valves operable to deactivate the manifold;

deactivate said second valve group;

loading a plurality of samples into the reaction site via a second portion of the second set of input lines; and

A fourth set of valves operable to separate the reaction sites is actuated.

71. The method of item 70, wherein actuating the first set of valves includes blocking the first set of valves.

72. The method of item 70, wherein actuating said first set of valves and actuating said second set of valves comprises applying a first pressure to a first pressure source, said first pressure source being in contact with said first set of valves and said second set of valves. The second valve group is in fluid communication.

73. The method of item 72, wherein actuating the third set of valves comprises applying a second pressure to a second pressure source in fluid communication with the second set of valves.

74. The method of item 73, wherein said second pressure is less than said first pressure.

75. The method of item 70, wherein deactuating said second set of valves comprises reducing said first pressure applied to said first pressure source.

76. The method of item 70, wherein actuating the third set of valves includes blocking the third set of valves.

77. The method of item 70, further comprising actuating a valve to prevent closure of a fifth set of valves operable to close said second portion of said second set of input lines.

78. The method of item 77, wherein said valve is in fluid communication with a first pressure source, said first pressure source being in fluid communication with said first valve set.

79. The method of item 70, wherein a first portion of each of said second set of input lines is in fluid communication with an input port configured to receive one of said plurality of samples.

80. The method of item 70, wherein said reaction site comprises a plurality of reaction chambers, each of said plurality of reaction chambers being in fluid communication with a second portion of one of said second set of input fluid lines via a through hole.

81. The method of item 70, wherein the first portion of each of said second set of input lines is in fluid communication with a different input port.

82. A method of operating a programmable microfluidic device having an array of reaction sites in fluid communication with a first set of input lines and a second set of input lines, the method comprising:

actuating a first set of valves operable to block the first set of input lines;

actuating a second set of valves operable to separate the reaction sites;

deactivate said second valve group;

loading a plurality of samples into the reaction site via a second set of input lines; and

Actuate the second valve group.

83. The method of item 82, wherein actuating the first set of valves includes blocking the first set of valves.

84. The method of item 82, wherein actuating the first set of valves comprises applying a first pressure to a first pressure source in fluid communication with the first set of valves.

85. The method of item 82, further comprising actuating a third set of valves simultaneously with actuating the first set of valves.

86. The method of item 85, wherein actuating the third set of valves includes blocking the third set of valves.

87. The method of item 82, wherein deactuating said first set of valves comprises reducing said first pressure applied to said first pressure source.

88. The method of item 82, wherein each of said second set of input lines is in fluid communication with a different input port.

89. The method of item 82, wherein actuating said first set of valves and actuating said second set of valves are performed simultaneously.

90. The method of item 82, wherein said reaction sites comprise reaction chambers defined in an elastomeric layer of said microfluidic device.

91. A microfluidic device comprising:

multiple reaction sites;

a first set of input lines providing fluid communication between a predetermined number of first input ports and the plurality of reaction sites, wherein the number of the first set is the predetermined number;

A second set of input lines providing fluid communication between a predetermined number of second input ports and the plurality of reaction chambers, wherein each of the second set of input lines includes a stem portion and a branch portion, and the first the number of two groups is less than said predetermined number; and

A programmable input device operable to fill the reaction chamber with the first set of input lines or the second set of input lines.

92. The microfluidic device of item 91, wherein said plurality of reaction sites are arranged in an array configuration.

93. The microfluidic device of item 91, wherein each of said plurality of reaction sites comprises a reaction chamber in fluid communication with one of said first set of input fluid lines through a through hole.

94. The microfluidic device of item 91, wherein each of said plurality of reaction sites comprises a reaction chamber in fluid communication with one of the branched portions of said second set of input fluid lines through a through hole.

95. The microfluidic device of item 91, wherein said branch portions of each of said set of input lines form four fluidic channels from said stem portion.

96. The microfluidic device of item 91, wherein said programmable input device comprises a valve set operable to close said first set of input fluid lines.

97. The microfluidic device of item 96, wherein said valve assembly is in fluid communication with a one-way valve operable to latch said valve assembly in a closed state.

98. The microfluidic device of item 91, wherein said programmable input means comprises a valve set operable to close said branch portion of said second set of input fluid lines.

99. The microfluidic device of item 98, wherein said valve set is in fluid communication with a one-way valve operable to latch said valve set in a closed state.

100. The microfluidic device of item 98, further comprising a separate valve disposed adjacent to said valve set and operable to prevent said valve set from closing.

101. A method of providing a microfluidic device having a plurality of control lines, the method comprising:

Start the first control line;

placing the valve in the first state;

Thereafter, a second control line operable to place the valve set in a second state, wherein the valves in the first state prevent the valve set from being placed in the second state, is actuated.

102. The method of item 101, wherein said first state is off.

103. The method of item 101, wherein said second state is off.

104. The method of item 101, wherein actuating the first control line comprises applying a first pressure to a first pressure source.

105. The method of item 104, wherein actuating the second control line includes applying a second pressure to a second pressure source.

106. The method of item 105, wherein said second pressure source is independent of said first pressure source.

107. A method of providing a microfluidic device having a plurality of control lines, the method comprising:

establishing a first state of the microfluidic device by actuating a first control line and then a second control line; and

The second state of the fluidic device is established by actuating the second control line followed by actuation of the first control line.

108. The method of item 107, wherein said first state comprises a first set of input lines in a closed state and a second set of input lines in fluid communication with a plurality of reaction chambers disposed on said microfluidic device.

109. The method of item 108, wherein said second state comprises said first set of input lines in fluid communication with said plurality of reaction chambers disposed on said microfluidic device and said second input line being in a closed state. line group.

110. The method of item 108, wherein said second state further comprises a set of valves in a closed state preventing fluid flow through a manifold connected to said second set of input lines.

111. The method of item 107, wherein said first control line is in fluid communication with a first pressure source.

112. The method of item 111, wherein said second control line is in fluid communication with a second pressure source.

113. The method of item 107, wherein said microfluidic device comprises an elastomeric material.

114. A microfluidic system comprising:

carrier, which contains:

a plurality of first input ports;

a plurality of first input lines each in fluid communication with one of the plurality of first input ports;

a plurality of second input ports;

a plurality of second input lines each in fluid communication with one of the plurality of second input ports;

primary stressor; and

secondary stressor

A microfluidic device mounted on the carrier, the microfluidic device comprising:

a plurality of third input lines each in fluid communication with one of the plurality of first input lines;

a plurality of fourth input lines each in fluid communication with one of the plurality of second input lines;

a first control line in fluid communication with said first pressure source;

a one-way valve operable to block at least a portion of said first control line; and

A second control line in fluid communication with the second pressure source.

115. The microfluidic system of item 114, wherein said plurality of first input ports is operable to receive a common pressure source.

116. The microfluidic system of item 114, wherein said plurality of second input ports is operable to receive a common pressure source.

117. The microfluidic system of item 114, further comprising a second one-way valve operable to block at least a portion of said second control line.

118. The microfluidic system of item 114, wherein said microfluidic device further comprises a manifold that connects said plurality of second input lines in said carrier to said plurality of fourth input lines in said microfluidic device connected.

119. The microfluidic system of item 118, further comprising a valve set operable to block a portion of said manifold.

120. The microfluidic system of item 114, wherein said microfluidic device further comprises a plurality of sets of reaction chambers, each reaction chamber of each set of reaction chambers being connected to one of said plurality of third input lines and said plurality of first One of the four input lines is in fluid communication.

Claims (11)

1. A microfluidic device comprising: first valve; second valve; a control line in fluid communication with the first valve and the second valve; a pressure accumulator in fluid communication with the control line; and a one-way valve positioned adjacent the control line and between the pressure accumulator and the second valve. 2. A microfluidic device comprising: a plurality of reaction chambers arranged in an array configuration, each of the plurality of reaction chambers having a first valve in fluid communication with one of the plurality of reaction chambers and a first valve in fluid communication with the one of the plurality of reaction chambers a connected second valve; a first control line operable to actuate the first valve and the second valve; an input line set in fluid communication with the plurality of reaction chambers; a plurality of sample inlets in fluid communication with the set of input lines; and a one-way valve disposed in the first control line. 3. A microfluidic device comprising: Multiple reaction chambers; a plurality of first input ports, wherein each of the plurality of first input ports is in fluid communication with one or more of the plurality of reaction chambers via one of the first plurality of input lines; a plurality of second input ports, wherein each of the plurality of second input ports is in fluid communication with one or more of the plurality of reaction chambers via one of the second plurality of input lines; a first pressure accumulator in fluid communication with a first control line, wherein the first control line is configured to close the first plurality of input lines; a second pressure accumulator in fluid communication with a second control line, wherein the second control line is configured to close the second plurality of input lines; a first one-way valve disposed in the first control line between the first pressure accumulator and the first plurality of input lines; and a second one-way valve disposed in the second control line between the second pressure accumulator and the second plurality of input lines. 4. A method of operating a microfluidic device having a plurality of valves and a one-way valve, the method comprising: applying a first fluid pressure to a control line of the microfluidic device; closing the plurality of valves in response to application of the first pressure; closing the one-way valve in response to application of the first pressure; and A second fluid pressure is applied to a second control line of the microfluidic device. 5. A method of operating a microfluidic device having a plurality of input ports, the method comprising: providing input fluid to one of the plurality of input ports; actuating a valve set to close a first portion of an input line connected to a subset of the plurality of input ports, wherein the subset does not include the one of the plurality of input ports; flowing said input fluid through an input line connected to said one of said plurality of input ports; flowing the input fluid through the input line to a second portion of the input line; and Close the second set of valves to separate multiple reaction chambers. 6. A method of operating a programmable microfluidic device having an array of reaction sites in fluid communication with a first set of input lines and a second set of input lines, the method comprising: actuating a first set of valves operable to block the first set of input lines; actuating a second set of valves operable to block a first portion of a subset of the second set of input lines; loading a sample into the reaction site via a second portion of the second set of input lines; and A third set of valves operable to separate the reaction sites is actuated. 7. A microfluidic device comprising: a predetermined number of input ports, each operable to receive one of a plurality of input fluids; a plurality of input fluid lines, each of the plurality of input fluid lines is in fluid communication with one of the predetermined number of input ports; a set of valves each operable to close one of the plurality of input fluid lines, wherein the number of sets of valves is less than the predetermined number; a manifold in fluid communication with each of the plurality of input fluid lines; and A second set of valves each operable to close a portion of the manifold. 8. A method of operating a programmable microfluidic device having an array of reaction sites in fluid communication with a first set of input lines, a second set of input lines, and a manifold connecting the second set of input lines, The methods include: actuating a first set of valves operable to close the first set of input lines; actuating a second set of valves operable to close a first portion of a subset of the second set of input lines; actuating a third set of valves operable to deactivate the manifold; deactivate said second valve group; loading a plurality of samples into the reaction site via a second portion of the second set of input lines; and A fourth set of valves operable to separate the reaction sites is actuated. 9. A method of operating a programmable microfluidic device having an array of reaction sites in fluid communication with a first set of input lines and a second set of input lines, the method comprising: actuating a first set of valves operable to block the first set of input lines; actuating a second set of valves operable to separate the reaction sites; deactivate said second valve group; loading a plurality of samples into the reaction site via a second set of input lines; and Actuate the second valve group. 10. A microfluidic device comprising: multiple reaction sites; a first set of input lines providing fluid communication between a predetermined number of first input ports and the plurality of reaction sites, wherein the number of the first set is the predetermined number; A second set of input lines providing fluid communication between a predetermined number of second input ports and the plurality of reaction chambers, wherein each of the second set of input lines includes a stem portion and a branch portion, and the first the number of two groups is less than said predetermined number; and A programmable input device operable to fill the reaction chamber with the first set of input lines or the second set of input lines. 11. A microfluidic system comprising: carrier, which contains: a plurality of first input ports; a plurality of first input lines each in fluid communication with one of the plurality of first input ports; a plurality of second input ports; a plurality of second input lines each in fluid communication with one of the plurality of second input ports; primary stressor; and secondary stressor A microfluidic device mounted on the carrier, the microfluidic device comprising: a plurality of third input lines each in fluid communication with one of the plurality of first input lines; a plurality of fourth input lines each in fluid communication with one of the plurality of second input lines; a first control line in fluid communication with said first pressure source; a one-way valve operable to block at least a portion of said first control line; and A second control line in fluid communication with the second pressure source.