Patent Publication Number: US-2023143960-A1

Title: Devices and methods for mesofluidic and/or microfluidic processes

Description:
This application is Continuation of Ser. No. 15/757,642 filed on Mar. 5, 2018 which is a 371 National stage application of International Application no. PCT/US2016/050246, filed Sep. 2, 2016, which claims priority to U.S. Provisional Application No. 62/214,630, filed Sep. 4, 2015, all of which are herein incorporated by reference in their entirety. 
    
    
     BACKGROUND 
     The present disclosure relates generally to devices and methods for mesofluidic and/or microfluidic processes, such as polymerase chain reactions and/or DNA sequencing. More specifically, this disclosure relates to devices and methods for mesofluidics and/or microfluidics to perform biomolecular processes, assays, and workflows, such as: nucleic acid extraction; PCR; preparation of biological samples for isolation, separation, and detection of biomolecules including nucleic acids and proteins; DNA sequencing; qPCR; immunoassays; preparation and genetic manipulation of cells; and immunotherapy. 
     The polymerase chain reaction (PCR) identifies and replicates strands of deoxyribonucleic acid (DNA) and is employed in modern techniques of genetic analysis. The principle of PCR is in vitro multiplication of strands of DNA using enzymatic polymerases. 
     The duplication of a DNA strand in PCR is carried out in three principle steps. First, the original double-stranded DNA is split in two single-strands of DNA, a process known as “denaturation” or “melting.” Second, primers attach to defined sites of the single-strands of DNA. Third, starting from the primer, a polymerase attaches to the single-strand DNA and forms a complementary DNA strand, thereby forming an identical (or almost identical) copy of the original double-stranded DNA. The process is then repeated multiple times to achieve an exponential growth of the number of identical DNA strands. Analysis of the resultant pool of DNA can then be performed. 
     PCR is classically performed in a device called a thermal cycler. Specifically, a small plastic tube contains the DNA sample together with the primer and polymerase molecules in a suitable buffer, and the thermal cycler repeatedly temperature cycles the tube through the PCR phases described above, for example between 15 and 25 times in typical applications. 
     SUMMARY 
     The present inventors recognized several problems with known methods and devices for mesofluidic and/or microfluidic processes such as continuous-flow PCR and DNA sequencing. One or more of these problems can be addressed by devices in a plurality of stages of fluid handling and processing which are automated, using an instrument and a complimentary customized disposable cartridge. The cartridge includes one or more integral flow paths that allow fluids or reagents to pass from one processing stage to the next without need for manual user handling or risk of contamination by employing a multitude of different laboratory techniques. The instrument includes several mechanical components that act on different portions of the disposable cartridge to accomplish the processing stages desired by the user. 
     One such disposable cartridge includes portions of a first elastomeric membrane that abut or are proximate to portions of a second elastomeric membrane, and these portions of the first and second elastomeric membranes can be sequentially pushed apart by a fluid to form a channel for the fluid with minimal or no dead volume. Preferably these channel-forming portions of the first and second elastomeric membranes are circumscribed by sealed portions of the first elastomeric membrane that are fixedly attached to corresponding sealed portions of the second elastomeric membrane. In other embodiments, a single elastomeric membrane can be employed. 
     Biomolecular assays and workflows can require complex manipulations of samples and reagents. A user benefits from simplifying such complex manipulations by automation and integration. Automating and integrating makes the processes more efficient, more robust, more easily repeatable, simpler to use, and more cost effective. 
     Therefore, the present disclosure provides a system which integrates the following functionality to enable a vast array of potential workflows and assays, including but not limited to, for example: pumping and valving workflows; integrated storage and release of wet and dry reagents across a broad range of volumes; precise thermal control of reagents and samples; solid phase isolation of reactants, intermediates, reaction products, and biomolecules via magnetic, paramagnetic, non-magnetic beads and coated films or substrates; and optical tools to detect reactions and intermediates. Finally, such an automated system has the ability to link each of the above exemplary workflows and associated functionality together. Linking functionality together as combinatorial building blocks enables rapid deployment of workflows that span from simple add and mix to complex fully integrates sample-to-answer systems that include processing samples through multiple sequential rounds of each of the above building blocks. 
     Additional features are described herein and will be apparent from the following Detailed Description and the Figures. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG.  1    depicts a cross-sectional view of one embodiment of the disclosure. 
         FIG.  2 A  depicts a cross-sectional view of a groove of one embodiment of the disclosure. 
         FIG.  2 B  depicts a cross-sectional view of a groove of one embodiment of the disclosure. 
         FIG.  2 C  depicts a cross-sectional view of a groove of one embodiment of the disclosure. 
         FIG.  3 A  illustrates an isometric view of one embodiment of the disclosure. 
         FIG.  3 B  illustrates a bottom view of one embodiment of the disclosure. 
         FIG.  4 A  illustrates an isometric view of one embodiment of the disclosure. 
         FIG.  4 B  illustrates a bottom view of one embodiment of the disclosure. 
         FIG.  5    depicts different views of the edge of a groove of one embodiment of the disclosure. 
         FIG.  6    depicts a cross-sectional view of one embodiment of the disclosure. 
         FIG.  7    illustrates a picture of a thermal plastic sheet sealing in an embodiment of the disclosure. 
         FIG.  8    depicts a cross-sectional view of one embodiment of the disclosure. 
         FIG.  9 A  depicts a cross-sectional view of one embodiment of the disclosure. 
         FIG.  9 B  depicts a cross-sectional view of a groove of one embodiment of the disclosure. 
         FIG.  9 C  depicts a top view of chambers of one embodiment the disclosure. 
         FIG.  9 D  depicts a cross-sectional view of a lane assembly of one embodiment of the disclosure. 
         FIG.  10 A  depicts a cross-sectional view of one embodiment of the disclosure. 
         FIG.  10 B  depicts a cross-sectional view of a groove of one embodiment of the disclosure. 
         FIG.  10 C  depicts a top view of chambers of one embodiment the disclosure. 
         FIG.  10 D  depicts a cross-sectional view of a lane assembly of one embodiment of the disclosure. 
         FIG.  11 A  depicts a cross-sectional view of one embodiment of the disclosure. 
         FIG.  11 B  depicts a cross-sectional view of a groove of one embodiment of the disclosure. 
         FIG.  11 C  depicts a top view of chambers of one embodiment the disclosure. 
         FIG.  12 A  depicts a cross-sectional view of one embodiment of the disclosure. 
         FIG.  12 B  depicts a cross-sectional view of a groove of one embodiment of the disclosure. 
         FIG.  12 C  depicts a top view of chambers of one embodiment the disclosure. 
         FIG.  13    depicts a cross-sectional view of one embodiment of the disclosure. 
         FIG.  14    depicts a cross-sectional view of a flow path of one embodiment of the disclosure. 
         FIG.  15    depicts a cross-sectional view a flow path of one embodiment of the disclosure. 
         FIG.  16    illustrates an isometric exploded view of an assembly of one embodiment of the disclosure. 
         FIG.  17    illustrates an isometric exploded view of an assembly of one embodiment of the disclosure. 
         FIG.  18 A  illustrates a cross-sectional exploded view of an assembly of one embodiment of the disclosure. 
         FIG.  18 B  illustrates a cross-sectional assembled view of one embodiment of the disclosure. 
         FIG.  19    illustrates a cross-sectional view of one embodiment of the disclosure. 
         FIG.  20 A  illustrates a cross-sectional view of the membrane fluid path of one embodiment of the disclosure. 
         FIG.  20 B  illustrates a cross-sectional view of one embodiment of the disclosure. 
         FIG.  21    illustrates a cross-sectional view of a flow path of one embodiment of the disclosure. 
         FIG.  22 A  illustrates a photograph of a heater block of one embodiment of the disclosure. 
         FIG.  22 B  illustrates a representation of a heater block of one embodiment of the disclosure. 
         FIG.  22 C  illustrates a representation of a heater block of one embodiment of the disclosure. 
         FIG.  23 A  illustrates a cross-sectional view of one embodiment of the disclosure. 
         FIG.  23 B  illustrates a cross-sectional view of one embodiment of the disclosure. 
         FIG.  23 C  illustrates a cross-sectional view of one embodiment of the disclosure. 
         FIG.  24 A  illustrates a cross-sectional view of one embodiment of the disclosure. 
         FIG.  24 B  illustrates a cross-sectional view of one embodiment of the disclosure. 
         FIG.  25 A  illustrates a side view of the elastomeric membranes of one embodiment of the disclosure. 
         FIG.  25 B  illustrates a side view of the elastomeric membranes of one embodiment of the disclosure. 
         FIG.  26 A  illustrates a photograph of a mixing chamber of the membranes of one embodiment of the disclosure. 
         FIG.  26 B  illustrates a photograph of a mixing chamber of the membranes of one embodiment of the disclosure. 
         FIG.  26 C  illustrates a photograph of a mixing chamber of the membranes of one embodiment of the disclosure. 
         FIG.  26 D  illustrates a photograph of a mixing chamber of the membranes of one embodiment of the disclosure. 
         FIG.  27 A  illustrates a cross-section of an elastomeric membrane of one embodiment of the disclosure. 
         FIG.  27 B  illustrates a cross-section of an elastomeric membrane of one embodiment of the disclosure. 
         FIG.  27 C  illustrates a cross-section of an elastomeric membrane of one embodiment of the disclosure. 
         FIG.  28 A  illustrates a cross-section of an elastomeric membrane of one embodiment of the disclosure. 
         FIG.  28 B  illustrates a cross-section of an elastomeric membrane of one embodiment of the disclosure. 
         FIG.  29    depicts the flow distribution through various mixing chamber geometry embodiments of the disclosure. 
         FIG.  30 A  illustrates a cross-section of a flow path of one embodiment of the disclosure. 
         FIG.  30 B  illustrates a cross-section of a flow path of one embodiment of the disclosure. 
         FIG.  30 C  depicts a schematic of a flow path of one embodiment of the disclosure. 
         FIG.  30 D  depicts a schematic of a flow path of one embodiment of the disclosure. 
         FIG.  30 E  depicts a schematic of a mixing chamber with mixing beads of one embodiment of the disclosure. 
         FIG.  31    illustrates a photograph of a mixing chamber embodiment of the disclosure. 
         FIG.  32 A  illustrates a view of elastomeric membranes of one embodiment of the disclosure. 
         FIG.  32 B  illustrates a view of elastomeric membranes having elastomeric patches of one embodiment of the disclosure. 
         FIG.  32 C  illustrates a cross-sectional view of one embodiment of the disclosure. 
         FIG.  33 A  illustrates a photograph of one embodiment of the disclosure. 
         FIG.  33 B  illustrates a photograph of one embodiment of the disclosure. 
         FIG.  34 A  illustrates a photograph of a cross-sectional view of one embodiment of the disclosure. 
         FIG.  34 B  illustrates a photograph of one embodiment of the disclosure. 
         FIG.  35    illustrates a photograph of one embodiment of the disclosure. 
         FIG.  36    illustrates a series of photographs showing fluid moving through one embodiment of the disclosure. 
         FIG.  37    illustrates a photograph of reagents flowing through the elastomeric membranes of one embodiment of the disclosure. 
         FIG.  38    illustrates an isometric view of an exploded assembly of one embodiment of the disclosure. 
         FIG.  39    illustrates a view of a partial assembly of one embodiment of the disclosure. 
         FIG.  40    illustrates a view of a partial assembly of one embodiment of the disclosure. 
         FIG.  41    illustrates a schematic of a partial assembly of one embodiment of the disclosure. 
         FIG.  42    illustrates a view of a bubble trap embodiment of the disclosure 
         FIG.  43 A  illustrates a photograph of one embodiment of the disclosure. 
         FIG.  43 B  illustrates a photograph of one embodiment of the disclosure. 
         FIG.  44    illustrates a volumetric schematic of one embodiment of the disclosure. 
         FIG.  45    illustrates a side cross-section exploded view of twin blisters in one embodiment of the disclosure. 
         FIG.  46    illustrates a side cross-section view of manufacture of twin blisters in one embodiment of the disclosure. 
         FIG.  47    illustrates a side cross-section view of use of twin blisters in one embodiment of the disclosure. 
         FIG.  48    illustrates a side cross-section view of use of twin blisters in one embodiment of the disclosure. 
         FIG.  49 A  is a flowchart of workflow in known preparation and processing methods. 
         FIG.  49 B  is a flowchart of workflow in one embodiment of the disclosure. 
         FIG.  50    illustrates an instrument with cartridges in one embodiment of the disclosure. 
     
    
    
     DETAILED DESCRIPTION 
     As used in this disclosure and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a material” or “the material” includes two or more materials. The term “and/or” used in the context of “X and/or Y” should be interpreted as “X,” or “Y,” or “X and Y.” Where used herein, the term “example,” particularly when followed by a listing of terms, is merely exemplary and illustrative, and should not be deemed to be exclusive or comprehensive. 
     As used herein, “about,” “approximately” and “substantially” are understood to refer to numbers in a range of numerals, for example the range of −10% to +10% of the referenced number, preferably within −5% to +5% of the referenced number, more preferably within −1% to +1% of the referenced number, most preferably within −0.1% to +0.1% of the referenced number. “Similarly dimensioned” means that the referenced components have at least two dimensions that are substantially the same as the corresponding dimensions of the other components, and in some embodiments three of such dimensions. 
     All numerical ranges herein should be understood to include all integers, whole or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth. 
     Numerical adjectives, such as “first” and “second,” are merely used to distinguish components. These numerical adjectives do not imply the presence of other components, a relative positioning, or any chronological implementation. In this regard, the presence of a “second opening” does not imply that a “first opening” is necessarily present. Further in this regard, a “second opening” can be upstream from, downstream from or co-located with a “first opening,” if any; and a “second opening” can be used before, after, and/or simultaneously with a “first opening,” if any. 
     The devices disclosed herein may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of” and “consisting of” the components identified. Similarly, the methods disclosed herein may lack any step that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of” and “consisting of” the steps identified. 
     Various embodiments of a mesofluidic and/or microfluidic device are disclosed herein, and any embodiment can be combined with any other embodiment unless explicitly and directly stated otherwise. In some embodiments, the device is a cartridge configured for a continuous-flow process that is PCR, DNA sequencing, or a combination thereof. As used herein, “continuous-flow” means that the initial sample is added to the cartridge and undergoes the process to completion without the need to be transferred from the cartridge to a different device. 
     Known Sample Prep Methods and Designs 
     Known systems for sample prep, PCR, and DNA sequencing typically involve several independent modules or instruments depending upon the particular stage of the process. In most known systems, each individual module or step of the process requires a separate instrument acting on the fluid, which therefore necessitates multiple phases of manual instrument set-up and handling of the fluid by an operator. For example, in one instance of sequencing prep, the typical known methods include a combination of lab bench workflow and instrument use. 
       FIG.  49 A  is a schematic flow-chart representative of known methods for sample preparation and processing. The workflow includes four stages: A, B, C and D, each of which requires the use of a separate respective instrument: Instrument #1, #2, #3 and #4. In one example embodiment, Stage A requires DNA extraction from a raw sample. The Instrument #1 to accomplish such extraction includes largely manual work including pipettes, tubes/plates, a vortexter, a centrifuge, and a magnet rack for bead handling. Following the DNA extraction of Stage A, the sample must undergo thermal cycling in Stage B, using Instrument #2, which would typically be a commercially available thermal cycler. Stage C also includes the use of a thermal cycler, but requires the user to add reagents to the plate after the thermal cycling of Stage B and before thermal cycling of Stage C begins. Finally, Stage D can be a sequencing clean-up workflow, which is typically accomplished manually. Instrument #4 used in Stage D can involve many of the same tools as Instrument #1 in Stage A, such as pipettes, a centrifuge, a vortexter, and a magnetic rack. 
     It should be appreciated that the manual workflows described in Stages A and D, as well as the manual reagent supplementation required between Stages B and C, are accompanied by a plurality of inefficiencies and variables. A process such as that described above and illustrated in  FIG.  49 A  requires very precise and consistent user execution that must be done efficiently, reliably, and without damaging or contaminating the sample during the process. 
     Overview of Automated Sample Prep Methods and Devices 
     In various embodiments provided by the present disclosure, each of Stages A to D, as well as all the Instruments required to execute Stages A to D, are integrated into a single automated system that includes an instrument and a complimentary corresponding cartridge. As depicted in  FIG.  49 B , the new workflow disclosed herein encapsulates each of Stages A, B, C, and D within a single instrument, thus eliminating the risks of error, inconsistency, or contamination of the sample, and therefore decreasing cost of producing repeatable samples. 
     In various embodiments of the present disclosure, as generally illustrated in  FIGS.  14 ,  21 , and  50   , a device for processes such as sample preparation, PCR, and DNA sequencing includes an integrated flow path with a plurality of different modules for processing the subject fluid. In various embodiments discussed herein and generally illustrated in  FIG.  50   , such a device includes an instrument  8  with a plurality of mechanical components and a complementary disposable cartridge  10  configured to contain any fluid or reagents to be processed. Exemplary flow paths and processes of a disposable cartridge are illustrated in detail in  FIGS.  14  and  21   . In various embodiments, the mechanical components of the instrument  8  are configured to act upon appropriate locations of the cartridge  10  to enable fluid flow in a fluid flow path, extract DNA from a raw sample, provide thermal cycling, introduce and mix reagents, perform sequencing clean-up, as well as other tasks while minimizing user handling in between stages of the process. By customizing the cartridge  10  to include the selected reagents, processes, fluid pathways, and specifications of a user&#39;s defined goal, the instrument  8  can be programmed to automate the entire workflow without requiring individual manual instrument set-up and unnecessary fluid handling by the user. 
     It should be appreciated that an automated system, as illustrated generally in  FIGS.  14 ,  21  and  50    and discussed throughout, would be more cost effective than known multi-instrument systems by providing greater sample quality, repeatability, and predictability. Further, enabling customization of the cartridge  10  to be used in a common instrument gives the user increased flexibility not available in current systems. The cartridge  10  can perform at least equivalently to the manual lab techniques discussed above and shown in  FIG.  49 A . 
     Another advantage of one or more embodiments of the system provided by the present disclosure is that the disposable cartridge  10  has all necessary reagents on-board. The cartridge  10  can enable multiplex, concurrent testing of multiple samples, for example 8-12 samples. Moreover, the cartridge  10  can comprise multiple individual pathways, each of which can process a single sample. The cartridge  10  can be configured by the user to process a desired number of samples. Notably, the cartridge  10  is not limited to one specific type of processing, and the cartridge  10  can be easily configured for any type of mesofluidic and/or microfluidic processing. 
     Preferably the cartridge  10  cooperatively connects to the instrument  8  to avoid the need for accurate alignment between the instrument  8  and the lanes of the cartridge  10 . In an embodiment, pneumatic connection of the cartridge  10  to the instrument  8  is not required. Eliminating pneumatic connection avoids the risk of cross-contamination and aerosolizing fluids, the poor reliability of air connections, and the need for calibration of the air source (pressure). Generally, the instrument  8  in which the cartridge is used  10  is simplified relative to known mesofluidic and/or microfluidic devices, to maximize reliability and to reduce cost. 
     In an embodiment generally illustrated in  FIG.  14   , partial cross sectional views of both the cartridge  10  and the instrument  8  are displayed. The cartridge  10 , for example, can include at least a first chassis  11 ; a support block  12  having a compliant layer  20 ; first and second elastomeric membranes  21 ,  22 ; and first and second foil layers  31 ,  32 . In various embodiments, the support block  12  can form one or more valves in the cartridge  10 . The mechanical elements of the instrument  8  can include at least plungers  51 ,  151  and  152 , a heater block  52 , pistons  53 , and upper and lower magnets  301  and  302  respectively. 
     As seen in  FIG.  14   , four example stages are illustrated and each stage is separated by vertical dashed lines. In the illustrated embodiment, the first stage is the fluid storage/release, wherein the initial fluid is forced into the flow path of the cartridge as discussed in greater detail below. As the flow continues toward the right of the illustration, the fluid undergoes thermal processing in the second stage. During thermal processing, the permanent heater block or heater blocks that are disposed within the instrument act upon the fluid flow path of the disposable cartridge to enable seamless transition from the first fluid storage/release stage to the thermal processing stage. The third exemplary stage illustrated in  FIG.  14    is the addition and reconstitution of a dry reagent. As seen in  FIG.  14   , the dry reagent release and reconstitution is enabled by the bursting of foil layers on the cartridge using plungers  151 ,  152 , which are permanent mechanical components integrated into the instrument  8 . 
     Finally, the fourth exemplary stage illustrated in  FIG.  14    is the magnetic bead handling of the sample. The magnetic bead handling mechanism  300  can be configured to bind, wash and elute the amplified DNA. The magnetic bead handling mechanism  300  can comprise one or more magnets, for example the upper magnet  301  and/or the lower magnet  302 . Part of the instrument in various embodiments, the magnets  301 ,  302  can be motor-driven. Each of the stages illustrated in  FIG.  14    is discussed in greater detail below. 
       FIG.  21    illustrates a similar configuration of an overview of the automated process as the arrangement discussed above and illustrated in  FIG.  14   . Among other distinctions,  FIG.  21    includes a lyophilized reagent bead pot  213  having a plunger  155  for introduction of a lyophilized reagent bead into the flow path between membranes  21  and  22 . The reagent within pot  213  is put into fluid communication with the flow path defined between elastomeric membranes  21  and  22 , preferably fluid communication through a foil layer or layers which are burst by the plunger  152  which can be provided by the instrument  8 . Additional variations and discussion of the embodiment depicted in  FIG.  21    are discussed in greater detail later herein. 
     Referring now to  FIG.  38   , an exploded view of a portion of the manual system is illustrated and described, however one skilled in the art would recognize that such a system can also be automated. One embodiment of cartridge  10  is illustrated with its several pots and a plurality of associated plungers to be disposed in each of said pot. The cartridge  10  is situated on top of a suitable clamping frame. It should be appreciated that in several embodiments, one or more of the clamping frame, the instrument chassis, and all of the individual clamps and plungers on the underside thereof are permanent parts of the instrument  8 . The layout on the cartridge  10  of the flow path(s), the pot(s) for reagent and/or fluid introduction, the mixing chamber(s), the heater blocks for thermal cycling and PCR, the magnetic bead handling mechanisms and sample cleanup can be designed in many contemplated configurations due to the plurality of toggle clamps, plungers and other permanent mechanisms of the instrument  8 . It should be appreciated that, in various embodiments discussed below, the toggle clamps of the instrument  8  act as valve actuators, which impart pressure upon different portions of the cartridge to define or obstruct flow paths, and to aid in pumping and directing fluid flow from initial introduction of the sample, through sample prep, and final clean-up. 
     As can be seen from  FIGS.  14 ,  21  and  38   , the system comprising the instrument  8  and the cartridge  10  simplifies the interfaces between the cartridge  10  and the instrument  8  in which the cartridge  10  is used (e.g. minimizes or avoids fluid/gas connections, avoids complex mechanical attachments to the lanes of the cartridge  10 , etc.). Moreover, the number of types of each interface is minimized (e.g. one type of mechanical interface). 
     Cartridge Construction and Design 
     In an embodiment generally illustrated in  FIGS.  1 ,  2 A- 2 C,  3 A,  3 B,  4 A and  4 B , a detailed view of one example of the cartridge  10  is disclosed. The cartridge  10  can comprise a first chassis  11 . The first chassis  11  can be formed at least partially by a polypropylene material, a cyclic olefin copolymer (COC), or any other suitable material or combination thereof. The first chassis  11  can form at least part of a frame of the cartridge  10 . 
     A first foil layer  31  can be positioned under the first chassis  11 . At least a portion of the upper surface of the first foil layer  31  can abut the first chassis  11 , preferably the entirety of the upper surface of the first foil layer  31 . The upper surface of the first foil layer  31  can comprise sealed portions  31   a  that are thermally welded, laser-welded, bonded by pressure-sensitive adhesive, or otherwise fixedly attached to the first chassis  11 . The first foil layer  31  can comprise aluminum or a plastic, such as polypropylene (PP), and can be smooth or corrugated. As a non-limiting example, the first foil layer  31  can have a thickness of about 37 μm. 
     A compliant layer  20  can be positioned under the first foil layer  31 . The compliant layer  20  can reversibly move between a closed position abutting the first foil layer  31  and an open position out of contact from the first foil layer  31 , for example, by moving a support block  12  to which the compliant layer  20  can be fixedly attached. In a preferred embodiment, the instrument  8  in which the cartridge  10  is used provides the support block  12  and the compliant layer  20 . 
     The upper surface of the first foil layer  31  comprises unsealed portions  31   b  circumscribed by the sealed portions  31   a . The sealed portions  31   a  of the first foil layer  31  can thus define a periphery of the unsealed portions  31   b  of the first foil layer  31  such that the distance between the sealed portions  31   a  in any particular direction is the width of the unsealed portions  31   b  in the same direction. The unsealed portions  31   b  can extend approximately the entire length of the cartridge  10 , continuous with each other and uninterrupted by the sealed portions  31   a , such that the unsealed portions  31   b  are configured to form a channel, as discussed in greater detail hereafter. As a non-limiting example, the channel may have a length of about 10 mm. 
     Fluid, for example fluid containing one or more reagents for PCR and/or DNA sequencing, can be positioned between the first chassis  11  and a section of the unsealed portions  31   b  of the first foil layer  31 . Advantageously, the cartridge  10  can enable an initial sample type that is a pipetted liquid. The fluid can be urged onto an adjacent downstream unsealed portion  31   b  of the first foil layer  31 , for example by positive displacement and preferably without pneumatic displacement, thereby pushing the downstream unsealed portion  31   b  of the first foil layer  31  away from the first chassis  11 . Consequently, the unsealed portions  31   b  of the first foil layer  31  can be sequentially pushed away from the first chassis  11  to form a channel between the first foil layer  31  and the first chassis  11 . 
     The sealed portions  31   a  of the first foil layer  31  can thus define a periphery of the channel formed between the first chassis  11  and the unsealed portions  31   b  of the first foil layer  31 ; for example, if the sealed portions  31   a  of the first foil layer  31  are about 2 mm apart, the channel can have a width of about 2 mm. 
     As the fluid progresses downstream, the upstream unsealed portions  31   b  of the channel which the fluid has evacuated can return to the resting state in which the first foil layer  31  is substantially flat. In a preferred embodiment, the compliant layer  20  is pushed into the unsealed portions  31   b  which contain the fluid to positively displace the fluid downstream and close the channel behind the fluid, for example by pushing the unsealed portions  31   b  containing the fluid to be substantially flat. If the upstream unsealed portions  31   b  are also held substantially flat by the compliant layer  20 , the fluid can travel downstream. The pressure required to close the channel can depend on the stiffness and thickness of the compliant layer  20 , the geometry of the compliant layer  20 , and the channel dimensions. 
     A groove  133  can facilitate formation of the channel, for example by allowing the unsealed portions  31   b  of the first foil layer  31  to expand. Preferably the width of the groove  133  should be about equal to the width of the unsealed portions  132  of the first foil layer  31  and thus about equal to the width of the resultant channel. As a non-limiting example, the groove  133  can have a width of about 2 mm to about 3 mm and a depth of at least about 20 μm, for example about 50 μm to about 100 μm. 
     In an embodiment depicted in  FIG.  2 A , the support block  12  can comprise the groove  133 . In an embodiment depicted in  FIGS.  2 B and  2 C , the first chassis  11  can comprise the groove  133 , and the groove  133  can form an opposite side of the channel from the first foil layer  31 . In  FIG.  2 B , the groove  133  is square or filleted, and in  FIG.  2 C  the groove  133  is rounded. In these embodiments, pressure applied through the compliant layer  20  can force the unsealed portions  132  of the first foil layer  31  to conform to the groove  133  and thereby close the channel. 
       FIG.  3 A  is a representation of an embodiment of the cartridge  10  using the configuration shown in  FIG.  1   , and  FIG.  3 B  is a schematic diagram showing the bottom surface of the first chassis  11  in such an embodiment.  FIG.  4 A  is an illustration of another embodiment of the cartridge  10  using the configuration shown in  FIG.  1   , and  FIG.  4 B  is a schematic diagram showing the bottom surface of the first chassis  11  in such an embodiment. 
     As generally illustrated in these figures and discussed in greater detail later herein, the first chassis  11  can have openings  11   a ; encapsulated reagents can be positioned in the openings  11   a , and/or one or more elastomeric seals can be positioned on the first chassis  11  over the openings  11   a . The encapsulated reagents can access the adjacent section of the channel and/or can be accessed from the adjacent section of the channel. The first chassis  11  can comprise one or more pots  11   b  positioned above one or more of the openings  11   a , and reagents within the one or more pots  11   b  can access the adjacent section of the channel and/or can be accessed from the adjacent section of the channel through the corresponding opening  11   a.    
     The fluid can be housed initially in a holding chamber (e.g., a foil “blister”) by a barrier membrane. For example, the barrier membrane can be a weaker weld or seal between the first foil layer  31  and the first chassis  11  and can be positioned within the unsealed portions  31   b  of the first foil layer  31 . In an embodiment, the barrier membrane can be spaced a distance inward from the intersection of the sealed portions  31   a  with the unsealed portions  31   b  of the first foil layer  31 . The barrier membrane can be broken to allow the fluid therein to initiate formation of the channel. 
     Pressure, such as pressure from a piston pushed down within an adjacent pot  11   b  and/or pressure from an adjacent section of the compliant layer  20  pushed upward into the first foil layer  31 , can break the barrier membrane and allow the fluid to exit the holding chamber and initiate formation of the channel. The breakable barrier membrane and process for breaking it is discussed in greater detail below. 
       FIG.  5    shows detailed views of the shape of the side of the groove  133  in the support block  12 . Alternatively or additionally, the groove  133  can be positioned in the first chassis  11 . In a preferred embodiment, the side of the groove  133  has a circular shape and can enable a cylindrical channel (bottom panel). Where beads such as magnetic beads are employed (discussed in more detail later herein), a filleted profile is preferred (middle panel). 
       FIG.  6    generally illustrates another configuration provided by the present disclosure. A first elastomeric membrane  21  can be positioned under the first chassis  11  and can comprise sealed portions  21   a  that are thermally welded, laser welded, bonded by pressure-sensitive adhesive, or otherwise fixedly attached to the first chassis  11 . As a non-limiting example, the first elastomeric membrane  21  can have a thickness from about 25 μm to about 200 μm. 
     The compliant layer  20  can be positioned under the first elastomeric membrane  21 . The compliant layer  20  can reversibly move between a closed position abutting the first elastomeric membrane  21  and an open position out of contact from the first elastomeric membrane  21 , for example, by moving the support block  12  to which the compliant layer  20  can be fixedly attached. 
     The sealed portions  21   a  of the first elastomeric membrane  21  can circumscribe unsealed portions  21   b  of the first elastomeric membrane  21 . The sealed portions  21   a  of the first elastomeric membrane  21  can thus define a periphery of the unsealed portions  21   b  of the first elastomeric membrane  21  such that the distance between the sealed portions  21   a  in any particular direction is the width of the unsealed portions  21   b  in the same direction. 
     The unsealed portions  21   b  can be continuous with each other such that they are configured to form the channel. Fluid, for example fluid containing one or more PCR reagents, can be directed between the first chassis  11  and a section of the unsealed portions  21   b  of the first elastomeric membrane  21 . The fluid can be urged onto an adjacent downstream unsealed portion  21   b  of the first elastomeric membrane  21 , for example by positive displacement and preferably without pneumatic displacement, thereby pushing the downstream unsealed portion  21   b  of the first elastomeric membrane  21  away from the first chassis  11 . Consequently, the unsealed portions  21   b  of the first elastomeric membrane  21  can be sequentially pushed away from the first chassis  11  to form a channel between the first elastomeric membrane  21  and the first chassis  11 . 
     The sealed portions  21   a  of the first elastomeric membrane  21  can thus define a periphery of the channel formed between the first chassis  11  and the unsealed portions  21   b  of the first elastomeric membrane  21 ; for example, if the sealed portions  21   a  of the first elastomeric membrane  21  are about 2 mm apart, the channel can have a width of about 2 mm. As discussed above and illustrated in  FIG.  38   , a plurality of actuators within the instrument  8  (in one embodiment, toggle clamps) can be disposed along the channel to permit and restrict fluid from flowing along the fluid flow path. It should be appreciated that any suitable known actuator can be used to create valves along the elastomeric membrane(s) and the fluid flow path. In discussions of embodiments having one or more elastomeric membrane(s) that act to define a fluid flow path, any disclosed valving can be accomplished using the toggle clamps of  FIG.  38    or other known actuation elements. 
     As the fluid progresses downstream, the upstream unsealed portions  21   b  of the channel which the fluid has evacuated can return to the resting state in which the first elastomeric membrane  21  is substantially flat. In a preferred embodiment, the compliant layer  20  is pushed into the unsealed portions  21   b  which contain the fluid to positively displace the fluid downstream and close the channel behind the fluid, for example by pushing the unsealed portions  21   b  containing the fluid to be substantially flat. If the upstream unsealed portions  21   b  are also held substantially flat by the compliant layer  20 , the fluid can travel downstream. 
       FIG.  7    shows a photograph of a non-limiting example of a thermal plastic sheet sealing in an embodiment of the cartridge  10 . In this embodiment, a low-density polyethylene (LDPE) sheet is used as a barrier to achieve a bond to a high-density polyethylene (HDPE) substrate. 
     Reagent Storage and Introduction 
     In various embodiments of the present disclosure, the reagents required for user-prescribed processes are stored within the cartridge, and either reconstituted, lyophilized, or simply added to the progressing sample in proper order during prep. As shown in  FIG.  8   , an embodiment of the cartridge  10  comprises a foil membrane  18  covering one of the pots  11   b , namely a first pot  13 , in the first chassis  11 . The first pot  13  can contain one or more reagents  16 , for example one or more reagents for PCR and/or DNA sequencing. The first pot  13  can be a structure having a lower opening  14  and an upper opening  15  on an opposite end of the first pot  13  from the lower opening  14 . The upper opening  15  of the first pot  13  can be sealed by the foil membrane  18  to prevent contamination of the interior of the first pot  13 . In various embodiments, the foil membrane  18  also acts as a barrier to minimize fluid loss through a bung. It should be appreciated that, in several embodiments, the bung is an optional member that acts upon a plunger within a pot. In such an embodiment, the bung is depressed by an external force, pushing upon the associated plunger within the pot, which then forces the one or more reagents  16  through a newly established fluid pathway between the membranes of the fluid flow path. 
     In a preferred embodiment, the first pot  13  has a cylindrical shape, but the first pot  13  is not limited to a specific shape. Furthermore, any number of first pots  13  can be used, and the cartridge  10  is not limited to a specific number of the first pot  13 . 
     The first foil layer  31  can be welded to the first chassis  11  to seal the one or more reagents  16  in the first pot  13 . The first elastomeric membrane  21  can be positioned under the first foil layer  31 . At least a portion of the upper surface of the first elastomeric membrane  21  can abut the first foil layer  31 . The first elastomeric membrane  21  can be thermally welded to the first foil layer  31 , laser-welded to the first foil layer  31 , attached to the first foil layer  31  by a pressure sensitive adhesive, or affixed to the first foil layer  31  using any other suitable means known to the skilled artisan. 
     The compliant layer  20  can be positioned under the first elastomeric membrane  21 , and at least a portion of the upper surface of the compliant layer  20  can abut the first elastomeric membrane  21 , preferably the entirety of the upper surface of the compliant layer  20 . The compliant layer  20  is preferably thicker than the first elastomeric membrane  21 , for example at least twice as thick. 
     A lower passage  50  through the support block  12  and the compliant layer  20  can be positioned for a first lower plunger  51  to burst the first foil layer  31  without breaching the first elastomeric membrane  21 . For example, the first lower plunger  51  can move upward in the lower passage  50  to push a vertically aligned section of the first elastomeric membrane  21  against a vertically aligned section of the first foil layer  31 , thereby breaking the vertically aligned section of the first foil layer  31 . 
     The first foil layer  31  in various embodiments is comprised of a foil sheet covering the entirety of the first elastomeric membrane  21 . In embodiments, a second elastomeric membrane  22  is situated adjacent to the first elastomeric membrane  21 . The first elastomeric membrane  21  can include one or more predefined openings, perforations, or points of weakness, each aligning with one or more associated pots  11   b . As discussed in more detail throughout this disclosure, the first and second membranes  21 , 22  can be connected together to form a fluid pathway. In various embodiments, the opening in the first elastomeric membrane  21  provides an entry point for fluid or other material to enter the fluid pathway from the one or more associated pots  11   b . In one dual-membrane embodiment, the second elastomeric membrane  22  is continuous and includes no openings. 
     In an embodiment, a foil layer or a foil patch is aligned with and attached near each opening in the first elastomeric membrane  21 . Some embodiments include individual foil patches covering each respective first elastomeric membrane opening, and other embodiments include one or more larger foil sheets that cover two or more first elastomeric membrane openings. The foil layer  31  can be welded over each individual first elastomeric membrane opening. 
     In embodiments with one or more foil layers sealed to the first elastomeric membrane  21 , the material of the foil layer  31  is chosen to enable a controlled breaking upon activation of one or more lower plungers  51 . In various embodiments, the foil layer  31  is made of a material more brittle than that of the second elastomeric membrane  22 . For each first elastomeric membrane opening, upon activation with one or more lower plungers  51 , the second elastomeric membrane  22  flexes and transfers pressure to the more brittle foil layer  31  disposed on the first elastomeric membrane opening until the foil layer  31  reaches a breaking point. Upon reaching a threshold pressure level, the plunger  51  causes the foil layer  31  to exceed its breaking point, and the fluid in a pot  11   b  associated with the first elastomeric membrane opening is fluidly communicable with the fluid pathway defined between the first and second elastomeric membranes  21 , 22  below the foil layer  31 . Due to the selection of material for the first and second elastomeric membranes  21 , 22 , the threshold pressure level that causes the foil layer  31  to reach its breaking point is insufficient to also cause the second elastomeric membrane  22  to be broken, pierced, or otherwise compromised. The second elastomeric membrane  22  will be flexed, but not broken. In various embodiments, the first and second elastomeric membranes  21 , 22  have the same or similar flexible properties. In alternative embodiments, the second elastomeric membrane  22  may have a different tolerance for flexion than the first elastomeric membrane  21 . 
     The first pot  13  can comprise a first upper plunger  55  that can be moved downward in the first chassis  11 , for example by being pressed upon by another component such as a piston. The first upper plunger  55  can thereby force the one or more reagents  16  through the broken section of the first foil layer  31 . 
     The above-noted features, along with other reagent storage features discussed later herein, allow the cartridge  10  to advantageously provide independent storage of multiple different reagents and washes on-board a single lane of the cartridge  10 . Moreover, the cartridge  10  prevents cross-contamination and excessive fluid loss during reagent storage. 
     Thermal Processing and Mixing of Reagents 
       FIGS.  9 A- 9 D  generally illustrate an embodiment of the cartridge  10  which utilizes the structure depicted in  FIG.  1   . As shown in  FIG.  9 C , the one or more reagents  16  can be restricted from flowing downstream in the cartridge  10  by a barrier membrane  33 . This embodiment can be employed by peeling or piercing the foil membrane  18 , depressing the first upper plunger  55  to actuate the one or more reagents  16 , and then the fluid pressure during the dispensing of the one or more reagents  16  can break the barrier membrane  33 . 
     As shown in  FIG.  9 A , the cartridge  10  and/or the instrument  8  in which the cartridge  10  is used can subject the one or more reagents  16  to thermal processing. For example, the instrument  8  in which the cartridge  10  is used can comprise a heater block  52 . Inserting the cartridge  10  into the instrument  8  can position the heater block  52  downstream from the barrier membrane  33 . 
     The heater block  52  can be configured to heat the one or more reagents  16  when the one or more reagents  16  are positioned adjacent the heater block  52  (e.g., above or below). In some embodiments, the heater block  52  extends through holes in the compliant layer  20 . 
     As discussed above, the unsealed portions  31   b  of the first foil layer  31  can form a channel when pushed away from the first chassis  11  by the one or more reagents  16 . The unsealed portions  31   b  can be configured so that the channel directs the one or more reagents  16  to the position adjacent the heater block  52 . As shown in  FIG.  9 B , the support block  12  can comprise the groove  133  that allows the unsealed portions  31   b  of the first foil layer  31  to form the channel, and the channel is then closed by the compliant layer  20 . 
     Referring to  FIGS.  9 A,  9 C and  9 D , the cartridge  10  can comprise a first mixing chamber  111  and/or a second mixing chamber  112 , and the first and second mixing chambers  111 , 112  can be positioned above or below the heater block  52 . The first and second mixing chambers  111 , 112  can be connected to each other by one or more mixing channels  113  that preferably have a width smaller than the width of the chamber preceding the first mixing chamber  111  and/or the width of the channel subsequent to the second mixing chamber  112 , for example less than half in relation thereto. 
     As shown in  FIG.  9 A , a second elastomeric membrane  22  can be positioned over openings in the first chassis  11  and on an opposite side of the openings from the heater block  52 . In an embodiment, the first elastomeric membrane  21  can be absent. The first and second mixing chambers  111 , 112  can be at least partially formed by the second elastomeric membrane  22  and the first foil layer  31 . One or both of the first and second mixing chambers  111 , 112  can be deformable to facilitate mixing of the one or more reagents  16  therein and/or to enable the one or more reagents  16  therein to be pumped through the cartridge  10 . 
     For example, the instrument  8  in which the cartridge  10  is used can comprise one or more pistons  53  that reversibly depress and release the first mixing chamber  111  and/or the second mixing chamber  112  to control fluid flow. The first mixing chamber  111  can be depressed to urge the one or more reagents  16  therein into the one or more mixing channels  113  and/or the second mixing chamber  112 . As a result, the cartridge  10  can provide on-board pumps that facilitate less complexity and easier use in the system (e.g. the instrument  8  can have a simpler design).  FIGS.  9 A,  10 A,  11 A and  12 A  textually identify the component labeled with reference numeral  53  as “Plungers (Instrument)”, while this detailed description refers to this component as piston(s)  53 . 
     A portion of the compliant layer  20  can reversibly close the section of the channel upstream from the first and second mixing chambers  111 , 112  and thus function as a first valve. Another portion of the compliant layer  20  can reversibly close the section of the channel downstream from the first and second mixing chambers  111 , 112  and thus function as a second valve. 
     If the first valve prevents access to the unsealed portions  31   b  of the first foil layer  31  that are upstream from the first mixing chamber  111 , the first mixing chamber  111  can be depressed by a corresponding piston  53  to urge the one or more reagents  16  from the first mixing chamber  111  into the one or more mixing channels  113  and/or the second mixing chamber  112 . If the second valve prevents access to the unsealed portions  31   b  of the first foil layer  31  that are subsequent to the second mixing chamber  112 , the second mixing chamber  112  can be depressed by a corresponding piston  53  to urge the one or more reagents  16  therein back into the one or more mixing channels  113  and/or the first mixing chamber  111  (preferably with the first mixing chamber  111  released). These steps can be repeated to achieve mixing of the one or more reagents  16  with each other and any additional reagents added thereto. 
     If the unsealed portions  31   b  of the first foil layer  31  that are subsequent to the second mixing chamber  112  are accessible and the first mixing chamber  11  is depressed by a corresponding piston  53 , the second mixing chamber  112  can be depressed to urge the one or more reagents  16  from the second mixing chamber  112  into these unsealed portions  31   b  of the first foil layer  31  to form a channel therein. 
     It should be appreciated that  FIGS.  9 A- 9 D ,  FIGS.  10 A- 10 D ,  FIGS.  11 A- 11 C  and  FIGS.  12 A- 12 C  illustrate similar respective views of different embodiments utilizing the structure depicted in  FIG.  8   . Specifically,  FIGS.  10 A- 10 D  generally illustrate an embodiment of the cartridge  10  that can have one or more components similar to those in the embodiment shown in  FIGS.  9 A- 9 D , although preferably the first lower plunger  51  is utilized instead of the barrier membrane  33 .  FIGS.  10 A,  11 A and  12 A  textually identify the component labeled with reference numeral  51  as “Piercer (breaks foil seal thru elastomer layer)”, while this detailed description refers to this component as lower plunger(s)  51 . 
     The first and second mixing chambers  111 , 112  can be at least partially formed by the first and second elastomeric membranes  21 , 22 . The embodiment in  FIGS.  10 A- 10 D  can use a second foil layer  32  to provide thermal contact of the heater block  52  with the first and second mixing chambers  111 , 112 , for example as a patch between the heater block  52  and the first elastomeric membrane  21 . 
       FIGS.  11 A- 11 C  generally illustrate an embodiment of the cartridge  10  that can have one or more components similar to those in the embodiment shown in  FIGS.  10 A- 10 D , although preferably the first and second mixing chambers  111 , 112  are formed by the first elastomeric membrane  21  and the first foil layer  31 . In some embodiments, the second elastomeric membrane  22  can be absent. 
     The heater block  52  can be located above the first and second mixing chambers  111 , 112 ; and the pistons  53  that reversibly depress and release the first mixing chamber  111  and/or the second mixing chamber  112  can be located below the first and second mixing chambers  111 , 112 . In such an embodiment, the first elastomeric membrane  21  can have holes that enable the movable components  53  to reversibly extend and retract therethrough. 
       FIGS.  12 A- 12 C  generally illustrate an embodiment of the cartridge  10  which can have one or more components similar to those in the embodiment shown in  FIGS.  11 A- 11 C , although preferably a second foil layer  32  is positioned between the first foil layer  31  and the compliant layer  20 . The second foil layer  32  can be attached to the first foil layer  31  by welding, pressure sensitive adhesive, or any other means known to the skilled artisan. The first elastomeric membrane  21  can be in the form of a patch that enables the first and second mixing chambers  111 , 112  to undergo the fluid mixing discussed previously. 
     Preferably the upper surface of the first foil layer  31  is fixedly attached to the first chassis  11 , preferably over substantially the entirety of the upper surface of the first foil layer  31 . In this embodiment, the channel can form between the first and second foil layers  31 , 32 . For example, the sealed portions  31   a  of the first foil layer  31  can be thermally welded, laser-welded, bonded by pressure-sensitive adhesive, or otherwise fixedly attached to portions  32   b  of the second foil layer  32 . The first and second foil layers  31 , 32  comprise unsealed portions  31   b , 32   b  circumscribed by the sealed portions  31   b , 32   b . The sealed portions  31   a , 32   a  of the first and the second foil layers  31 , 32  can thus define a periphery of the unsealed portions  31   b , 32   b  of the first and second foil layers  31 , 32  such that the distance between the sealed portions  31   a , 32   a  in any particular direction is the width of the unsealed portions  32   a , 32   b  in the same direction. The unsealed portions  32   a , 32   b  of the first and second foil layers  31 , 32  can be continuous such that they are configured to form the channel. 
     The one or more reagents  16  can be directed between the unsealed portions  31   b , 32   b  of the first and second foil layers  31 , 32 . The fluid can be urged onto adjacent downstream unsealed portions  31   b , 32   b  of the first and second foil layers  31 , 32 , for example by positive displacement and preferably without pneumatic displacement, thereby pushing the downstream unsealed portion  31   b  of the first foil layer  31  and the downstream unsealed portion  32   b  of the second foil layer  32  away from each other. Consequently, the unsealed portions  31   b , 32   b  of the first and second foil layers  31 , 32  can be sequentially pushed apart to form a channel between the first and second foil layers  31 , 32 . 
     The sealed portions  31   a , 32   a  of the first and second foil layers  31 , 32  can thus define a periphery of the channel formed between the unsealed portions  31   b , 32   b  of the first and second foil layers  31 , 32 . As the fluid progresses downstream, the upstream unsealed portions  31   b , 32   b  of the channel which the fluid has evacuated can return to the resting state in which the unsealed portions  31   b , 32   b  of the first and second foil layers  31 , 32  are substantially flat. 
     Construction of Foil Layer and Elastomeric Membrane Defining Fluid Pathway 
     In an embodiment of the cartridge  10  generally illustrated in  FIGS.  13 - 16   , the first elastomeric membrane  21  can be positioned under the first chassis  11 , and at least a portion of the upper surface of the first elastomeric membrane  21  can abut the first chassis  11 . The first foil layer  31  can be positioned under the first elastomeric membrane  21 , and at least a portion of the upper surface of the first foil layer  31  can abut the first elastomeric membrane  21 . The second foil layer  32  can be positioned under the first foil layer  31 , and at least a portion of the upper surface of the second foil layer  32  can abut the first foil layer  31 . The second elastomeric membrane  22  can be positioned under the second foil layer  32 , and at least a portion of the upper surface of the second elastomeric membrane  22  can abut the second foil layer  32 . 
     The compliant layer  20  can be positioned under the second elastomeric membrane  22 , and the compliant layer  20  can reversibly move between a closed position abutting the second elastomeric membrane  22  and an open position out of contact with the second elastomeric membrane  22 . The support block  12 , which can be provided by the instrument  8  in which the cartridge  10  is used, can be positioned under the compliant layer  20 ; the support block  12  can move the compliant layer  20 . 
     In this embodiment, the first elastomeric membrane  21  preferably is a layer of polypropylene/thermoplastic elastomer (PP/TPE) and preferably has a thickness of about 50 μm to about 200 μm, for example about 180 μm. The first foil layer  31  preferably is hard temper aluminum foil and preferably has a thickness of about 20 μm. The second foil layer  32  preferably is soft annealed aluminum foil and preferably has a thickness of about 25 μm. The second elastomeric membrane  22  preferably is a layer of polyurethane elastomer and preferably has a thickness of about 25 μm. Nevertheless, these components are not limited to a specific material or a specific thickness. 
     The first chassis  11  can comprise the first pot  13  that can contain the one or more reagents  16 . The sample can be any size, as non-limiting examples about 10 μl, about 25 μl or about 50 μl. In the embodiment of the cartridge  10  shown in  FIG.  16   , the cartridge  10  can comprise a cap  19  that connects to the first chassis  11  and covers the first pot  13 . 
     Referring again to  FIGS.  13 - 15   , the first pot  13  can contain a first ball  17  for urging the one or more reagents  16  through the lower opening  14 . In a preferred embodiment, the first pot  13  has a passage extending from the upper opening  15  to the lower opening  14 , and the first ball  17  is sized to move through at least part of the passage. Pushing the first ball  17  toward the lower opening  14  can push the one or more reagents  16  toward the lower opening  14  and through the lower opening  14 . In an embodiment, the passage has a lower section with a narrower diameter than the first ball  17  to prevent the first ball  17  from reaching the lower opening  14 . The first ball  17  is not limited to a specific shape, and the first ball  17  can be any component that can be moved within the passage to urge the one or more reagents  16  through the lower opening  14 . 
     In an embodiment, the first ball  17  acts as the first upper plunger  55 . In another embodiment, the first upper plunger  55  is a different component that is used to push the first ball  17 . 
     The first elastomeric membrane  21  can comprise a first weakened portion  23 , and the first pot  13  can be vertically aligned with the first weakened portion  23  of the first elastomeric membrane  21 , such that the lower opening  14  of the first pot  13  is directly above the first weakened portion  23 . The first weakened portion  23  can seal the lower opening  14  of the first pot  13  such that the one or more reagents  16  is held within the first pot  13 , and then the first weakened portion  23  can be broken to allow the one or more reagents  16  to evacuate the first pot  13 . 
     The first weakened portion  23  can be one or more slits, for example two slits intersecting each other to form an “X”, a hole punched in the first elastomeric membrane  21 ; a section of the first elastomeric membrane  21  that has a smaller thickness than the adjacent sections of the first elastomeric membrane  21 ; or any structure that allows pressure applied to this section of the first elastomeric membrane  21  to break the first weakened portion  23  of the first elastomeric membrane  21  without damaging the adjacent portions of the first elastomeric membrane  21 . The first weakened portion  23  is not limited to a specific structure. 
     In a preferred embodiment, the first weakened portion  23  is broken by the first lower plunger  51  being urged upward through the support block  12 . The first lower plunger  51  can push upward against the section of the compliant layer  20  that is vertically aligned with the first lower plunger  51  such that the compliant layer  20  pushes against the first weakened portion  23  of the first elastomeric membrane  21  and thereby breaks the first weakened portion  23 . Then the one or more reagents  16  in the first pot  13  can exit the first pot  13  through the broken first weakened portion  23 , for example by using the first ball  17  and/or the first upper plunger  55 . 
     As shown in  FIG.  14    and discussed briefly above, the first foil layer  31  and/or the second foil layer  32  can comprise one or more holes vertically aligned with the first weakened portion  23  and/or the first lower plunger  51 . In such an embodiment, the one or more reagents  16  exiting the first pot  13  through the lower opening  14  can then travel through the broken first weakened portion  23  and the one or more holes of the first foil layer  31  and/or the second foil layer  32  to reach the upper surface of the second elastomeric membrane  22 . The one or more reagents  16  which exited the first pot  13  to be positioned between the second foil layer  32  and the 2nd elastomeric membrane  22  can then proceed to downstream unsealed portions of the second foil layer  32  and the second elastomeric membrane  22 , thereby forming the channel between the second foil layer  32  and the second elastomeric membrane  22 . As discussed above, the fluid flow along the channel is controlled at least in part by the applied pressure of various actuators or clamps within the instrument  8  disposed along the channel. Such clamps can act as valves to selectively obstruct or permit passage of fluid as desired. 
     Assembly of the foil layers and elastomeric membranes can be accomplished in different ways. For example, as shown in  FIGS.  17 ,  18 A,  18 B,  19 ,  20 A,  20 B and  21   , various assembled configurations of the layers vis-à-vis the cartridge  10  and its other components are contemplated. In one such preferred embodiment, the first foil layer  31  can be positioned under the first chassis  11 , and a pressure-sensitive adhesive (PSA) layer  34  can be positioned under the first foil layer  31 . The first and second elastomeric membranes  21 , 22  can be positioned under the PSA layer  34  and above the compliant layer  20 . As shown in  FIG.  18 B , the first pot  13  can comprise the first ball  17  and/or the first upper plunger  55 . 
     The first foil layer  31  can seal the lower opening  14  of the first pot  13 . As discussed above, in some embodiments, the first foil layer  31  is provided as one or more patches that each cover only a portion of the lower surface of the first chassis  11 , although in other embodiments the first foil layer  31  can be one or more continuous pieces that substantially covers the entirety of the lower surface of the first chassis  11 . 
     In this preferred embodiment, the first elastomeric membrane  21  can have one or more holes  25 , and the PSA layer  34  can have one or more holes  35 . As shown in  FIG.  17   , the holes  25 , 35  can be vertically aligned with the one or more pots  11   b  such that breaking the portion of the first foil layer  31  under the corresponding pot  11   b  can allow the one or more reagents  16  in the corresponding pot  11   b  to travel through the broken portion of the first foil layer  31 , the one or more holes  35  of the PSA layer  34 , and the one or more holes  25  of the first elastomeric membrane  21 . As a result, the one or more reagents  16  can reach the upper surface of the second elastomeric membrane  22 . The one or more reagents  16  which exited the first pot  13  to be positioned between the first and second elastomeric membranes  21 , 22  can then proceed to the downstream unsealed portions of the first and second elastomeric membranes  21 , 22 . 
     For example, as shown in  FIGS.  19 ,  20 A and  20 B , the first elastomeric membrane  21  can comprise sealed portions  21   a  that are thermally welded, laser-welded, bonded by pressure-sensitive adhesive, or otherwise fixedly attached to portions  22   a  of the second elastomeric membrane  22 . The first and second elastomeric membranes  21 , 22  comprise unsealed portions  21   b , 22   b  circumscribed by the sealed portions  21   a , 22   a . The one or more reagents  16  can push an adjacent unsealed portion  22   b  of the second elastomeric membrane and the corresponding unsealed portion  21   b  of the first elastomeric membrane  21  away from each other to form the channel. The one or more reagents  16  can be urged between adjacent downstream unsealed portions  21   b , 22   b  of the first and second elastomeric membranes  21 , 22 , for example by positive displacement and preferably without pneumatic displacement, thereby pushing the downstream unsealed portions  21   b , 22   b  of the first and second elastomeric membranes  21 , 22  apart. Consequently, the unsealed portions  21   b , 22   b  of the first and second elastomeric membranes  21 , 22  can be sequentially pushed apart to form the channel between the first and second elastomeric membranes  21 , 22 . 
     The sealed portions  21   a , 22   a  of the first and second elastomeric membranes  21 , 22  can thus define a periphery of the channel; for example, if the sealed portions  21   a , 22   a  of the first and second elastomeric membranes  21 , 22  are about 2 mm apart, the channel can have a width of about 2 mm. As the fluid progresses downstream, the upstream portions of the channel which the fluid has evacuated can return to the resting state in which the first and second elastomeric membranes  21 , 22  are substantially flat, for example under pressure applied through the compliant layer  20  which forces the unsealed portion  21   b  of the first elastomeric membrane  21  and the unsealed portion  22   b  of the second elastomeric membrane  22  together to close the channel ( FIG.  20 B ). 
     In this embodiment, a section of the unsealed portions  21   b , 22   b  of the first and second elastomeric membranes  21 , 22  preferably forms a chamber ( FIG.  20 A ), such as one or both of the first and second mixing chambers  111 , 112 . The first part of the chamber can be formed by the first elastomeric membrane  21  and can be symmetrical to a second part of the chamber formed by the second elastomeric membrane  22 . Preferably these chamber-forming sections abut or are proximate to each other before the one or more reagents  16  reach them, for example 0.0-50 μm apart, preferably 0.0-10 μm apart. The chamber can be formed by the one or more reagents  16  forcing the chamber-forming section of the first elastomeric membrane  21  apart from the corresponding chamber-forming section of the second elastomeric membrane  22 . The chamber preferably has an ovoid shape but is not limited to a specific shape. 
       FIG.  21    generally illustrates an embodiment of the cartridge  10 , and in this embodiment the first and second membranes  21 , 22  can be configured as shown in  FIGS.  17 ,  18 A,  18 B,  19 ,  20 A and  20 B . The first foil layer  31  can be positioned under one of the pots  11   b  of the chassis  11 , for example a second pot  103 . The PSA layer  34  can be positioned under the first foil layer  31 , and the first and second elastomeric membranes  21 , 22  can be positioned under the PSA layer  34 . 
     In a resting state, the unsealed portion  21   b  of the first elastomeric membrane  21  abuts or is proximate to the unsealed portion  22   b  of the second elastomeric membrane  22 , for example 0.0-50 μm apart, preferably 0.0-10 μm apart. As discussed above, the one or more reagents  16  can be positively displaced between the unsealed portion  21   b  of the first elastomeric membrane  21  and the unsealed portion  22   b  of the second elastomeric membrane  22  in a region of the first and second elastomeric membranes  21 , 22  to push the unsealed portions  21   b , 22   b  away from each other in this region. 
     Then the fluid can be urged into an adjacent region of the first and second elastomeric membranes  21 , 22 , for example by continued positive displacement, thereby pushing the unsealed portions  21   b , 22   b  apart in the adjacent region. Consequently, the unsealed portions  21   b , 22   b  can be sequentially pushed apart to form a channel between the first and second elastomeric membranes  21 , 22 . As the fluid progresses through the unsealed portions  21   b , 22   b , the upstream unsealed portions  21   b , 22   b  which the fluid has evacuated can return to the resting state in which they abut or are proximate to each other. Preferably, the channel does not contain any pressure sensitive adhesive, and thus the fluid does not contact such material. 
     For example, the unsealed portions  21   b  of the first elastomeric membrane  21  can face similarly dimensioned unsealed portions  22   b  of the second elastomeric membrane  22 , and the unsealed portions  21   b , 22   b  can extend continuously from a point proximate to one end of the cartridge  10  to a point proximate to the opposite end of the cartridge  10  without being interrupted by the sealed portions  21   a , 22   a.    
     Without being bound by theory, the present inventors believe that the fluid pushing the unsealed elastomeric membrane portions apart to a volume that substantially conforms to the volume of the fluid minimizes or prevents dead volume in the path travelled by the fluid and thus minimizes or prevents air bubbles in the fluid. In the devices provided by the present disclosure, the fluids can be confined between two elastomeric membranes; the chambers and the fluid paths can be defined by weld lines; zero-volume chambers and fluid channels can be achieved; sample insertion pressures can be used to inflate chambers and channels; and material performance can be assessed, for example during thermal cycling, by modeling the flow and the expansion. 
     Accordingly, the first and second elastomeric membranes  21 , 22  minimize or prevent dead volume in a channel traversed by a mesofluidic and/or microfluidic sample. Moreover, this configuration minimizes the number of fluidic connections in the cartridge  10  by providing a channel that continuously extends between processing steps without the need for transfer to another device and without the need for transfer into a different channel. For example, as discussed in greater detail hereafter, the cartridge  10  is capable of carrying out manual chamber processes, for example both magnetic bead handling and thermal cycling, and in some embodiments using similar materials and chamber designs for each process relative to the other processes. 
     As shown in  FIGS.  14  and  21   , embodiments of the cartridge  10  can subject the one or more reagents  16  traversing the cartridge  10  to thermal processing. For example, the heater block  52 , which can be provided by the instrument  8  in which the cartridge  10  is used, can be positioned downstream from the first pot  13 . The heater block  52  can be configured to heat the one or more reagents  16  when the one or more reagents  16  is positioned between the first and second elastomeric membranes  21 , 22 , above or below the heater block  52 . 
     The unsealed portions  21   b , 22   b  of the first and second elastomeric membranes  21 , 22  can be configured to form a first chamber  24  above the heater block  52 . The first chamber  24  preferably has an ovoid shape with a 0-75 μL volume and a height of about 0.0-2.0 mm, but the first chamber  24  is not limited to a specific shape or specific dimensions. The first chamber  24  is shown in  FIGS.  17  and  21    in an at least partially inflated state. 
     As generally illustrated in  FIG.  22 A , the heater block  52  of one embodiment is convex, and the support block  12  is spaced apart from the lateral sides of the heater block  52  to reduce conduction and improve thermal cycling performance. As shown in  FIGS.  22 B and  22 C , the two circular heater blocks which form the heater block  52  in the embodiment shown in  FIG.  17    are preferably merged into one oval block that forms the heater block  52  in the embodiment shown in  FIG.  21   . In various alternative embodiments, the heater blocks  52  can have a flat, concave or convex contact surface. 
       FIG.  22 C  illustrates that the heater block  52  can extend past the edges of the first chamber  24  to reduce the heat conducted away from the first chamber  24  by the adjacent components of the cartridge  10 . For example, the heater block  52  can have a length greater than the length of the first chamber  24  and/or a width greater than the width of the first chamber  24 . The first chamber  24  can be a single chamber with an external separating valve  252 . 
     Referring again to  FIGS.  14  and  21   , preferably the first chamber  24  can be depressed to evacuate the first chamber  24  of the one or more reagents  16  and urge the one or more reagents  16  from the first chamber  24  to the downstream unsealed portions  21   b , 22   b  of the first and second elastomeric membranes  21 , 22 . For example, one of the pistons  53  can depress the first chamber  24  to urge the one or more reagents  16  from the first chamber  24  to the downstream unsealed portions  21   b ,  22   b  of the first and second elastomeric membranes  21 , 22 . In an embodiment, the one or more pistons  53  can be pressed against one of the first elastomeric membrane  21  and the second elastomeric membrane  22  to push inward and thereby at least partially deflate the first chamber  24 . As a result, the first chamber  24  can be used as a pump. 
     The one or more reagents  16  that have been thermally treated can travel downstream from the heater block  52  through the unsealed portions  21   b ,  22   b  of the first and second membranes  21 , 22 . The cartridge  10  can comprise a second pot  103  that can contain at least a portion of a composition  116  to be mixed with the one or more reagents  16  that have been thermally treated. In an embodiment, the composition  116  comprises at least one reagent for amplification of DNA that is present in the one or more reagents  16  that have been thermally treated. 
     The second pot  103  can be a structure having a lower opening  114  and an upper opening  115  on an opposite end of the second pot  103  from the lower opening  114 . In a preferred embodiment, the second pot  103  has a cylindrical shape, but the second pot  103  is not limited to a specific shape. Furthermore, any number of second pots  103  can be used, and the cartridge  10  is not limited to a specific number of the second pot  103 . In the embodiment of the cartridge  10  shown in  FIG.  16   , the cap  19  that can connect to the first chassis  11  can cover the second pot  103 . 
     As discussed above, the various components of the cartridge enable a fluid passage of reagents from introduction through PCR and to clean-up. The cartridge facilitates superior sample prep by creating an integrated fluid pathway, through which the sample can travel from one process to another, and by incorporating mechanisms allowing external introduction of reagents throughout, as well as magnetic bead handling and thermal processing as necessary. 
     As discussed above,  FIGS.  14  and  21    illustrate various embodiments of the entire integrated workflow contemplated by the automated device of the present disclosure. In  FIGS.  14  and  21   , the upper opening  115  of the second pot  103  can be sealed, for example with a foil membrane  128 , to prevent contamination of the interior of the second pot  103 . The second pot  103  can contain a second ball  118  for urging the composition  116  through the lower opening  114  of the second pot  103 . In a preferred embodiment, the second pot  103  has a passage extending from the upper opening  115  to the lower opening  114 , and the second ball  118  is sized to move through at least part of the passage. Pushing the second ball  118  toward the lower opening  114  can push the composition  116  toward the lower opening  114  and through the lower opening  114 . In an embodiment, the passage has a lower section with a narrower diameter than the second ball  118  to prevent the second ball  118  from reaching the lower opening  114 . The second ball  118  is not limited to a specific shape, and the second ball  118  can be any component that can be moved within the passage to urge composition  116  through the lower opening  114 . 
     In the embodiment shown in  FIGS.  14  and  15   , the lower opening  114  of the second pot  103  can be adjacent to or abut the first elastomeric membrane  21 , for example in a sealing engagement. The first elastomeric membrane  21  can comprise a second weakened portion  123 , and the second pot  103  can be vertically aligned with the second weakened portion  123  of the first elastomeric membrane  21  such that the lower opening  114  of the second pot  103  is directly above the second weakened portion  123 . The second weakened portion  123  can seal the lower opening  114  of the second pot  103  such that the composition  116  is held within the second pot  103 , and then the first weakened portion  23  can be broken to allow the one or more reagents  16  to evacuate the first pot  13 . 
     The second weakened portion  123  can be one or more slits, for example two slits intersecting each other to form an “X”, a hole punched in the first elastomeric membrane  21 ; a section of the first elastomeric membrane  21  that has a smaller thickness than the adjacent sections of the first elastomeric membrane  21 ; or any structure that allows pressure applied to this section of the first elastomeric membrane  21  to break the second weakened portion  123  of the first elastomeric membrane  21  without damaging the adjacent portions of the first elastomeric membrane  21 . The second weakened portion  123  is not limited to a specific structure. 
     In a preferred embodiment, the second weakened portion  123  is broken by a second lower plunger  151  urged upward through the support block  12 . The second lower plunger  151  can push upward against the section of the second elastomeric membrane  22  that is vertically aligned with the second lower plunger  151  such that the second elastomeric membrane  22  pushes against the second weakened portion  123  of the first elastomeric membrane  21  and thereby breaks the second weakened portion  123 . Then the composition  116  in the second pot  103  can exit the second pot  103  through the broken second weakened portion  123 . 
     The first foil layer  31  and/or the second foil layer  32  can comprise one or more holes vertically aligned with the second weakened portion  123  and/or the second lower plunger  151 . In such an embodiment, the composition  116  exiting the second pot  103  through the lower opening  114  can then travel through the broken second weakened portion  123  and the one or more holes of the first and second foil layers  31 ,  32  to reach the upper surface of the second elastomeric membrane  22 . 
     Introduction of Solid Reagents 
     As shown in  FIGS.  23 A- 23 C , the composition  116  can be at least partially formed from one or more solid reagents  117 , and the solid reagents  117  can be positioned in the second pot  103 . For example, the one or more solid reagents  117  can be a lyophilized bead. As another example, the one or more solid reagents  117  can be a freeze-dried cake, and the cake can be dried within the pot or dried between foil layers and a seal. Different foil layers can be used with the cake; in an embodiment, the upper foil layer is weaker than the lower foil layer to ensure that only the upper foil layer breaks when external force is applied and/or the upper foil layer breaks before the lower foil layer when external force is applied. 
     The composition  116  can be formed by reconstituting the lyophilized bead  117 , for example by using diluent  119 . Reconstitution of the lyophilized bead  117  by the diluent  119  can form the composition  116 , and the composition  116  can mix with the one or more reagents  16  that have been thermally treated. At least a portion of the diluent  119  can be positioned in the second pot  103 . The second pot  103  can comprise a second upper plunger  155 , and the second upper plunger  155  can facilitate reconstitution of the lyophilized bead  117  by the diluent  119 . As a result, the cartridge  10  is advantageously capable of on-board storage and reconstitution of dried reagents alongside wet reagents, unlike known mesofluidic and/or microfluidic processing devices. 
     The lyophilized bead  117  can be covered by an upper membrane and a lower membrane. As shown in  FIGS.  23 A and  23 B , the second upper plunger  155  can comprise a downward-pointing spike at a lower end of the second upper plunger  155 . The second upper plunger  155  can be moved downward in the second pot  103 , for example by being pressed by a bung, and thereby force the downward-pointing spike through the upper membrane, allowing the diluent  119  to reach the lyophilized bead  117 . 
     As shown in  FIG.  23 A , the second upper plunger  155  can comprise an extension on an upper end of the second upper plunger  155 , and the extension can be depressed to move the second upper plunger  155  downward in the second pot  103 . The downward-pointing spike can break the upper membrane, allowing the diluent  119  to reach the lyophilized bead  117 , and can break the lower membrane, allowing the composition  116  formed by reconstitution of the lyophilized bead  117  by the diluent  119  to reach the one or more reagents  16  that has been thermally treated. The first and second elastomeric membranes  21 ,  22  can be configured to accommodate the downward-pointing spike. 
     As shown in  FIGS.  23 B and  23 C , a component (e.g. one or both of the first and second elastomeric membranes  21 ,  22 ) can comprise an upward-pointing spike. The upward-pointing spike can be pushed toward the second pot  103 , for example by being pressed by a bung, to force the upward-pointing spike upward through the lower membrane covering the lyophilized bead  117 . Preferably, the upward-pointing spike is employed and then the second upper plunger  155  such that the second upper plunger  155  pushes the composition  116  out of the second pot  103  through the opening created by the upward-pointing spike ( FIG.  23 B ). In another embodiment shown in  FIG.  23 C , the second upper plunger  155  can not have the downward-pointing spike, and the upward-pointing spike can puncture both the lower and upper membranes covering the lyophilized bead  117 . 
     In the embodiment shown in  FIG.  21   , the lower opening  114  of the second pot  103  can be adjacent to or abut the first foil layer  31 , for example in sealing engagement. The first foil layer  31  can be positioned under a third pot  213 , and the pressure-sensitive adhesive (PSA) layer  34  can be positioned under the first foil layer  31 . 
     The second pot  103  can contain the diluent  119 , and a third pot  213  can contain the lyophilized bead  117 . The third pot  213  can be a structure having a lower opening  214  and an upper opening  215  on an opposite end of the third pot  213  from the lower opening  214 . In a preferred embodiment, the third pot  213  has a cylindrical shape, but the third pot  213  is not limited to a specific shape. Furthermore, any number of third pots  213  can be used, and the cartridge  10  is not limited to a specific number of the third pot  213 . In some embodiments, a third lower plunger  152  can be used to break the section of the first foil layer  31  adjacent to the lower opening  214  of the third pot  213 . 
     The third pot  213  can be a separate piece which connects to the first chassis  11  and thus can enable the cartridge  10  to be modular (e.g., a specific third pot  213  can be selected from a plurality of third pots  213  based on the desired material contained by the selected third pot  213 ). For example, a portion of the third pot  213  can have a shape that is similarly dimensioned to an opening in the first chassis  11  ( FIG.  24 A ). Alternatively, the third pot  213  can be integral with the first chassis  11  such that at least a portion of the third pot  213  and at least a portion of the first chassis  11  are formed by one single piece of material ( FIG.  24 B ). 
     The upper opening  215  of the third pot  213  can be sealed, for example with a foil membrane  218 , to prevent contamination of the interior of the third pot  213 . The third pot  213  can contain a third upper plunger  255  for urging the lyophilized bead  117  through the lower opening  214  after reconstitution of the lyophilized bead  117 , as discussed in more detail hereafter. In a preferred embodiment, the third pot  213  has a passage extending from the upper opening  215  to the lower opening  214 , and the third upper plunger  255  is sized to move through at least part of the passage. In an embodiment, the passage has a lower section with a narrower diameter than the third upper plunger  255  to prevent the third upper plunger  255  from reaching the lower opening  214 . 
     The first elastomeric membrane  21  can have at least one of the one or more holes  25  vertically aligned with the lower opening  114  of the second pot  103  such that breaking the portion of the first foil layer  31  under the second pot  103  can allow the diluent  119  in the second pot  103  to travel through the broken portion of the first foil layer  31  and the one or more holes  25  of the first elastomeric membrane  21  to reach the upper surface of the second elastomeric membrane  22 . 
     In a preferred embodiment, the PSA layer  34  can have one or more pre-formed holes. The portion of the first foil layer  31  under the second pot  103  is broken by the second lower plunger  151  which is urged upward through the support block  12 . The second lower plunger  151  can push upward against the section of the first and second elastomeric membranes  21 ,  22  that is vertically aligned with the second lower plunger  151  such that the second elastomeric membranes  22  push against the portion of the first foil layer  31  under the second pot  103  and thereby breaks the portion of the first foil layer  31  under the second pot  103 . Additionally or alternatively, the PSA layer  34  can have weakened structure which can be broken together with the portion of the first foil layer  31 . 
     The first elastomeric membrane  21  and the PSA layer  34  can have at least one of the one or more holes  25  vertically aligned with the lower opening  214  of the third pot  213  such that breaking the portion of the first foil layer  31  under the third pot  213  can allow the diluent  119  which has exited the second pot  103  to travel through the one or more holes  25  of the first elastomeric membrane  21  and the PSA layer  34  and the broken portion of the first foil layer  31  to reach the interior of the third pot  213 . The diluent  119  entering the third pot  213  can reconstitute the lyophilized bead  117 . The third upper plunger  255  can be pushed downward to urge the composition  116  formed by reconstitution of the lyophilized bead  117  by the diluent  119  through the lower opening  214  of the third pot  213 . The composition  116  can travel through the broken portion of the first foil layer  31  and the one or more holes  25  of the first elastomeric membrane  21  and the PSA layer  34  to reach the upper surface of the second elastomeric membrane  22 . 
       FIGS.  45 - 48    generally illustrate another embodiment in which the one or more solid reagents  117  (e.g., a lyophilized bead) can be mixed with the one or more reagents  16  that are being processed or handled in the cartridge  10 . As shown in  FIG.  45   , the components of this embodiment can comprise one or more of: (i) a blister layer  124  that can comprise a layer of amine-terminated polyaniline (OPA)  124   a , an aluminum layer  12   b  and a polypropylene layer  124   c ; (ii) a pierceable foil layer  131  that can comprise a heat seal lacquer  131   a  and a pierceable aluminum layer  131   b ; and (iii) the first and second elastomeric membranes  21 ,  22 . 
     As shown in  FIG.  46   , the one or more solid reagents  117  can be positioned in the blister layer  124 , for example by filling the one or more solid reagents  117  into the blister layer  124  while the one or more solid reagents  117  are in liquid form and then lyophilizing the blister layer  124  with the one or more solid reagents therein. After the one or more solid reagents  117  are positioned in the blister layer  124 , the pierceable foil layer  131  can be heat-sealed to the blister layer  124  to encapsulate the one or more solid reagents  117 . Then, the blister layer  124  with the pierceable foil layer  131  thereon can be heat-sealed to the upper surface of the first chassis  11  to form the twin blisters. 
     The first and second elastomeric membranes  21 ,  22  can be heat-sealed to the bottom surface of the first chassis  11 . Additionally or alternatively, a pressure-sensitive adhesive can be used to attach the first and second elastomeric membranes  21 , 22  to the bottom surface of the first chassis  11 . The first and second elastomeric membranes  21 , 22  can be attached to the bottom surface of the first chassis  11  before the blister layer  124  with the pierceable foil layer  131  thereon is heat-sealed to the upper surface of the first chassis  11 , after the blister layer  124  with the pierceable foil layer  131  thereon is heat-sealed to the upper surface of the first chassis  11 , and/or during the blister layer  124  with the pierceable foil layer  131  thereon being heat-sealed to the upper surface of the first chassis  11 . 
     The one or more solid reagents  117  (e.g., a lyophilized bead) can be vertically aligned with the openings  11   a  in the first chassis  11  such that the one or more solid reagents  117  can be accessed by the one or more reagents travelling through the first and second elastomeric membranes  21 , 22 . For example, as shown in  FIG.  47   , the twin blister can be used by compressing the blisters such that the pierceable foil layer  131  is broken in the areas adjacent the openings  11   a . The section of the first chassis  11  between the openings  11   a  can be valved close, for example by a clamp or another device that can press at least one of the first and second elastomeric membranes  21 , 22  against the first chassis  11 , to prevent fluid flow therethrough. Then the one or more reagents  16  can travel through the first one of the openings  11   a  to reach the one or more solid reagents  117  and mix with the one or more solid reagents  117 . The resultant mixture can travel between the blister layer  124  and the upper surface of the first chassis  11  and then go through the second one of the openings  11   a  to return to a position between the first and second elastomeric membranes  21 , 22 . 
     As shown in  FIG.  48   , the cartridge  10  can be used without the one or more solid reagents  117  positioned thereon. For example, the areas of the blister layer  124  that cover the openings  11   a  can be compressed downward such that the one or more reagents  16  are maintained below the first chassis  11 . The one or more reagents  16  can travel downstream by continuing along the second elastomeric membrane  22 . 
     The twin blister embodiment generally illustrated in  FIGS.  45 - 48    can provide storage stability by fulling enclosing the one or more solid reagents  117  (e.g., a lyophilized bead). Furthermore, the manufacturability can be advantageous because this embodiment is amenable to strip format processing and separates blister fabrication from chassis assembly. Moreover, this embodiment minimizes dead volume significantly due to blister compression. 
     As shown in  FIGS.  14  and  21   , the one or more reagents  16  that has been thermally treated can be urged downstream through the unsealed portions  21   b , 22   b  of the first and second elastomeric membranes  21 , 22  so that the one or more reagents  16  that has been thermally treated mixes with the composition  116 . Preferably the reagents in the composition  116  amplify the single-stranded DNA in the one or more reagents  16 . The one or more reagents  16  that has been thermally treated can mix with the composition  116  within the unsealed portions  21   b ,  22   b  of the first and second elastomeric membranes  21 , 22 . 
     Magnetic Bead Handling Mechanism 
     As shown in  FIGS.  14  and  21   , the cartridge  10  can comprise a magnetic bead handling mechanism  300  that can be configured to bind, wash and elute the amplified DNA. Thus the cartridge  10  can perform processes such as PCR and/or DNA sequencing without the need for a centrifuge. The magnetic bead handling mechanism  300  can comprise one or more magnets, for example an upper magnet  301  and/or a lower magnet  302 , and the magnets  301 , 302  can be motor-driven. For example, a processor, optionally communicatively connected to a graphic user interface, can control movement of the magnets  301 , 302  (e.g. vertical movement to change a height of the magnet relative to the first and second elastomeric membranes  21 , 22 ; horizontal movement to change a distance of the magnet along the length and/or the width of the cartridge  10 ; and rotation movement of the magnet). Each of the magnets  301 ,  302  can be a permanent magnet and/or can be an electromagnet which can be activated, deactivated, and have its field strength adjusted, for example by the processor. Any number of magnets may be used, and disclosures herein regarding the first and second magnets  301 , 302  also apply to additional magnets, e.g. a third magnet, a fourth magnet, etc. 
     The magnetic bead handling mechanism  300  can comprise one or more chambers  310  formed by the first and second elastomeric membranes  21 , 22 , and the one or more reagents  16  can enter the one or more chambers  310  after being mixed with the composition  116 . One or more washes of the bead-bound DNA can be performed while the magnets  301 ,  302  maintain the position of the beads in the chambers  310 , for example by the magnets  301 ,  302  keeping the bead-bound DNA clumped together. Preferably the magnets  301 ,  302  are held stationary while the beads are washed. In a preferred embodiment, the one or more chambers  310  comprise the first and second mixing chambers  111 , 112  and the one or more mixing channels  113  discussed previously herein. 
     The magnetic bead handling mechanism  300  can comprise temperature sensors and temperature control devices (not shown), which can be controlled by the processor in the instrument  8  in which the cartridge  10  is used. 
     In various alternative embodiments, dye exterminator beads may be employed to extract dye from the reagent before it continues in the process. In some embodiments, such beads can be non-magnetic dye sponges encapsulated in a chamber along the flexible membrane, accessible via a pierceable bypass valve. In some embodiments, the non-magnetic dye exterminator beads are employed instead of the magnetic bead handling mechanism discussed above. In other embodiments, the non-magnetic dye exterminator beads are used as a supplement to the magnetic bead handling mechanism. 
     Referring again to  FIG.  21   , the compliant layer  20  and the support block  12  can form one or more valves at various positions of the cartridge  10 . A closed position of the valve can close a section of the channel formed by the unsealed portions  21   b , 22   b  of the first and second elastomeric membranes  21 , 22  and force the fluid in the channel upstream and/or downstream (depending on the position of the other valves and pistons). An open position of the valve can allow a section of the channel to open under pressure from the one or more reagents  16 . 
     For example, a closed position of the valve can hold a section of the unsealed portions  21   b  of the first elastomeric membrane  21  against the corresponding (e.g., vertically aligned) section of the unsealed portions  22   b  of the second elastomeric membrane  22 . Consequently, the one or more reagents  16  cannot move into or through these sections of the unsealed portions  21   b , 22   b  of the first and second elastomeric membranes  21 , 22 . The open position of the valve can allow the section of the unsealed portions  21   b  of the first elastomeric membrane  21  to move away from the corresponding section of the unsealed portions  22   b  of the second elastomeric membrane  22  when the one or more reagents  16  reaches these sections. 
     As a non-limiting example, one of the valves can be positioned between the first pot  13  and the heater block  52 , can be in a closed position to prevent the one or more reagents  16  injected from the first pot  13  from reaching the first chamber  24  adjacent the heater block  52 , then can be in an open position to allow the one or more reagents  16  to reach the first chamber  24  adjacent the heater block  52 , and then can return to the closed position to prevent the one or more reagents  16  in the first chamber  24  adjacent the heater block  52  from travelling upstream in the cartridge  10  (e.g., back toward the first pot  13 ). 
     As another non-limiting example, one of the valves can be positioned between the heater block  52  and the second pot  103 , can be in a closed position to prevent the one or more reagents  16  in the first chamber  24  adjacent the heater block  52  from reaching the second pot  103 , then can be in an open position to allow the one or more reagents  16  to reach the second pot  103 , and then can return to the closed position to prevent the one or more reagents  16  adjacent the second pot  103  from travelling upstream in the cartridge  10  (e.g., back toward the first chamber  24  above the heater block  52 ). 
     As yet another non-limiting example, one of the valves can be positioned between the third pot  213 , can be in a closed position to prevent the one or more reagents  16  adjacent the third pot  213  from reaching the magnetic bead handling mechanism  300 , then can be in an open position to allow the one or more reagents  16  to reach the magnetic bead handling mechanism  300 , and then can return to the closed position to prevent the one or more reagents  16  adjacent the magnetic bead handling mechanism  300  from travelling upstream in the cartridge  10  (e.g., back toward the third pot  213 ). 
     As shown in  FIGS.  14  and  21   , the one or more chambers  310  of the magnetic bead handling mechanism  300  can comprise the first mixing chamber  111 , the second mixing chamber  112 , and the one or more mixing channels  113  that provide fluid communication between the first and second mixing chambers  111 , 112 . 
     In an embodiment, the sections of the first and second elastomeric membranes  21 , 22  that correspond to the first and second mixing chambers  111 , 112  can be pre-formed with the desired shape ( FIG.  25 A ). In such an embodiment, before the one or more reagents  16  reaches these pre-formed sections of the first and second elastomeric membranes  21 , 22 , the pre-formed section of the first elastomeric membrane  21  can curve downward and abut the pre-formed section of the second elastomeric membrane  22 . Then, when the one or more reagents  16  reaches these sections, the one or more reagents  16  can push the pre-formed section of the first elastomeric membrane  21  away from the pre-formed section of the second elastomeric membrane  22  such that the first and second mixing chambers  111 , 112  are formed. Advantages of such an embodiment can include ease of inflation of the first and second mixing chambers  111 , 112  and a minimized pressure inside the first and second mixing chambers  111 , 112 . 
     In another embodiment, the sections of the first and second elastomeric membranes  21 , 22  that correspond to the first and second mixing chambers  111 , 112  are flat before the one or more reagents  16  reaches these sections ( FIG.  25 B ). Then, when the one or more reagents  16  reaches these sections, the one or more reagents  16  can push them apart such that the first and second mixing chambers  111 , 112  are formed. Advantages of such an embodiment can include improved thermal contact and a reduced chance of air bubbles inside the first and second mixing chambers  111 , 112 . 
     In other embodiments, one or both of the first and second mixing chambers  111 , 112  are not pre-formed sections of the first and second elastomeric membranes  21 , 22 . 
       FIGS.  26 A- 26 D  are photographs from an initial chamber fluid handling test performed in an embodiment in which the first and second mixing chambers  111 , 112  comprise pre-formed sections of the first and second elastomeric membranes  21 , 22 . 
       FIGS.  27 A- 27 C  show a cross-section of an embodiment of the first elastomeric membrane  21  and the second elastomeric membrane  22  attached to each other. An inlet port  224  and/or a chamber port  225  can be positioned on the first elastomeric membrane  21 . The inlet port  224  and the chamber port  225  can be formed of any suitable material, for example cyclic olefin copolymer (COC), and can be provided by a single piece of material or be distinct pieces of material. In an embodiment, the first chassis  11  can provide the inlet port  224  and/or the chamber port  225 , preferably as part of the magnetic bead handling mechanism  300 , for example in vertical alignment with the first mixing chamber  111  and the second mixing chamber  112  respectively. The pistons  53  can move in the inlet port  224  and/or the chamber port  225  to push against the first elastomeric membrane  21  and/or the second elastomeric membrane  22  to urge the one or more reagents  16  therein to the other mixing chamber or a downstream position. 
       FIGS.  28 A and  28 B  show displacement of the unsealed portions  21   b , 22   b  of the first and second elastomeric membranes  21 , 22  in a cross-section thereof. As shown in these figures, the fluid displaces the elastomeric membranes  21 , 22  from each other to form a channel and exerts even greater displacement to form a chamber.  FIG.  29    shows the flow distribution in such a “Bow-Tie” configuration relative to other geometries. 
     The first and second mixing chambers  111 , 112  can be utilized for one or more steps of a DNA purification process performed in the cartridge  10 . For example, magnetic beads can be employed in the first and second mixing chambers  111 , 112 . As used herein in reference to the magnetic beads, “dispersing” refers to re-suspending beads in fluid so they can be transported, and “mixing” refers to re-suspending beads and then performing steps to ensure sufficient bead surface area contacts a particular reagent (sample/wash) for the required timescale. When the magnetic beads are transported, the magnetic beads must reach the correct chamber rather than diffusing or drifting beyond this point. Therefore, in an embodiment, the magnetic beads may be fixedly positioned (“trapped”) by one or more of the magnets  301 ,  302  as a solution of the magnetic beads is injected into the first mixing chamber  111 . 
     The magnetic beads can disperse far into the first and second mixing chambers  111 , 112  when injected but can be trapped precisely if the magnetic beads are injected while one or more of the magnets  301 ,  302  are present. In an embodiment, the magnetic beads can be collected in less than one minute, and collection of the magnetic beads can be assisted if the one or more of the magnets  301 ,  302  are moved relative to the first and second mixing chambers  111 , 112  rather than maintained in a stationary position. 
     Flow within the first and second mixing chambers  111 , 112  can be generated by an inlet to one of the first and second mixing chambers  111 , 112  and/or by pumping fluid by depressing and releasing one or more of the first and second elastomeric membranes  21 , 22 . 
     A round shape of the first and second mixing chambers  111 , 112  can result in stagnant areas at the edges thereof, where velocity is low. Magnetic beads positioned in these areas may be difficult to dislodge. Therefore, in a preferred embodiment, the one or more mixing channels  113  can be narrow, for example having a width of 1-2 mm, and a length of 3-5 mm, to concentrate fluid flow and ensure a high velocity therein. Magnetic beads can be collected in the one or more mixing channels  113  and readily dispersed when the magnets  301 ,  302  are removed. 
     The magnetic beads can be substantially uniformly dispersed and/or mixed by spreading the magnetic beads with a moving magnet and/or using oscillating flow. The oscillating flow can be generated by depressing and releasing one or more of the first and second elastomeric membranes  21 , 22  (e.g., using the pistons  53 ) and/or by directing flow using a syringe connected to an inlet or an outlet of the first and second mixing chambers  111 , 112 . 
     As a non-limiting example, the Sanger method may be used in the cartridge  10 . In this embodiment, magnetic beads can be released in solution, the magnetic beads can be transferred to the first mixing chamber  111 , the sample can be transferred into the first mixing chamber  111 , the magnetic beads can be re-suspended if needed and/or desired, the magnetic beads can be fixedly positioned in the first mixing chamber  111  by one or more magnets while subjected to a first wash, the magnetic beads can be re-suspended if needed and/or desired, the magnetic beads can be fixedly positioned in the first mixing chamber  111  by one or more of the magnets  301 ,  302  while subjected to a second wash, the magnetic beads can be re-suspended if needed and/or desired, elution buffer can be added to the first mixing chamber  111 , the magnetic beads can be re-suspended if needed and/or desired, and the elution buffer can be transferred from the first mixing chamber  111  to the second mixing chamber  112 . In this non-limiting example, these steps are preferably performed in the order they are listed, but one or more of the steps may be omitted, and additional steps may be added. 
     In an embodiment, the magnets  301 ,  302  can trap the magnetic beads in the first and second mixing chambers  111 , 112  during ethanol washes and can trap the magnetic beads in the one or more mixing channels  113  prior to elution steps with elution buffer. In an embodiment, the magnets  301 ,  302  can be positioned within the pistons  53  and can be retractable within the pistons  53 . This configuration of the magnets  301 ,  302  can trap the magnetic beads rapidly inside the first and second mixing chambers  111 , 112 . After the magnetic beads are collected by the magnets  301 ,  302 , the collected magnetic beads can be drawn into the one or more mixing channels  113 . 
       FIGS.  30 A- 30 E  generally illustrate various configurations that can be employed in the magnetic bead handling mechanism  300 . As shown in  FIGS.  30 A- 30 D , the mixing channel  113  can comprise a pierceable bypass valve which can contain pre-loaded dry reagent or liquid beads. As shown in  FIG.  30 E , the first and second mixing chambers  111 , 112  can form a wide “kidney-shaped” chamber, and the mixing channel  113  can be absent. The magnetic beads can be trapped in a valved-off region at an end of the chambers  111 , 112 . 
     In some embodiments, an additional chamber  114  can be connected to one or both of the first and second mixing chambers  111 , 112 . A valve can selectively block the connection to the additional chamber  114  to prevent the contents of the additional chamber  114  from exiting the chamber  114 , such as during washing of the corresponding one of the first and second mixing chambers  111 , 112 . A non-limiting example of an experiment utilizing the additional chamber  114  is shown in  FIG.  31   . 
     Elastomeric Membrane Physical Properties 
     Preferably the material of the first and second elastomeric membranes  21 , 22  has one or more of the following characteristics: capable of piercing foil by being pushed against the foil be a plunger, as discussed previously herein; capable of being inflated to form a chamber, as discussed previously herein; formable; weldable; biologically compatible; capable of maintaining integrity at a 100° C. operating temperature; and/or performing as a high temperature vapor barrier. For example, some thermoplastic elastomers such as polyvinyl chloride are capable of continued functionality at higher temperatures, and some high barrier films such as a co-extrusion of polyethylene and polyurethane can be employed. Therefore, in a preferred embodiment generally illustrated in  FIG.  32 A , the first elastomeric membrane  21  is formed from a single piece of material, and the second elastomeric membrane  22  is formed from a single piece of material. 
     As generally illustrated in  FIGS.  32 B and  32 C , an embodiment of the cartridge  10  uses one or more first elastomeric patches  121  on the first elastomeric membrane  21 . The first elastomeric membrane  21  can comprise open sections  21   c  with which the one or more first elastomeric patches  121  can be substantially co-extensive such that the combination of the one or more first elastomeric patches  121  and the first elastomeric membrane  21  form a continuous layer. The one or more first elastomeric patches  121  are preferably a different material than the first elastomeric membrane  21 , for example a material with higher temperature tolerance and/or greater vapor barrier characteristics relative to the material of the first elastomeric membrane  21 . The one or more first elastomeric patches  121  can be used in areas of the cartridge  10  where the fluid traversing the cartridge  10  is subjected to higher temperatures. The cartridge  10  can comprise one or more second elastomeric patches (not shown) which can cooperate with the second elastomeric membrane  22  similarly to the one or more first elastomeric patches  121  and the first elastomeric membrane  21  as discussed above. 
     As a non-limiting example,  FIGS.  33 A and  33 B  show an embodiment of the cartridge  10  in which an elastomer is bonded directly to a moulded lane, for example a lane moulded into polypropylene. The elastomer can be directly bonded by heat-sealing, a pressure-sensitive adhesive, or any other suitable means known to the skilled artisan. Additionally or alternatively to the direct bonding, the elastomer can be bonded to a foil layer that is on the moulded lane, and the foil layer can form the lids of the chambers, optionally with a pressure sensitive adhesive layer. An externally applied feature can be used to evacuate fluid from the channels. 
       FIGS.  34 A and  34 B  show this embodiment of the cartridge  10  clamped into a fixture  80  that can comprise a clamping plate  81 , with a valve comprising a screw that can rotated one direction to move the valve to the open position and can be rotated an opposite direction to move the valve to the closed position. Preferably, the clamping plate  81  comprises recesses configured to allow the unsealed portions  21   b , 22   b  of the first and second elastomeric membranes  21 , 22  to inflate when not closed by the valve and when reached by the one or more reagents  16 . The clamping plate  81  can prevent the layers in the cartridge  10 , such as the first and second elastomeric membranes  21 , 22 , from delaminating. 
       FIG.  35    shows fluid loading into this embodiment of the cartridge  10 .  FIG.  36    shows progression of fluid through the cartridge  10 , for example fluid dosing and mixing.  FIG.  37    shows the one or more reagents  16  flowing between the unsealed portions  21   b , 22   b  of the first and second membranes  21 , 22 , thereby forming a channel in the cartridge  10 . 
       FIGS.  38 - 41    show an embodiment of a system  100  comprising the cartridge  10 . The system  100  can comprise plungers  101  that can be used to dose the one or more reagents  16  and/or other materials into the cartridge  10 , a clamping frame  102  to which the cartridge  10  can reversibly connect, an intermediate piece  104  to which the clamping frame  102  can reversibly connect, and a base  105  to which the intermediate piece  103  can reversibly connect. The base  105  can comprise one or more legs  106  configured to position the cartridge  10  at an elevated location. 
     In an embodiment, the base  106  can comprise toggle clamps  107  that are configured to be separately operated to thereby actuate the channels in the cartridge  10 , for example by moving between a closed and an open position. Moving one of the toggle clamps  106  into a closed position can urge the one or more reagents  16  positioned in the corresponding section of the cartridge  10  to a downstream location in the cartridge  10 . For example, each of the toggle clamps  107  can comprise a section of the support block  12  and a section of the compliant layer  20  affixed on the section of the support block  12 . 
       FIG.  42    generally illustrates an embodiment of the cartridge  10  in which bubble traps are used, preferably between the first and second elastomeric membranes  21 , 22 . The bubble traps can lead to dead volume, thus the bubble traps can be configured to balance the dead volume created and the effectiveness at scavenging bubbles.  FIG.  42    shows that inserts, for example stereolithography inserts, can be employed as bubble traps. Alternative geometries and materials can be used for the inserts. In an embodiment, the inserts can be formed or fabricated plastic foils. 
       FIG.  42    contains schematic drawings showing that the welding techniques used to laminate the first and second elastomeric membranes  21 , 22  can form the bubble traps; for example, sections of the first elastomeric membrane  21  welded to sections of the second elastomeric membrane  22  can form the bubble traps.  FIG.  43 A  is a photograph of an embodiment in which sections of the first elastomeric membrane  21  are welded to sections of the second elastomeric membrane  22  can form the bubble traps. 
       FIG.  43 B  is a photograph of a de-bubbler channel  253 . In various embodiments, the de-bubbler channel  253  includes a fluid channel where one wall is a porous membrane. As a fluid containing air bubbles is pushed along the channel under pressure, the air is vented through the membrane and is thereby removed from the fluid. 
       FIG.  44    is a schematic diagram of a non-limiting example of reagents and their positioning in the cartridge  10  in an embodiment of the cartridge  10 . 
     Various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. Therefore, such changes and modifications are covered by the appended claims.