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.
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.
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.
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
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
In various embodiments of the present disclosure, as generally illustrated in
It should be appreciated that an automated system, as illustrated generally in
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
As seen in
Finally, the fourth exemplary stage illustrated in
Referring now to
As can be seen from
Cartridge Construction and Design
In an embodiment generally illustrated in
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 31a 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 31b circumscribed by the sealed portions 31a. The sealed portions 31a of the first foil layer 31 can thus define a periphery of the unsealed portions 31b of the first foil layer 31 such that the distance between the sealed portions 31a in any particular direction is the width of the unsealed portions 31b in the same direction. The unsealed portions 31b can extend approximately the entire length of the cartridge 10, continuous with each other and uninterrupted by the sealed portions 31a, such that the unsealed portions 31b 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 31b 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 31b of the first foil layer 31, for example by positive displacement and preferably without pneumatic displacement, thereby pushing the downstream unsealed portion 31b of the first foil layer 31 away from the first chassis 11. Consequently, the unsealed portions 31b 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 31a of the first foil layer 31 can thus define a periphery of the channel formed between the first chassis 11 and the unsealed portions 31b of the first foil layer 31; for example, if the sealed portions 31a 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 31b 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 31b which contain the fluid to positively displace the fluid downstream and close the channel behind the fluid, for example by pushing the unsealed portions 31b containing the fluid to be substantially flat. If the upstream unsealed portions 31b 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 31b 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
As generally illustrated in these figures and discussed in greater detail later herein, the first chassis 11 can have openings 11a; encapsulated reagents can be positioned in the openings 11a, and/or one or more elastomeric seals can be positioned on the first chassis 11 over the openings 11a. 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 11b positioned above one or more of the openings 11a, and reagents within the one or more pots 11b can access the adjacent section of the channel and/or can be accessed from the adjacent section of the channel through the corresponding opening 11a.
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 31b 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 31a with the unsealed portions 31b 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 11b 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.
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 21a of the first elastomeric membrane 21 can circumscribe unsealed portions 21b of the first elastomeric membrane 21. The sealed portions 21a of the first elastomeric membrane 21 can thus define a periphery of the unsealed portions 21b of the first elastomeric membrane 21 such that the distance between the sealed portions 21a in any particular direction is the width of the unsealed portions 21b in the same direction.
The unsealed portions 21b 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 21b of the first elastomeric membrane 21. The fluid can be urged onto an adjacent downstream unsealed portion 21b of the first elastomeric membrane 21, for example by positive displacement and preferably without pneumatic displacement, thereby pushing the downstream unsealed portion 21b of the first elastomeric membrane 21 away from the first chassis 11. Consequently, the unsealed portions 21b 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 21a of the first elastomeric membrane 21 can thus define a periphery of the channel formed between the first chassis 11 and the unsealed portions 21b of the first elastomeric membrane 21; for example, if the sealed portions 21a 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
As the fluid progresses downstream, the upstream unsealed portions 21b 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 21b which contain the fluid to positively displace the fluid downstream and close the channel behind the fluid, for example by pushing the unsealed portions 21b containing the fluid to be substantially flat. If the upstream unsealed portions 21b are also held substantially flat by the compliant layer 20, the fluid can travel downstream.
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
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 11b. 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 11b. 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 11b 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
As shown in
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 31b 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 31b 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
Referring to
As shown in
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).
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 31b 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 31b 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 31b 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 31b of the first foil layer 31 to form a channel therein.
It should be appreciated that
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
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.
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 31a of the first foil layer 31 can be thermally welded, laser-welded, bonded by pressure-sensitive adhesive, or otherwise fixedly attached to portions 32b of the second foil layer 32. The first and second foil layers 31,32 comprise unsealed portions 31b,32b circumscribed by the sealed portions 31b,32b. The sealed portions 31a,32a of the first and the second foil layers 31,32 can thus define a periphery of the unsealed portions 31b,32b of the first and second foil layers 31,32 such that the distance between the sealed portions 31a,32a in any particular direction is the width of the unsealed portions 32a,32b in the same direction. The unsealed portions 32a,32b 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 31b,32b of the first and second foil layers 31,32. The fluid can be urged onto adjacent downstream unsealed portions 31b,32b 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 31b of the first foil layer 31 and the downstream unsealed portion 32b of the second foil layer 32 away from each other. Consequently, the unsealed portions 31b,32b 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 31a,32a of the first and second foil layers 31,32 can thus define a periphery of the channel formed between the unsealed portions 31b,32b of the first and second foil layers 31,32. As the fluid progresses downstream, the upstream unsealed portions 31b,32b of the channel which the fluid has evacuated can return to the resting state in which the unsealed portions 31b,32b 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
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
Referring again to
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
Assembly of the foil layers and elastomeric membranes can be accomplished in different ways. For example, as shown in
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
For example, as shown in
The sealed portions 21a,22a of the first and second elastomeric membranes 21,22 can thus define a periphery of the channel; for example, if the sealed portions 21a,22a 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 21b of the first elastomeric membrane 21 and the unsealed portion 22b of the second elastomeric membrane 22 together to close the channel (
In this embodiment, a section of the unsealed portions 21b,22b of the first and second elastomeric membranes 21,22 preferably forms a chamber (
In a resting state, the unsealed portion 21b of the first elastomeric membrane 21 abuts or is proximate to the unsealed portion 22b 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 21b of the first elastomeric membrane 21 and the unsealed portion 22b of the second elastomeric membrane 22 in a region of the first and second elastomeric membranes 21,22 to push the unsealed portions 21b,22b 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 21b,22b apart in the adjacent region. Consequently, the unsealed portions 21b,22b 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 21b,22b, the upstream unsealed portions 21b,22b 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 21b of the first elastomeric membrane 21 can face similarly dimensioned unsealed portions 22b of the second elastomeric membrane 22, and the unsealed portions 21b,22b 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 21a,22a.
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
The unsealed portions 21b,22b 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
As generally illustrated in
Referring again to
The one or more reagents 16 that have been thermally treated can travel downstream from the heater block 52 through the unsealed portions 21b, 22b 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
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,
In the embodiment shown in
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
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
As shown in
As shown in
In the embodiment shown in
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 (
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.
As shown in
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 11a 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
As shown in
The twin blister embodiment generally illustrated in
As shown in
Magnetic Bead Handling Mechanism
As shown in
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
For example, a closed position of the valve can hold a section of the unsealed portions 21b of the first elastomeric membrane 21 against the corresponding (e.g., vertically aligned) section of the unsealed portions 22b of the second elastomeric membrane 22. Consequently, the one or more reagents 16 cannot move into or through these sections of the unsealed portions 21b,22b of the first and second elastomeric membranes 21,22. The open position of the valve can allow the section of the unsealed portions 21b of the first elastomeric membrane 21 to move away from the corresponding section of the unsealed portions 22b 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
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 (
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 (
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.
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.
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
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
As generally illustrated in
As a non-limiting example,
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.
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.
Filing Document | Filing Date | Country | Kind |
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PCT/US2016/050246 | 9/2/2016 | WO | 00 |