阅读说明：本技术 用于反应容器的原位密封的设备、方法和系统 (Apparatus, method and system for in situ sealing of reaction vessels ) 是由 C·W·麦克纳尔 M·A·约翰逊 于 2019-12-20 设计创作，主要内容包括：提供了用于原位密封反应孔的系统、方法和设备。本发明提供了用于原位密封说明性地封闭反应容器中的各个反应孔的反应容器、方法和系统,其使用反应(例如,热循环反应)中已经存在的条件来使密封材料变形,从而密封反应孔,并产生在反应期间存在且在反应完成之后保留的密封。(Systems, methods, and apparatus for sealing reaction wells in situ are provided. The present invention provides reaction vessels, methods and systems for in situ sealing of illustratively closed individual reaction wells in a reaction vessel, which use conditions already present in the reaction (e.g., thermal cycling reaction) to deform a sealing material, thereby sealing the reaction wells and producing a seal that is present during the reaction and remains after the reaction is complete.)

1. A method for in situ sealing of fluid samples in a plurality of reaction wells, comprising:

providing a reaction vessel comprising an array having a plurality of reaction wells, wherein the array is disposed between a lower layer and an upper layer, the lower layer being bonded to a first end of the array to seal the first end of the reaction wells, and a second end of the array or an inner surface of the upper layer being provided with a sealing material for sealing the second end of the reaction wells in situ,

introducing a fluid sample into the reaction vessel such that each of the plurality of reaction wells is filled with a portion of the fluid sample, an

Exposing the array to reaction conditions comprising heat and/or pressure such that the sealing material seals the second ends of the reaction wells in situ, thereby substantially preventing the fluid sample from flowing out of the plurality of reaction wells during or after exposure to the reaction conditions.

2. The method of claim 1, wherein exposing the array to the reaction conditions comprises applying heat or pressure to the array, and wherein the reaction conditions comprise applying heat or pressure substantially only to the array and without requiring in situ addition of additional heat or pressure to seal the second end of the reaction well with the sealing material.

3. The method of claim 1, wherein exposing the array to the reaction conditions comprises applying both heat and pressure to the array.

4. The method of claim 1, wherein exposing the array to reaction conditions comprises exposing the array to thermal cycling conditions.

5. The method of claim 4, wherein exposing the array to the thermal cycling conditions comprises applying heat adjacent to the lower layer and applying pressure adjacent to the upper layer.

6. The method of claim 1, wherein the upper layer is a flexible film layer that can be pressed against the array to seal a portion of the sample in each of the plurality of reaction wells.

7. The method of claim 6, wherein the sealing material comprises a film layer bonded to an inner surface of the upper layer adjacent to the second end of the reaction well,

the film layer comprises a sealing material selected from the group comprising: heat and pressure activated adhesives, swelling materials that swell in an aqueous environment, waxes, and combinations thereof, and

the method also includes bonding the sealing material under reactive conditions to seal each of the plurality of reactive pores.

8. The method of claim 7, wherein the heat and pressure activated adhesive is selected from the group comprising: ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA), Ethylene Methyl Acetate (EMA), ethylene n-butyl acrylate (EnBA), Ethylene Acrylic Acid (EAA), Thermoplastic Polyurethane (TPU), polycaprolactone, silicone rubber, thermoplastic elastomers, waxes, polyethylene, polypropylene, low density polyethylene, copolymers thereof, and combinations thereof.

9. The method of claim 8, wherein the heat and pressure activated adhesive has a melting point in a range of about 60 ℃ to about 100 ℃ and exposing the array to the reaction conditions comprises deforming the sealing material, and wherein deforming the sealing material comprises softening or at least partially melting the heat and pressure activated adhesive in situ under thermal cycling conditions to deform the heat and pressure activated adhesive into the openings of the plurality of reaction wells.

10. The method of claim 1, wherein the array further comprises a perforated layer bonded to the array adjacent to the second end of the upper layer, the perforated layer having one or more perforations for each reaction well, wherein the one or more perforations for each reaction well allow fluid sample to enter into each of the plurality of reaction wells but prevent fluid sample from flowing back out of the reaction well.

11. The method of claim 10, wherein the perforated layer further comprises a sealing material selected from the group consisting of: heat and pressure activated adhesives, swelling materials that swell in an aqueous environment, oils, waxes, and combinations thereof, and wherein the sealing material of the perforated layer deforms in situ under thermal cycling conditions to seal each of the plurality of reaction pores.

12. The method of claim 11, wherein the heat and pressure activated adhesive is selected from the group comprising: ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA), Ethylene Methyl Acetate (EMA), ethylene n-butyl acrylate (EnBA), Ethylene Acrylic Acid (EAA), Thermoplastic Polyurethane (TPU), polycaprolactone, silicone rubber, thermoplastic elastomers, waxes, polyethylene, polypropylene, low density polyethylene, copolymers thereof, and combinations thereof.

13. The method of claim 1, wherein the array is disposed in a closed reaction vessel, the reaction vessel further comprising:

a sample injection port for introducing a sample into the container,

a cell lysis zone configured for lysing cells, viruses, or spores located in the sample, the cell lysis zone being in fluid connection with a sample injection port,

a nucleic acid preparation zone in fluid connection with the cell lysis zone, the nucleic acid preparation zone configured for purifying nucleic acids, and

a first stage reaction zone fluidly connected to the nucleic acid preparation zone and the array, the first stage reaction zone comprising a first stage reaction bubble cap configured for a first stage amplification of a sample,

wherein the cell lysis zone, the nucleic acid preparation zone and the first stage reaction zone are all disposed within the closed reaction vessel, and

the method further comprises the steps of:

injecting a fluid sample into the container via the sample injection port and sealing the sample injection port after injecting the fluid sample,

introducing the fluid sample into the cell lysis zone and performing cell lysis in the cell lysis zone to produce a cell lysate,

extracting nucleic acid from the cell lysate and moving the extracted nucleic acid to the first-stage reaction zone,

subjecting the nucleic acid in the first reaction zone to amplification conditions,

fluidically moving a portion of the nucleic acid from the first stage reaction zone to each of the plurality of reaction wells of the array, an

Performing a second stage amplification in a plurality of reaction wells of the array.

14. The method of claim 13, wherein the first stage reaction zone comprises a set of primers for PCR amplification of nucleic acids in the fluid sample, and wherein each of the plurality of reaction wells of the array comprises a pair of primers for PCR amplification of a unique nucleic acid.

15. The method of claim 1, wherein the seal is formed using heat and pressure supplied during or generated by reaction conditions, and wherein the formation of the seal does not include a separate heating or pressure step.

16. A container for performing a reaction with a fluid sample in a closed system, the container comprising:

a reaction region including a plurality of layers including an array layer in which a plurality of reaction wells are formed, a first outer layer coupled to a first end of the array layer to seal the first end of the reaction wells, a second outer layer disposed adjacent to a second end of the reaction wells opposite to the first end of the reaction wells such that a fluid sample introduced into the reaction region can flow into each of the reaction wells, and

a sealing layer bonded to the second outer layer disposed adjacent to the second end of the reaction wells or bonded to the second end of the array layer adjacent to the second outer layer, wherein the sealing layer substantially seals the reaction wells in situ under at least one of heat and pressure to prevent backflow of the fluid sample from the reaction wells during or after the reaction.

17. The container of claim 16, wherein the sealing layer comprises a sealing material selected from the group consisting of: heat and pressure activated adhesives, swelling materials that swell in an aqueous environment, waxes, and combinations thereof.

18. The container of claim 17, wherein the heat and pressure activated adhesive and/or wax at least softens and deforms under thermal cycling conditions to substantially seal the second end of the reaction well.

19. The container of claim 18, wherein the heat and pressure activated adhesive is selected from the group comprising: ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA), Ethylene Methyl Acetate (EMA), ethylene n-butyl acrylate (EnBA), Ethylene Acrylic Acid (EAA), Thermoplastic Polyurethane (TPU), polycaprolactone, silicone rubber, thermoplastic elastomers, waxes, polyethylene, polypropylene, low density polyethylene, copolymers thereof, and combinations thereof.

20. The container of claim 19, wherein the heat and pressure activated adhesive and/or wax has a melting point in the range of about 60 ℃ to about 100 ℃.

21. The container of claim 16, further comprising a perforated layer bonded to the array layer adjacent to the second outer layer, wherein the perforated layer has one or more perforations for each reaction well, and the one or more perforations extend through the perforated layer and are large enough to allow the fluid sample to enter into each of the plurality of reaction wells, but small enough to prevent backflow of the fluid sample from the reaction wells.

22. The container of claim 21, wherein the perforated layer further comprises a sealing material selected from the group consisting of: heat and pressure activated adhesives, swelling materials that swell in an aqueous environment, oils, waxes, and combinations thereof.

23. The container of claim 22, wherein the heat and pressure activated adhesive is selected from the group consisting of: ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA), Ethylene Methyl Acetate (EMA), ethylene n-butyl acrylate (EnBA), Ethylene Acrylic Acid (EAA), Thermoplastic Polyurethane (TPU), polycaprolactone, silicone rubber, thermoplastic elastomers, waxes, polyethylene, polypropylene, low density polyethylene, copolymers thereof, and combinations thereof.

24. The container of claim 16, further comprising

A sample injection port for introducing a sample into the container,

a cell lysis zone configured for lysing cells or spores located in the sample, the cell lysis zone being in fluid connection with a sample injection port,

a nucleic acid preparation zone in fluid connection with the cell lysis zone, the nucleic acid preparation zone configured for purifying nucleic acids, and

a first stage reaction zone fluidly connected to the nucleic acid preparation zone and the reaction zone, the first stage reaction zone comprising a first stage reaction bubble cap configured for a first stage amplification of a sample.

25. The container of claim 24, wherein the cell lysis zone, nucleic acid preparation zone, first stage reaction zone, and reaction zone are all disposed within the closed system.

26. A heat cycle system comprises

A sample container for containing a fluid sample to be thermally cycled, the sample container comprising:

a high density reaction zone comprising an array having a plurality of reaction wells, wherein the high density reaction zone is disposed in a closed system between an upper layer and a lower layer bonded to the array to seal one end of the reaction wells, and a sealing material for sealing a second end of reaction wells in situ,

wherein the fluid sample received in the high-density reaction region flows into each of the reaction wells, and

wherein the sealing material deforms under thermal cycling conditions to seal the second end of the reaction well to substantially prevent backflow of the fluid sample from the reaction well,

an instrument configured to receive the sample container and subject a sample therein to thermal cycling conditions, wherein the instrument comprises:

a heater unit for thermally cycling a fluid sample in the high density reaction zone between at least a first temperature and a second temperature at a cycle time, the sample container being received in the instrument with an underlying layer adjacent to the heater unit,

a pressure transducer for compressing the high density reaction zone adjacent to an upper layer; and

a controller for controlling the heater unit and the pressure transducer.

27. The system of claim 26, wherein the controller comprises one or both of an internal computing device or an external computing device.

28. The system of claim 26, wherein the sample container is part of a closed reaction vessel having at least one additional fluidly connected sample container therein.

29. The system of claim 26, wherein the controller is programmed to perform a method of sealing fluid samples in the plurality of reaction wells in situ, the method comprising:

providing a sample container, wherein the sample container is provided,

introducing the fluid sample into the high-density reaction zone such that each of the plurality of reaction wells is filled with a portion of the fluid sample, an

Exposing the array to reaction conditions comprising heat and/or pressure such that the sealing material seals the second ends of the reaction wells in situ, thereby substantially preventing fluid sample from flowing out of the plurality of reaction wells during or after exposure to the reaction conditions.

Background

Infectious diseases account for approximately 7% of human mortality in the united states, canada, and western europe, while infectious diseases account for over 40% of human mortality in developing regions. Infectious diseases lead to various clinical manifestations. Common manifestations include fever, pneumonia, meningitis, diarrhea, and bloody diarrhea. Although physical manifestations suggest that the disease is caused by some pathogens and eliminates others as pathogens, there are a variety of potential pathogens and a definitive diagnosis often requires the performance of various assays. Traditional microbiological techniques for identifying pathogens in clinical specimens can take days or weeks, often delaying the appropriate course of treatment.

In recent years, Polymerase Chain Reaction (PCR) has become the method of choice for rapid identification of infectious agents. PCR is a rapid, sensitive, and specific tool for diagnosing infectious diseases. However, the challenge of using PCR as a primary diagnostic tool is the variety of possible pathogenic organisms or viruses and the low levels of organisms or viruses present in some pathological samples. It is often impractical to run large PCR set assays, one for each possible pathogenic organism or virus, where most pathogenic organisms or viruses are expected to be negative. This problem is exacerbated when the pathogen nucleic acid is at low concentrations and requires large amounts of sample to collect sufficient reaction templates. In some cases, there are not enough samples to assay for all possible pathogens. One solution is to run a "multiplex PCR" in which the sample is assayed simultaneously for multiple targets in a single reaction. While multiplex PCR has proven valuable in some systems, there are disadvantages associated with the robustness of high-level multiplex reactions and the difficulty of washing assays for multiple products. To address these issues, the assay can then be divided into multiple secondary PCRs. Nesting secondary reactions within the primary product increases robustness. Closed systems such as FilmArray (BioFire Diagnostics, LLC, salt lake City, Utah) reduce throughput and thereby reduce the risk of contamination.

The array of micropores contained within the FilmArray bag provides a platform for performing a plurality of analytical tests on small liquid samples. It is necessary to properly seal the liquid within each microwell in this and other systems to isolate the reaction and produce accurate results. A permanent seal may also be desirable to maintain the integrity of the wells, allowing for subsequent evaluation and analysis after the initial reaction period, illustratively for further analysis performed sometime after the bag is removed from the instrument. Both pressure sensitive adhesives and heat seal adhesives present difficulties in performing this sealing function. Pressure sensitive adhesives risk prematurely adhering and sealing the micropore openings prior to filling the pores. Heat sealing can also be problematic because the temperature sensitivity of the reagents in the reaction wells can prevent the use of an additional heating step to seal the wells. The present invention addresses various improvements associated with sealing reaction wells in situ using conditions already present in thermal cycling.

Disclosure of Invention

Embodiments of the present disclosure solve one or more of the foregoing or other problems in the art. The present invention provides reaction vessels, methods and systems for in situ sealing of illustratively closed individual reaction wells in a reaction vessel, using conditions already present in the reaction (e.g., thermal cycling reaction) to deform a sealing material, thereby sealing the reaction wells and forming a seal that is present during the reaction and remains after the reaction is complete. Such a sealable reaction vessel, method and system does not risk prematurely adhering and sealing the micro-well opening prior to filling the well. Also, because the conditions required to form a seal are already present in a normal reaction, the containers, methods, and systems described herein do not require an additional heating step to form a seal. Reaction wells sealed according to the methods and systems described herein can be stored and readied on the same or different instruments. For example, such reaction wells may be used to compare well-to-well variability or instrument-to-instrument variability. In addition, reaction wells sealed according to the methods and systems described herein can be used to make standards (e.g., fluorescence standards), which can be used to calibrate an instrument. Because the sealing material is included with the reaction vessel and there is little risk of premature seal formation, the use of a sealable reaction vessel and the methods and systems described herein may not require any special handling or sample preparation on the part of the user. Although the embodiments described herein relate to in situ sealing of reaction wells, it should be understood that the principles and apparatus described herein may be used for in situ sealing of any portion of a reaction vessel, such as for in situ sealing of a reaction chamber (e.g., reaction bubble cap) or fluidic channel.

Described herein are:

1. a method for sealing a fluid sample in situ in a plurality of reaction wells, comprising:

providing a reaction vessel comprising an array having a plurality of reaction wells, wherein the array is disposed between a lower layer and an upper layer, the lower layer is bonded to a first end of the array to seal the first end of the reaction wells, and a second end of the array or an inner surface of the upper layer is provided with a sealing material for sealing the second end of the reaction wells in situ,

introducing a fluid sample into the reaction vessel such that each of the plurality of reaction wells is filled with a portion of the fluid sample, an

Exposing the array to reaction conditions comprising heat and/or pressure such that the sealing material seals the second ends of the reaction wells in situ, thereby substantially preventing the fluid sample from flowing out of the plurality of reaction wells during or after exposure to the reaction conditions.

2. The method according to clause 1, wherein exposing the array to the reaction conditions comprises applying heat or pressure to the array, and wherein the reaction conditions comprise applying heat or pressure substantially only to the array, and no additional heat or pressure needs to be added in situ to seal the second end of the reaction well with the sealing material.

3. The method according to one or more of clauses 1 or 2, wherein exposing the array to the reaction conditions comprises applying both heat and pressure to the array.

4. The method according to one or more of clauses 1-3, wherein exposing the array to reaction conditions comprises exposing the array to thermal cycling conditions.

5. The method according to one or more of clauses 1-4, wherein exposing the array to thermal cycling conditions comprises applying heat adjacent to the lower layer and applying pressure adjacent to the upper layer.

6. The method according to one or more of clauses 1-5, wherein the upper layer is a flexible membrane layer that can be pressed against the array to seal a portion of the sample in each of the plurality of reaction wells.

7. The method according to one or more of clauses 1-6, wherein the sealing material comprises a membrane layer bonded to the inner surface of the upper layer adjacent to the second end of the reaction well,

the film layer comprises a sealing material selected from the group comprising: heat and pressure activated adhesives, swelling materials that swell in an aqueous environment, waxes, and combinations thereof, and

the method also includes bonding a sealing material under the reaction conditions to seal each of the plurality of reaction wells.

8. The method according to one or more of clauses 1-7, wherein the heat-activated and pressure-activated adhesive is selected from the group comprising: ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA), Ethylene Methyl Acetate (EMA), ethylene n-butyl acrylate (EnBA), Ethylene Acrylic Acid (EAA), Thermoplastic Polyurethane (TPU), polycaprolactone, silicone rubber, thermoplastic elastomers, waxes, polyethylene, polypropylene, low density polyethylene, copolymers thereof, and combinations thereof.

9. The method according to one or more of clauses 1-8, wherein the heat and pressure activated adhesive has a melting point in the range of about 60 ℃ to about 100 ℃, and exposing the array to the reaction conditions comprises deforming the sealing material, and wherein deforming the sealing material comprises softening or at least partially melting the heat and pressure activated adhesive in situ under thermal cycling conditions to deform the heat and pressure activated adhesive into the openings of the plurality of reaction wells.

10. The method of one or more of clauses 1-9, wherein the array further comprises a perforated layer bonded to the array adjacent to the second end of the upper layer, the perforated layer having one or more perforations for each reaction well, wherein the one or more perforations for each reaction well allow fluid sample to enter each of the plurality of reaction wells but prevent fluid sample from flowing back from the reaction well.

11. The method of one or more of clauses 1-10, wherein the perforated layer further comprises a sealing material selected from the group consisting of: heat-activated and pressure-activated adhesives, swelling materials that swell in an aqueous environment, oils, waxes, and combinations thereof, and wherein the sealing material of the perforated layer deforms in situ under thermal cycling conditions to seal each of the plurality of reaction pores.

12. The method of one or more of clauses 1-11, wherein the heat-activated and pressure-activated adhesive is selected from the group consisting of: ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA), Ethylene Methyl Acetate (EMA), ethylene n-butyl acrylate (EnBA), Ethylene Acrylic Acid (EAA), Thermoplastic Polyurethane (TPU), polycaprolactone, silicone rubber, thermoplastic elastomers, waxes, polyethylene, polypropylene, low density polyethylene, copolymers thereof, and combinations thereof.

13. The method according to one or more of clauses 1-12, wherein the array is provided in a closed reaction vessel, the reaction vessel further comprising:

a sample injection port for introducing a sample into the container,

a cell lysis zone configured for lysing cells, viruses, or spores located in a sample, the cell lysis zone being in fluid connection with a sample injection port,

a nucleic acid preparation zone in fluid connection with the cell lysis zone, the nucleic acid preparation zone configured for purifying nucleic acids, and

a first stage reaction zone fluidly connected to the nucleic acid preparation zone and the array, the first stage reaction zone comprising a first stage reaction bubble cap configured for first stage amplification of the sample,

wherein the cell lysis zone, the nucleic acid preparation zone and the first-stage reaction zone are all arranged in a closed reaction vessel, and

the method further comprises the following steps:

injecting a fluid sample into the container via the sample injection port, and sealing the sample injection port after injecting the fluid sample,

introducing a fluid sample into the cell lysis zone and performing cell lysis in the cell lysis zone to produce a cell lysate,

extracting nucleic acid from the cell lysate, and moving the extracted nucleic acid to the first-stage reaction zone,

subjecting the nucleic acid in the first reaction zone to amplification conditions,

fluidically moving a portion of the nucleic acid from the first stage reaction zone to each of the plurality of reaction wells of the array, an

Performing a second stage amplification in the plurality of reaction wells of the array.

14. The method according to one or more of clauses 1-13, wherein the first stage reaction zone comprises a set of primers for PCR amplification of nucleic acids in the fluid sample, and wherein each of the plurality of reaction wells of the array comprises a pair of primers for PCR amplification of a unique nucleic acid.

15. The method according to one or more of clauses 1-14, wherein the seal is formed using heat and pressure supplied during or generated by the reaction conditions, and wherein forming the seal does not include a separate heating or pressure step.

16. A container for performing a plurality of reactions with a fluid sample, the container comprising:

an array having a plurality of reaction wells, wherein the array is disposed between an upper layer and a lower layer, the lower layer being bonded to a first end of the array to seal the first end of the reaction wells, an

At least one of the second end of the array or the upper layer is provided with a sealing material for sealing the second end of the reaction wells in situ, wherein after providing the fluid sample to the plurality of reaction wells, and the reaction conditions comprising heat and/or pressure cause the sealing material to seal the second end of the reaction wells to substantially prevent the fluid sample from flowing out of the reaction wells.

17. The vessel according to clause 16, wherein the reaction conditions include both heat and pressure applied to the array.

18. The vessel according to one or more of clauses 16-17, wherein the reaction conditions substantially only include heat or pressure applied to the array, and no additional heat or pressure needs to be added in situ to seal the reaction wells with the sealing material.

19. The container according to one or more of clauses 16-18, wherein the reaction conditions include heat applied adjacent to the lower layer and pressure applied adjacent to the upper layer.

20. The vessel according to one or more of clauses 16-19, wherein heat and pressure are applied to the array during the thermocycling reaction.

21. The container according to one or more of clauses 16-20, wherein the sealing material comprises a film layer bonded to an upper layer adjacent to the second end of the reaction well, wherein the film layer bonded to the upper layer comprises a sealing material selected from the group consisting of: heat and pressure activated adhesives, swelling materials that swell in an aqueous environment, waxes, and combinations thereof.

22. The container according to one or more of clauses 16-21, wherein the heat-activated and pressure-activated adhesive or wax at least partially softens or melts under thermal cycling conditions to adhere to and substantially seal the second end of the reaction well.

23. The container according to one or more of clauses 16-22, wherein the heat-activated and pressure-activated adhesive is selected from the group consisting of: ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA), Ethylene Methyl Acetate (EMA), ethylene n-butyl acrylate (EnBA), Ethylene Acrylic Acid (EAA), Thermoplastic Polyurethane (TPU), polycaprolactone, silicone rubber, thermoplastic elastomers, waxes, polyethylene, polypropylene, low density polyethylene, hydrophilic gels or gelling agents, polyvinyl alcohol, polyvinyl acetate, copolymers thereof, and combinations thereof.

24. The container according to one or more of clauses 16-23, wherein the heat-activated and pressure-activated adhesive has a melting point in the range of from about 60 ℃ to about 100 ℃.

25. The container of one or more of clauses 16-24, further comprising a perforated layer having one or more perforations for each reaction well, the perforated layer bonded to the array adjacent to the layer, wherein the one or more perforations extend through the perforated layer and are large enough to allow a fluid sample to enter into each of the plurality of reaction wells, but small enough to prevent backflow of a fluid sample from the reaction well.

26. The container according to one or more of clauses 16-25, wherein the perforated layer does not include a sealing material.

27. The container according to one or more of clauses 16-26, wherein the perforated layer further comprises a sealing material selected from the group consisting of: heat and pressure activated adhesives, swelling materials that swell in an aqueous environment, oils, waxes, and combinations thereof.

28. The container according to one or more of clauses 16-27, wherein the heat-activated and pressure-activated adhesive is selected from the group consisting of: ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA), Ethylene Methyl Acetate (EMA), ethylene n-butyl acrylate (EnBA), Ethylene Acrylic Acid (EAA), Thermoplastic Polyurethane (TPU), polycaprolactone, silicone rubber, thermoplastic elastomers, waxes, polyethylene, polypropylene, low density polyethylene, copolymers thereof, and combinations thereof.

29. The container according to one or more of clauses 16-28, wherein each of the plurality of reaction wells comprises one or more reagents, wherein the reagents comprise one or more of a pair of PCR primers, each of the plurality of reaction wells is provided with a different pair of PCR primers, or a control nucleic acid and a pair of primers configured to amplify a control nucleic acid, and at least another well comprises the same primers but does not comprise a control nucleic acid.

30. The container according to one or more of clauses 16-29, wherein the array is disposed in a closed system, the container further comprising

A sample injection port for introducing a sample into the container,

a cell lysis zone configured for lysing cells or spores located in a sample, the cell lysis zone being in fluid connection with a sample injection port,

a nucleic acid preparation zone in fluid connection with the cell lysis zone, the nucleic acid preparation zone configured for purifying nucleic acids, and

a first stage reaction zone in fluid connection with the nucleic acid preparation zone and the channel for receiving the fluid sample into the plurality of reaction wells, the first stage reaction zone comprising a first stage reaction bubble cap configured for first stage amplification of the sample, wherein the array is disposed in a second stage reaction zone, wherein each of the plurality of wells comprises means for further amplification of the sample.

31. The vessel according to one or more of clauses 16-30, wherein the cell lysis zone, the nucleic acid preparation zone, and the first stage reaction zone are all disposed within a closed system.

32. A vessel for performing a reaction with a fluid sample in a closed system, the vessel comprising:

a reaction zone including a stack of layers including an array layer having a plurality of reaction wells formed therein, a first outer layer bonded to the array layer to seal first ends of the reaction wells, a second outer layer disposed adjacent to second ends of the reaction wells opposite to the first ends of the reaction wells such that a fluid sample introduced into the reaction zone can flow into each reaction well, and

a sealing layer bonded to a second end of the second outer layer or the array layer disposed adjacent to the second end of the reaction wells adjacent to the second outer layer, wherein the sealing layer substantially seals the reaction wells in situ under at least one of heat and pressure to prevent backflow of the fluid sample from the reaction wells during or after the reaction.

33. The container of clause 32, wherein the sealing layer comprises a sealing material selected from the group consisting of: heat and pressure activated adhesives, swelling materials that swell in an aqueous environment, waxes, and combinations thereof.

34. The container according to one or more of clauses 32-33, wherein the heat-activated and pressure-activated adhesive and/or wax at least softens and deforms under thermal cycling conditions to substantially seal the second end of the reaction well.

35. The container according to one or more of clauses 32-34, wherein the heat-activated and pressure-activated adhesive is selected from the group consisting of: ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA), Ethylene Methyl Acetate (EMA), ethylene n-butyl acrylate (EnBA), Ethylene Acrylic Acid (EAA), Thermoplastic Polyurethane (TPU), polycaprolactone, silicone rubber, thermoplastic elastomers, waxes, polyethylene, polypropylene, low density polyethylene, copolymers thereof, and combinations thereof.

36. The container according to one or more of clauses 32-35, wherein the heat-activated and pressure-activated adhesive and/or wax has a melting point in the range of from about 60 ℃ to about 100 ℃.

37. The vessel according to one or more of clauses 32-36, wherein the stack of layers of the reaction region further comprises a perforated layer bonded to the array layer adjacent to the second outer layer, wherein for each reaction well perforated layer there is one or more perforations, and the one or more perforations extend through the perforated layer and are large enough to allow the fluid sample to enter into each of the plurality of reaction wells, but small enough to prevent backflow of the fluid sample from the reaction well.

38. The container according to one or more of clauses 32-37, wherein the perforated layer further comprises a sealing material selected from the group consisting of: heat and pressure activated adhesives, swelling materials that swell in an aqueous environment, oils, waxes, and combinations thereof.

39. The container according to one or more of clauses 32-38, wherein the heat-activated and pressure-activated adhesive is selected from the group consisting of: ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA), Ethylene Methyl Acetate (EMA), ethylene n-butyl acrylate (EnBA), Ethylene Acrylic Acid (EAA), Thermoplastic Polyurethane (TPU), polycaprolactone, silicone rubber, thermoplastic elastomers, waxes, polyethylene, polypropylene, low density polyethylene, copolymers thereof, and combinations thereof.

40. The container according to one or more of clauses 32-39 further comprising

A sample injection port for introducing a sample into the container,

a cell lysis zone configured for lysing cells or spores located in a sample, the cell lysis zone being in fluid connection with a sample injection port,

a nucleic acid preparation zone in fluid connection with the cell lysis zone, the nucleic acid preparation zone configured for purifying nucleic acids, and

a first stage reaction zone fluidly connected to the nucleic acid preparation zone and the reaction zone, the first stage reaction zone comprising a first stage reaction bubble cap configured for a first stage amplification of a sample.

41. The vessel according to one or more of clauses 32-40, wherein the cell lysis zone, the nucleic acid preparation zone, the first stage reaction zone, and the reaction zone are all disposed within a closed system.

42. A heat cycle system comprises

A sample container for containing a fluid sample to be thermally cycled, the sample container comprising:

a high-density reaction zone comprising an array having a plurality of reaction wells, wherein the high-density reaction zone is disposed in a closed system between an upper layer and a lower layer, the lower layer being bonded to the array to seal one end of the reaction wells, and a sealing material for sealing a second end of the reaction wells in situ,

wherein the fluid sample received in the high-density reaction region flows into each reaction well, and

wherein the sealing material deforms under thermal cycling conditions to seal the second end of the reaction well to substantially prevent backflow of the fluid sample from the reaction well,

an instrument configured to receive a sample container and subject a sample therein to thermal cycling conditions, wherein the instrument comprises:

a heater unit for thermally cycling a fluid sample in the high density reaction zone between at least a first temperature and a second temperature at a cycle time, the sample container being received in the instrument with the underlying layer adjacent to the heater unit,

a pressure transducer for laminating the high density reaction zone adjacent to the upper layer; and

a controller for controlling the heater unit and the pressure transducer.

43. The system of clause 42, wherein the controller comprises one or both of an internal computing device or an external computing device.

44. The system according to one or more of clauses 42-43, wherein the sample container is part of a closed reaction vessel having at least one additional fluidly connected sample container therein.

45. The system of one or more of clauses 42-44, wherein the controller is programmed to perform the method of one or more of clauses 1-15.

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

Additional features and advantages will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The features and advantages may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features will become more fully apparent from the following description and appended claims, or may be learned by the practice of the invention as set forth hereinafter.

Drawings

FIG. 1 shows a flexible bag for stand-alone PCR.

Fig. 2 is an exploded perspective view of an instrument for use with the bag of fig. 1, including the bag of fig. 1.

Fig. 3 shows a partial cross-section of the instrument of fig. 2 with the bag of fig. 1, including the bladder member of fig. 2.

FIG. 4 shows a motor used in one illustrative embodiment of the instrument of FIG. 2.

Fig. 5A illustrates a cross-sectional view of an embodiment of a high density reaction zone of a reaction vessel in which an in-situ sealing layer is disposed on an inner surface of an upper outer layer.

Fig. 5B illustrates the high-density reaction zone of fig. 5A, wherein an in-situ seal is formed to substantially seal the fluid sample in the high-density wells.

FIG. 6A illustrates a cross-sectional view of another embodiment of a high density reaction zone of a reaction vessel with in-situ sealing material disposed on the high density array.

Fig. 6B illustrates the high-density reaction zone of fig. 6A, wherein an in-situ seal is formed to substantially seal the fluid sample in the high-density wells.

Fig. 7A illustrates a cross-sectional view of another embodiment of a high density reaction zone of a reaction vessel in which an in-situ sealing layer is disposed on an inner surface of an upper outer layer.

Fig. 7B illustrates the high-density reaction zone of fig. 7A, wherein an in-situ seal is formed to substantially seal the fluid sample in the high-density wells.

FIG. 8A illustrates a cross-sectional view of another embodiment of a high density reaction zone of a reaction vessel in which in-situ sealing materials are associated with the high density array.

Fig. 8B illustrates the high-density reaction zone of fig. 8A, wherein an in-situ seal is formed to substantially seal the fluid sample in the high-density wells.

FIG. 9 illustrates a cross-sectional view of a film material that may be used to make an in-situ sealing material.

Fig. 10A-10C illustrate an embodiment of a thermal cycle system that may be used with a reaction vessel that includes a high density reaction zone and an in-situ sealing feature.

Fig. 10D illustrates a high density reaction zone similar to that shown in fig. 7A and 7B after an in situ seal is formed in the thermal cycler apparatus of fig. 10A-10C.

Fig. 11 illustrates a time course experiment at several time points (in the process, 1 week, 3 weeks) where the fluorescent material was retained in the wells of the high-density reaction zone with and without the in-situ sealing material.

Detailed Description

Example embodiments are described below with reference to the drawings. Many different forms and embodiments are possible without departing from the spirit and teachings of the present disclosure, and therefore the present disclosure should not be construed as limited to the example embodiments set forth herein. Rather, these example embodiments are provided so that this disclosure will be thorough and complete, and will convey the scope of the disclosure to those skilled in the art. In the drawings, the size and relative sizes of layers and regions may be exaggerated for clarity. Like reference numerals refer to like elements throughout the specification.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of this application and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Although a few methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, only certain illustrative materials and methods are described herein.

All publications, patent applications, patents, or other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification will control.

Various aspects of the disclosure, including apparatus, systems, methods, etc., may be described with reference to one or more illustrative embodiments. As used herein, the terms "exemplary" and "illustrative" mean "serving as an example, instance, or illustration," and should not be construed as preferred or advantageous over other embodiments disclosed herein. In addition, references to "an embodiment" or "an embodiment" of the present disclosure or invention include specific references to one or more embodiments thereof, and vice versa, and are intended to provide illustrative examples without limiting the scope of the invention, which is indicated by the appended claims rather than by the following description.

It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a sheet of material (tile)" includes one, two or more sheets of material. Similarly, references to multiple references are to be construed as including a single reference and/or multiple references unless the content and/or context clearly dictates otherwise. Thus, reference to "a sheet of material" does not necessarily require a plurality of such sheets of material. Rather, it is to be understood that the terminology is used independently of the conjunctions; one or more sheets of material are contemplated herein.

Further, as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination ("or") when interpreted in the alternative.

As used throughout this application, the words "may" and "may" are used in a permissive sense (i.e., meaning having the potential of … …), rather than the mandatory sense (i.e., meaning must). Furthermore, the terms "comprising," "having," "involving," "including," "characterized by," and variations thereof (e.g., "comprises," "having," "involves," "including," etc.), as well as similar terms used herein, including the claims, are intended to be inclusive and/or open-ended, should have the same meaning as the word "comprising" and variations thereof (e.g., "comprises" and "comprising"), and illustratively do not exclude additional, unrecited elements or method steps.

As used herein, directions and/or any terms, such as "top," "bottom," "left," "right," "upper," "lower," "inner," "outer," "proximal," "distal," "front," "rear," and the like, may be used solely to indicate relative directions and/or orientations, and are not intended to otherwise limit the scope of the disclosure, including the description, inventions, and/or claims.

It will be understood that when an element is referred to as being "coupled," "connected," or "responsive" to or "on" another element, it can be directly coupled, connected, or responsive to or on the other element or intervening elements may also be present. In contrast, when an element is referred to as being "directly coupled," "directly connected," "directly responsive" or "directly on" another element, there are no intervening elements present.

Illustrative embodiments of the inventive concept are described herein with reference to cross-sectional illustrations that are schematic illustrations of idealized embodiments (and intermediate structures) of the exemplary embodiments. Thus, variations from the shapes of the illustrations as a result, for example, of manufacturing techniques and/or tolerances, are to be expected. Thus, example embodiments of the inventive concept should not be construed as limited to the particular shapes of regions illustrated herein but are to include deviations in shapes that result, for example, from manufacturing. Thus, the regions illustrated in the figures are schematic in nature and their shapes are not intended to illustrate the actual shape of a region of a device and are not intended to limit the scope of example embodiments.

It will be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Thus, a "first" element may be termed a "second" element without departing from the teachings of the present embodiments.

It should also be understood that various embodiments described herein may be utilized in conjunction with any other embodiment described or disclosed without departing from the scope of the present disclosure. Thus, products, components, elements, devices, apparatus, systems, methods, processes, compositions, and/or kits according to certain embodiments of the present disclosure may contain, incorporate, or otherwise include the properties, features, components, elements, steps, and/or the like described in other embodiments disclosed herein (including systems, methods, apparatus, and/or the like) without departing from the scope of the present disclosure. Thus, references to particular features associated with an embodiment are not to be construed as limited to application within that embodiment.

The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims. To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. Further, where possible, like element numbers are used in the various figures. Further, alternative configurations of particular elements may each include a separate letter appended to the element number.

The term "about" as used herein means approximately, in the region of … …, approximately, or approximately. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify numerical values that are 5% variance above and below the stated value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

The word "or" as used herein means any one member of a particular list and also includes any combination of members of that list.

"sample" means an animal; a tissue or organ from an animal; cells (in a subject, taken directly from a subject, or cells maintained in culture, or cells taken from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; a solution (e.g., a polypeptide or nucleic acid) containing one or more molecules derived from a cell, cellular material, or viral material; or a solution containing non-naturally occurring nucleic acids, drugs or pharmaceuticals and drug processing precursors (e.g., biologicals, pharmaceuticals, injectants, bioreactor parts, etc.), which can be assayed as described herein. The sample may also be any bodily fluid or discharge (e.g., without limitation, blood, urine, feces, saliva, tears, bile, or cerebrospinal fluid) which may or may not contain host or pathogen cells, cellular components, or nucleic acids. Samples may also include environmental samples such as, but not limited to, soil, water (fresh water, waste water, etc.), air monitoring system samples (e.g., materials captured in an air filtration medium), surface swabs, and carriers (e.g., mosquitoes, ticks, fleas, etc.).

The phrase "nucleic acid" as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-or double-stranded, sense or antisense, capable of hybridizing to a complementary nucleic acid by watson-crick base pairing. The nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU) and non-phosphodiester internucleoside linkages (e.g., Peptide Nucleic Acids (PNAs) or thiodiester linkages). In particular, nucleic acids may include, but are not limited to, DNA, RNA, mRNA, rRNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

"Probe", "primer" or "oligonucleotide" means a single-stranded nucleic acid molecule of defined sequence that can base pair with a second nucleic acid molecule ("target") containing a complementary sequence. The stability of the resulting hybrid depends on the length, GC content and the extent to which base pairing occurs. The degree of base pairing is affected by parameters such as the degree of complementarity between the probe and target molecule and the stringency of the hybridization conditions. The degree of stringency of hybridization is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to those skilled in the art. Probes, primers and oligonucleotides may be detectably labeled, either radioactively, fluorescently or non-radioactively, by methods well known to those skilled in the art. dsDNA binding dyes can be used to detect dsDNA. It will be understood that a "primer" is specifically configured to be extended by a polymerase, while a "probe" or "oligonucleotide" may or may not be so configured.

By "dsDNA binding dye" is meant a dye that fluoresces differently when bound to double stranded DNA than when bound to single stranded DNA or when free in solution, typically by fluorescing more strongly. While reference is made to dsDNA binding dyes, it is to be understood that any suitable dye may be used herein, some non-limiting illustrative dyes of which are described in U.S. patent No. 7,387,887, which is incorporated herein by reference. Other signal-generating substances may be used to detect nucleic acid amplification and melting, illustratively, enzymes, antibodies, etc., as are known in the art.

By "specifically hybridizes" is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (i.e., base pairs) with substantially complementary nucleic acid (e.g., sample nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acid.

By "high stringency conditions" is meant conditions that typically occur at about the melting temperature (Tm) minus 5 ℃ (i.e., 5 ℃ below the Tm of the probe). Functionally, high stringency conditions are used to identify nucleic acid sequences having at least 80% sequence identity.

As used herein, the term "canonical sequence" (the term "consensus sequence" is synonymous and also commonly used in the art) refers to the calculated order of the most common nucleotide residues found at each position in a sequence alignment. Canonical sequences represent the results of multiple sequence alignments in which the related sequences are compared to each other and similar sequence motifs are calculated. The panels referred to herein are typically designed to detect a group of organisms. For each organism in a panel, known variants of that organism typically have some sequence differences in the amplicons amplified from that panel. Thus, for most assays, reference to a pathogen sequence is often inaccurate because each pathogen in the panel represents a population of closely related sequence variants. Thus, the amplicons of a given organism represent all variants in the test population-i.e., the canonical sequence. Although the term "canonical sequence" may often be more accurate, the term "pathogen sequence" is used herein as a synonym. Although many assays use canonical sequences, some assays may use native sequences, particularly where there is little difference between the inclusion lines of a particular target sequence. The term "canonical sequence" is also meant to include such sequences.

Although PCR is the amplification method used in the examples herein, it is understood that any amplification method using primers is suitable. Such suitable procedures include Polymerase Chain Reaction (PCR); strand Displacement Amplification (SDA); nucleic Acid Sequence Based Amplification (NASBA); tandem rolling circle amplification (CRCA), loop-mediated isothermal amplification of dna (lamp); isothermal and chimeric primer-primed nucleic acid amplification (ICAN); target-based helicase-dependent amplification (HDA); transcription-mediated amplification (TMA), and the like. Thus, when the term PCR is used, it is to be understood as including other alternative amplification methods. For amplification methods without discrete cycles, reaction times can be used, where measurements are taken as cycles, doubling times, or crossover points (Cp), and additional reaction times can be added when additional PCR cycles are added in the examples described herein. It should be understood that the scheme may need to be adjusted accordingly.

As used herein, the term "cross-point" (Cp) (or, alternatively, a cycle threshold (Ct), a quantitative cycle (Cq), or synonymous terms used in the art) refers to the number of PCR cycles required to obtain a fluorescence signal above a certain threshold for a given PCR product (e.g., target or internal standard), as determined experimentally. The cycle at which each reaction rises above the threshold depends on the amount of target (i.e., reaction template) present at the beginning of the PCR reaction. The threshold can generally be set at a detectable point where the fluorescence signal of the product is above background fluorescence; however, other thresholds may be employed. As an alternative to setting some arbitrary threshold, Cp may be determined by calculating the reaction point at which the first, second or nth derivative has its maximum, which determines the cycle at which the amplification curve curvature is maximum. One illustrative derivatization method is taught in U.S. Pat. No. 6,303,305, which is incorporated herein by reference in its entirety. Nevertheless, it is generally not important where or how the threshold is set, as long as the same threshold is used for all reactions being compared. Other points known in the art may also be used, and any such point may be substituted for Cp, Ct, or Cq in any of the methods discussed herein.

Although various examples herein refer to human targets and human pathogens, these examples are merely illustrative. The methods, kits, and devices described herein can be used to detect and sequence a wide variety of nucleic acid sequences from a wide variety of samples, including human, veterinary, industrial, and environmental samples. Furthermore, although nucleic acid amplification is discussed herein, the methods, kits, and devices described herein can be used for a wide variety of reactions using a variety of vessels that require in situ sealing.

Various embodiments disclosed herein use separate nucleic acid analysis bags to illustratively determine the presence of various biological substances (illustratively antigens and nucleic acid sequences) in a sample in a single closed system. Such systems, including bags and instruments for use with bags, are disclosed in more detail in U.S. patent nos. 8,394,608, 8,895,295, and 10,464,060, the entire contents of which are incorporated herein by reference. However, it should be understood that such bags are merely illustrative, and that the nucleic acid preparation and amplification reactions discussed herein may be performed in any of a variety of open or closed system sample vessels known in the art, including 96-well plates, plates of other configurations, arrays, carousels, and the like, using a variety of nucleic acid purification and amplification systems as known in the art.

Although the terms "sample well," "amplification container," "reaction chamber," "reaction zone," and the like are used herein, these terms are intended to encompass wells, tubes, and various other reaction vessels used in these amplification systems. In one embodiment, the bag may be an assay device comprising one or more reaction vessels or reaction zones. In one embodiment, the bag may be a flexible container. For example, the pouch/flexible container may include one or more sample wells, amplification containers, reaction chambers, reaction zones, etc., formed between two or more layers of flexible material. In one embodiment, the bag is used for assays against multiple pathogens. The bag may include one or more blisters that serve as sample wells, illustratively in a closed system. Illustratively, various steps may be performed in an optional disposable bag, including nucleic acid preparation, primary bulk multiplex PCR, primary amplification product dilution, and secondary PCR, eventually with optional real-time detection or post-amplification analysis, such as melting curve analysis. Further, it should be understood that while various steps may be performed in the bags of the present invention, for certain uses one or more steps may be omitted and the configuration of the bag may be altered accordingly.

Fig. 1 shows an illustrative bag 510 that may be used in or reconfigured for various embodiments. The pouch 510 is similar to fig. 15 of U.S. patent No. 8,895,295, wherein like items are numbered the same. Fitting 590 is provided with inlet channels 515a to 515l, which inlet channels 515a to 515l also serve as reagent reservoirs or waste reservoirs. Illustratively, the reagents may be lyophilized in fitting 590 and rehydrated prior to use. Blisters 522, 544, 546, 548, 564, and 566, and their respective channels 514, 538, 543, 552, 553, 562, and 565 are similar to the same numbered blisters of fig. 15 of U.S. patent No. 8,895,295. Second stage reaction zone 580 of FIG. 1 is similar to that of U.S. patent application No. 8,895,295, but second stage apertures 582 of high density array 581 are arranged in a slightly different pattern. The more circular pattern of high density array 581 of FIG. 1 eliminates holes in the corners and may result in a more uniform filling of second level holes 582. As shown, high density array 581 is provided with 102 second stage apertures 582. The bags 510 are suitable for use in FilmArray ® instruments (BioFire Diagnostics, LLC, salt lake City, UT). However, it should be understood that the bag embodiments are merely illustrative.

Illustratively, bag 510 may be formed from two layers of flexible plastic film or other flexible material, such as polyester, polyethylene terephthalate (PET), polycarbonate, polypropylene (PP), polymethyl methacrylate, mixtures, compositions, and layers thereof, which may be made by any method known in the art, including extrusion, plasma deposition, and lamination, although other containers may be used. For example, each layer may be composed of one or more layers of a single type or more than one type of material laminated together. One operational example is a two-layer plastic film comprising a PET layer and a PP layer. Metal foils or plastics with aluminium laminates may also be used. If plastic films are used, the layers may be bonded together, illustratively by laser welding and/or heat sealing. Illustratively, the material has low nucleic acid binding capacity. Similar materials (e.g., PET or polycarbonate) may be used for high density array 581.

In some embodiments, a barrier film may be used in one or more layers used to form the flexible bag 510. For example, barrier films may be desirable for certain applications because they have lower water vapor and/or oxygen transmission rates than conventional plastic films. For example, typical barrier films have a thickness of about 0.01 g/m²24 hours to about 3 g/m²In the range of 24 hours, preferably about 0.05 g/m²24 hours to about 2 g/m²24 hours (e.g., no more than about 1 g/m)²/24 hours) and a Water Vapor Transmission Rate (WVTR) in the range of about 0.01cc/m²24 hours to about 2cc/m²In the range of/24 hours, preferably in the range of about 0.05cc/m²24 hours to about 2cc/m²In the range of/24 hours (e.g., no more than about 1 cc/m)²24 hours). Examples of barrier films include, but are not limited to, films that can be metallized by metal (e.g., aluminum or another metal) vapor deposition or sputter coated with an oxide (e.g., Al₂O₃Or SiO_x) Or a film of another chemical composition. A common example of a metallized film is an aluminized polyester film, which is a metal-coated biaxially oriented pet (bopet). In some applications, the coated barrier film may be laminated with a layer of polyethylene, PP, or similar thermoplastic, which provides sealability and improved puncture resistance. As with conventional plastic films, the barrier film layers used to make the bags may be bonded together, illustratively by heat sealing. Illustratively, the material has low nucleic acid binding and low protein binding capacity. Other barrier materials known in the art may be sealed together to form the blister and channel.

For embodiments employing fluorescence monitoring, plastic films having sufficiently low absorbance and autofluorescence at the operating wavelength are preferred. Such materials can be identified by testing different plastics, different plasticizers and compound ratios, and different film thicknesses. For plastics with aluminum or other foil laminates, the portion of the bag to be read by the fluorescence detection device may be left without foil. For example, if fluorescence is monitored in second stage wells 582 of second stage reaction zone 580 of bag 510, one or both layers at wells 582 will be foil-free. In the example of PCR, a film laminate consisting of polyester (mylar, dupont, wilminton DE) about 0.0048 inches (0.1219 mm) thick and polypropylene film 0.001-0.003 inches (0.025-0.076 mm) thick performed well. Illustratively, the pouch 510 may be made of a transparent material capable of transmitting approximately 80% -90% of incident light.

In one embodiment, high-density array 581 and holes 582 are made of card material having a selected thickness such that holes 582 formed in the card material have a selected volume. In one embodiment, the card material may be disposed between two or more flexible film layers that respectively seal one end of the array apertures 582 and form channels or open spaces that allow the apertures 582 to be filled and then at least partially closed for performing reactions in a high density array. It should be understood that although pouch 510 is designed to be flexible, high density reaction zone 580 and high density array 581 optionally may be less flexible and may be rigid and still be part of a flexible sample container. Thus, it should be understood that a "flexible bag" need only be flexible in certain regions.

In the illustrative embodiment, the material is moved between the blisters by applying pressure on the blisters and the channels by a pressure actuator (illustratively a pneumatic pressure actuator). Thus, in embodiments employing pressure, the bag material is illustratively sufficiently flexible to allow the pressure to have the desired effect. The term "flexible" is used herein to describe the physical properties of the pouch material. The term "flexible" is defined herein as being easily deformed by the pressure levels used herein without cracking, breaking, crazing, etc. For example, thin plastic sheets (such as Saran @andZiploc @) and thin metal foils (such as aluminum foil) are flexible. However, even in embodiments employing pneumatic pressure, only certain areas of the blister and channel need be flexible. Furthermore, only one side of the blister and channel need be flexible, as long as the blister and channel can be easily deformed. Other areas of the bag 510 may be made of, or may be reinforced with, a rigid material. Thus, it should be understood that when the terms "flexible bag" or "flexible sample container" or the like are used, only portions of the bag or sample container need be flexible.

Illustratively, a plastic film may be used for the bag 510. A sheet of metal (such as aluminum or other suitable material) may be milled or otherwise cut to produce a mold having a raised surface pattern. When assembled into a pneumatic press (illustratively, Milton WI, Janesville Tool, a-5302-PDS), illustratively adjusted to an operating temperature of 195 ℃, the pneumatic press operates like a printing press, melting the sealing surface of the plastic film only where the mold contacts the film. Also, the plastic film for the bag 510 may be cut and welded together using a laser cutting and welding device. When the pouch 510 is formed, various components such as PCR primers (illustratively spot-coated onto the membrane and dried), antigen binding substrates, magnetic beads, and zirconium silicate beads can be sealed within various blisters. Reagents for sample processing may be applied to the membrane either collectively or individually prior to sealing. In one embodiment, Nucleotide Triphosphates (NTPs) are applied to the membrane separately from the polymerase and primers, thereby substantially eliminating the activity of the polymerase until the reaction can be hydrated by the aqueous sample. This allows for true hot start PCR and reduces or eliminates the need for expensive chemical hot start components if the aqueous sample has been heated prior to hydration. In another embodiment, the ingredients may be provided in powder or pellet form and placed in the blister prior to final sealing.

The pouch 510 may be used in a manner similar to that described in U.S. patent No. 8,895,295. In one illustrative embodiment, 300 μ l of a mixture comprising the sample to be tested (100 μ l) and lysis buffer (200 μ l) can be injected into fitting 590 near an injection port (not shown) of inlet channel 515a, and the sample mixture can be drawn into inlet channel 515 a. Water may also be injected into a second injection port (not shown) of fitting 590 adjacent to inlet channel 515l and dispensed via a channel (not shown) provided in fitting 590, thereby hydrating up to 11 different reagents, each of which was previously provided in dry form at inlet channels 515b through 515 l. Illustrative methods and devices for injecting sample and hydration fluid (e.g., water or buffer) are disclosed in U.S. patent No. 10,464,060, which is incorporated by reference, although it is understood that these methods and devices are illustrative only and that the only and other means of introducing sample and hydration fluid into the bag 510 are within the scope of the present disclosure. These reagents illustratively may include lyophilized PCR reagents, DNA extraction reagents, wash solutions, immunoassay reagents, or other chemical entities. Illustratively, the reagents are used for nucleic acid extraction, first-stage multiplex PCR, dilution of multiplex reactions, and preparation of second-stage PCR reagents and control reactions. In the embodiment shown in fig. 1, all that needs to be injected is the sample solution in one injection port and the water in the other injection port. After injection, both injection ports may be sealed. For more information on various configurations of bag 510 and fitment 590, see U.S. patent No. 8,895,295, which has been incorporated herein by reference.

After injection, the sample may move from injection channel 515a to lysis blister 522 via channel 514. The cracking blisters 522 are provided with beads or particles 534, such as ceramic beads or other abrasive elements, and are configured for swirling via impact using rotating blades or blades provided within a FilmArray @. Bead milling of the sample by shaking, vortexing, sonication, and the like in the presence of lysis particles, such as Zirconium Silicate (ZS) beads 534, is an effective method of forming the lysate. It is to be understood that as used herein, terms such as "lyse", "lysing", and "lysate" are not limited to rupturing cells, but that such terms include the rupturing of non-cellular particles, such as viruses.

Fig. 4 illustrates the bead motor 819 of the instrument 800 shown in fig. 2, the bead motor 819 including a vane 821 that can be mounted on a first side 811 of the support member 802. The blade may extend through the slot 804 to contact the pocket 510. However, it should be understood that the motor 819 may be mounted to other structures of the instrument 800. In one illustrative embodiment, the motor 819 is a Mabuchi RC-280SA-2865 DC motor (Japan kilo-lobe) mounted on the support member 802. In one illustrative embodiment, the motor rotates at a speed of 5000 to 25000 rpm, more illustratively 10000 to 20000 rpm, and still more illustratively approximately 15000 to 18000 rpm. For the Mabuchi motor, it has been found that 7.2V provides sufficient rpm for cleavage. However, it should be appreciated that the actual speed may be slightly slower when the blades 821 strike the pockets 510. Other voltages and speeds may be used for lysis, depending on the motor and paddle used. Optionally, a controlled small volume of air may be provided into the pouch 822 adjacent to the lysis blister 522. It has been found that in some embodiments, partially filling adjacent pockets with one or more small volumes of air helps to position and support the lysis blister during the lysis process. Alternatively, other structures (illustratively rigid or flexible gaskets or other retaining structures surrounding the lysis blister 522) may be used to restrain the pouch 510 during lysis. It should also be understood that motor 819 is merely illustrative and that other means may be used to grind, shake, or vortex a sample. In some embodiments, chemicals or heat may be used in addition to or in place of mechanical lysis.

Once the sample material has been sufficiently lysed, the sample is moved to the nucleic acid extraction zone, illustratively through channel 538, blister 544 and channel 543, to blister 546 where the sample is mixed with nucleic acid binding substances, such as silica-coated magnetic beads 533. Alternatively, magnetic beads 533 may be rehydrated, illustratively hydrated using a fluid provided from one of inlet channels 515c-515e, and then moved through channel 543 to blister 544 and then through channel 538 to blister 522. The mixture is allowed to incubate for an appropriate length of time, illustratively approximately 10 seconds to 10 minutes. A retractable magnet located within the instrument adjacent to blister 546 captures magnetic beads 533 from the solution, forming a bead against the inner surface of blister 546. If incubation is performed in blister 522, portions of the solution may need to be moved to blister 546 for capture. The liquid is then moved out of blister 546 and back through blister 544 and into blister 522, blister 522 now serving as a waste receptacle. One or more wash buffers from one or more of injection channels 515c through 515e are provided to blister 546 via blister 544 and channel 543. Optionally, the magnet is retracted and the magnetic beads 533 are washed by moving the magnetic beads back and forth from the blisters 544 and 546 via channel 543. Once the magnetic beads 533 are washed, the magnetic beads 533 are recaptured in the blister 546 by activation of the magnet, and then the wash solution is moved to the blister 522. This process can be repeated as necessary to wash the lysis buffer and sample debris from the nucleic acid-binding magnetic beads 533.

After washing, the elution buffer stored at injection channel 515f is moved to blister 548 and the magnet is retracted. The solution is circulated between blisters 546 and 548 via channel 552, breaking up the beads of magnetic beads 533 in blister 546 and allowing the captured nucleic acids to dissociate from the beads and into the solution. The magnet is again activated, capturing the magnetic beads 533 in the blister 546, and the eluted nucleic acid solution is moved into the blister 548.

The first stage PCR master mix from injection channel 515g is mixed with the nucleic acid sample in blister 548. Optionally, the mixture is mixed by forcing mixing between 548 and 564 via passage 553. After several mixing cycles, the solution is contained in the blister 564, a bead of first-stage PCR primers, at least one set of primers per target, is provided in the blister 564, and a first-stage multiplex PCR is performed. If RNA targets are present, RT steps can be performed prior to or simultaneously with the first multiplex PCR. The first stage multiple PCR temperature cycles in the FilmArray instrument are illustratively performed for 15-20 cycles, although other levels of amplification may be desired depending on the requirements of a particular application. The first stage PCR mastermix may be any of a variety of mastermixes known in the art. In one illustrative example, the first stage PCR master mix can be any of the chemicals disclosed in U.S. patent No. 9,932,634, which is incorporated herein by reference in its entirety for use with a PCR protocol that takes 20 seconds or less per cycle.

After the first stage PCR has been performed for the desired number of cycles, the sample can be diluted, illustratively by forcing most of the sample back into blister 548, leaving only a small amount in blister 564, and adding the second stage PCR master mix from injection channel 515 i. Alternatively, the dilution buffer from 515i can be moved to the blister 566 and then mixed with the amplified sample in the blister 564 by moving the fluid back and forth between the blisters 564 and 566. If desired, the dilution may be repeated several times using dilution buffers from injection channels 515j and 515k, or injection channel 515k may be retained illustratively for sequencing or other post-PCR analysis, and then the second stage PCR master mix from injection channel 515h is added to some or all of the diluted amplification samples. It will be appreciated that the dilution level may be adjusted by altering the number of dilution steps or by altering the percentage of sample that is discarded prior to mixing with the dilution buffer or second stage PCR master mix, which includes the components for amplification, illustratively polymerase, dNTPs and appropriate buffers, although other components may also be appropriate, particularly for non-PCR amplification methods. If desired, the mixture of sample and second stage PCR master mix may be preheated in blister 564 before moving to second stage well 582 for second stage amplification. This preheating may avoid the need for hot start components (antibodies, chemicals, or others) in the second stage PCR mixture.

The illustrative second stage PCR master mix is incomplete, lacks primer pairs, and each of the 102 second stage wells 582 is preloaded with a particular PCR primer pair. If desired, the second stage PCR mastermix may lack other reaction components, and these components may also be preloaded in the second stage wells 582. Each primer pair may be similar or identical to a primary PCR primer pair, or may be nested within a primary primer pair. Movement of the sample from the blister 564 to the second-stage well 582 completes the PCR reaction mixture. Once high density array 581 is filled, the individual second stage reactions are sealed in their respective second stage blisters by any number of means as are known in the art, as is known in the art. An illustrative method of filling and sealing high density array 581 without cross-contamination is discussed in U.S. patent No. 8,895,295, which has been incorporated herein by reference. Illustratively, the various reactions in wells 582 of high density array 581 are illustratively thermally cycled simultaneously or individually using one or more peltier devices, although other means for thermal cycling are known in the art.

In certain embodiments, the second stage PCR master mix contains the dsDNA binding dye LCGreen Plus (BioFire Diagnostics, LLC) to generate a signal indicative of amplification. However, it is understood that this dye is merely illustrative and that other signals may be used, including other dsDNA binding dyes and probes labeled by fluorescent, radioactive, chemiluminescent, enzymatic, etc., means known in the art. Alternatively, holes 582 of array 581 may be provided without a signal, with the results reported by subsequent processing.

When pressure applied to the pocket blister is used to move the material within the pocket 510, in one embodiment, a pneumatic "bladder" may be employed. In other embodiments, various mechanically driven piezo actuators may be used. The bladder assembly 810, a portion of which is shown in FIGS. 2-3, includes a bladder plate 824 that houses a plurality of inflatable bladders 822, 844, 846, 848, 864, and 866, each of which may be individually inflatable, illustratively by a source of pressurized gas. Because the bladder assembly 810 may be subjected to compressed gas and used multiple times, the bladder assembly 810 may be made of a tougher or thicker material than the bag. Alternatively, the bladders 822, 844, 846, 848, 864, and 866 may be formed from a series of plates secured together with gaskets, seals, valves, and pistons. Other arrangements are also within the scope of the invention. Alternatively, an array or mechanical actuator and seal may be used to seal the channels and direct the movement of fluid between the blisters. Mechanical seals and actuator systems that may be suitable for the instruments described herein are described in detail in WO 2018/022971, the entire contents of which are incorporated herein by reference.

The success of the secondary PCR depends on the template generated by the multiplex first-stage reaction. Generally, PCR is performed using high purity DNA. Methods such as phenol extraction or commercial DNA extraction kits provide high purity DNA. Samples processed through bag 510 may require adjustment to compensate for less pure formulations. The components of biological samples may inhibit PCR, which is a potential obstacle. Illustratively, hot start PCR, higher concentrations of Taq polymerase, adjusting magnesium chloride concentration, adjusting primer concentration, and adding adjuvants (such as DMS, TMSO, or glycerol) optionally can be used to compensate for lower nucleic acid purity. While purity issues are likely to be of greater concern for first stage amplification, it is understood that similar adjustments may be provided in second stage amplification as well.

When the bag 510 is placed within the instrument 800, the bladder assembly 810 is pressed against one face of the bag 510 such that if a particular bladder is inflated, the pressure will force liquid out of the corresponding blister in the bag 510. In one or more embodiments, one or more expandable bladders may be expanded in the instrument to enhance contact between the blister and one or more components of the instrument. For example, the pneumatic bladder 822 may be at least partially expanded to enhance contact between the blister 522 on one side and the lysis device on the other side. In another case, the pneumatic bladders 848 and 864 may at least partially expand over the blisters 548 and 564 to enhance contact between the blisters 548 and 564 and the heater assemblies for the first stage PCR. In addition to the pockets corresponding to the many blisters of bag 510, pocket assembly 810 may have additional pneumatic actuators, such as pockets or pneumatic pistons corresponding to the various channels of bag 510. Fig. 2-3 show illustrative piston or hard seals 838, 843, 852, 853, and 865 corresponding to channels 538, 543, 553, and 565 of the bag 510, and seals 871, 872, 873, 874 that minimize backflow into the fitting 590. When activated, the hard seals 838, 843, 852, 853, and 865 form pinch valves to pinch off and close the corresponding passages. To confine liquid within a particular blister of bag 510, the hard seal is activated on the passage to and from the blister so that the actuator functions as a pinch valve to pinch the passage closed. Illustratively, to mix two volumes of liquid in different blisters, a pinch valve actuator that seals the connecting channel is activated and alternately pressurizes a pneumatic bladder on the blister, thereby forcing the liquid back and forth through the channel connecting the blisters to mix the liquids therein. Pinch valve actuators can have various shapes and sizes, and can be configured to pinch off more than one channel at a time.

While pneumatic actuators are discussed herein, it should be understood that other types of pressure transducers for providing pressure to the bag are contemplated, including various electromechanical actuators, such as linear stepper motors, motor driven cams, rigid paddles driven by pneumatic, hydraulic, or electromagnetic forces, rollers, rocker arms, and in some cases cocking springs. In addition, there are various methods of closing the channel, either reversibly or irreversibly, in addition to applying pressure perpendicular to the channel axis. These include kinking the bag across the channel, heat sealing, rolling actuators, and various physical valves such as butterfly and ball valves that seal into the channel. Additionally, a small peltier device or other temperature regulator may be placed adjacent the channel and set at a temperature sufficient to freeze the fluid, effectively forming a seal. Furthermore, while the bag design of fig. 1 is suitable for automated instruments, characterized by actuator elements positioned on each blister and channel, it is also contemplated that the actuators may remain stationary and that the bag 510 may be switched so that a small number of actuators may be used in several processing stations, including sample disruption, nucleic acid capture, first and second stage PCR, and processing stations for other applications of the bag 510, such as immunoassays and immuno-PCR. Rollers acting on the channels and blisters may prove particularly useful in configurations where the bag 510 translates between stations. Thus, while a pneumatic actuator is used in the presently disclosed embodiments, when the term "pneumatic actuator" is used herein, it should be understood that other pressure transducers, actuators, and other ways of providing pressure may be used depending on the configuration of the bag and the instrument.

In addition to the pneumatic bladder and seal described previously, fig. 3 illustrates another configuration of pressure transducers 880, which pressure transducers 880 may be sized and positioned to apply pressure to the high density reaction zones 580 and the high density reaction apertures 582. The pressure transducer 880 may be sized and positioned to apply pressure generally to the high-density reaction zone 580, or the pressure transducer 880 may be or include a substructure 882 sized and positioned to apply pressure only to the high-density reaction apertures 582. In one embodiment, actuation of the pressure transducer 880 has the effect of lightly pressing the high-density reaction zone 580 and high-density reaction wells 582 against the second stage PCR heater (888 in fig. 2) to promote heat transfer from the heater 888 to the fluid in the reaction wells 582. In another embodiment, actuation of the pressure transducer 880 over the high density reaction zone 580 or the high density reaction wells 582 may compress the flexible layers 599 and 597 above and below the high density reaction wells 582 to seal closed the wells and clear excess fluid from the high density reaction zone 580.

The pressure transducer 880 may be mechanically or pneumatically actuated as described in detail herein above. Where fluorescence excitation of the high-density reaction wells 582 and detection of fluorescence from the high-density reaction wells 582 is desired, the pressure transducer 880 may comprise a transparent plastic balloon or the like that may be inflated over the high-density reaction wells 582 after the high-density reaction wells 582 are filled with a reaction mixture. In this case, the pressure transducer 880 may include a "window balloon" that expands over the high-density reaction well 582 while allowing excitation light from the light source 898 (fig. 2) to pass through to excite fluorescence and allow viewing through the camera 896 (fig. 2). Thus, in embodiments using fluorescence or other optical detection, it is preferred that the pressure transducer 880 be made of an optically transparent and minimally fluorescent material. Several such materials are known in the art.

Similarly, in addition to the foregoing, in one embodiment, the pressure transducer 880 may also efficiently and effectively purge excess fluid from the high-density reaction wells 582. For example, purging excess fluid from the second stage array may reduce PCR cycle time (i.e., smaller volumes of liquid may be cycled faster). In addition, removing excess fluid helps to inhibit intermixing (often referred to herein as "cross-talk") between adjacent wells of the second stage PCR array. As discussed in U.S. patent No. 8,895,295 (which patent is already incorporated herein by reference), the second level array may be provided with a perforated cover layer that allows filling of the second level holes and helps to suppress cross talk. After the reaction is complete, the pressure on the high density reaction zone 580 can be reduced to allow removal from the apparatus 800. In embodiments where further analysis is not required, it is no longer necessary to prevent cross-talk between the apertures 582. Where further analysis is desired, a more permanent sealing mechanism, illustratively any of the sealing layers described in connection with fig. 5-10, may be used.

Returning to fig. 2, each pneumatic actuator is connected to a source of compressed air 895 via a valve 899. Although only a few hoses 878 are shown in FIG. 2, it should be understood that each pneumatic fitting is connected to a source 895 of pressurized gas via hose 878. The compressed gas source 895 may be a compressor, or alternatively, the compressed gas source 895 may be a compressed gas bottle, such as a carbon dioxide bottle. Compressed gas bottles are particularly useful if portability is desired. Other sources of compressed gas are also within the scope of the present invention. Similar pneumatic control may be provided in the embodiment of fig. 12-16 for controlling the fluid in bag 1400, or other actuators, servo systems, etc. may be provided.

Several other components of the instrument 810 are also connected to a source 895 of pressurized gas. The magnet 850 mounted on the second side 814 of the support member 802 is illustratively deployed and retracted via a hose 878 using gas from a compressed gas source 895, although other methods of moving the magnet 850 are known in the art. The magnet 850 is seated in the recess 851 of the support member 802. It should be understood that the recess 851 may be a channel through the support member 802 such that the magnet 850 may contact the blister 546 of the pocket 510. However, depending on the material of the support member 802, it will be appreciated that the recess 851 need not extend all the way through the support member 802, so long as the magnet 850 is close enough to provide a sufficient magnetic field at the blister 546 when the magnet 850 is deployed, and the magnet 850 does not significantly affect any magnetic beads 533 present in the blister 546 when the magnet 850 is fully retracted. Although reference is made to a retracted magnet 850, it should be understood that an electromagnet may be used and may be activated and deactivated by controlling the current through the electromagnet. Thus, while this specification discusses withdrawing or retracting a magnet, it should be understood that these terms are broad enough to encompass other ways of withdrawing a magnetic field. It will be appreciated that the pneumatic connection may be a pneumatic hose or a pneumatic air manifold, thus reducing the number of hoses or valves required.

The various pneumatic pistons 868 of the pneumatic piston array 869 are also connected to a source 895 of pressurized gas via hoses 878. Although only two hoses 878 are shown connecting the pneumatic pistons 868 to the source of pressurized gas 895, it should be understood that each pneumatic piston 868 is connected to the source of pressurized gas 895. 12 pneumatic pistons 868 are shown.

A pair of temperature control elements are mounted on a second side 814 of support 802. As used herein, the term "temperature control element" refers to a device that adds or removes heat to or from a sample. Illustrative examples of temperature control elements include, but are not limited to, heaters, coolers, peltier devices, resistive heaters, inductive heaters, electromagnetic heaters, thin film heaters, printing element heaters, positive temperature coefficient heaters, and combinations thereof. The temperature control element may include a plurality of heaters, coolers, peltier devices, and the like. In one aspect, a given temperature control element may include more than one type of heater or cooler. For example, an illustrative example of a temperature control element may include a peltier device having a separate resistive heater applied to a top and/or bottom surface of the peltier device. Although the term "heater" is used throughout the specification, it should be understood that other temperature control elements may be used to regulate the temperature of the sample.

As discussed above, first stage heater 886 can be positioned to heat and cool blister 564 or the contents of blisters 548 and 564 for use in first stage PCR. As shown in fig. 2, second stage heater 888 may be positioned to heat and cool the contents of second stage bubble caps 582 of array 581 of bags 510 for second stage PCR. However, it should be understood that these heaters may also be used for other heating purposes, and may optionally include other heaters suitable for particular applications.

As discussed above, while peltier devices that thermally cycle between two or more temperatures are effective for PCR, in some embodiments it may be desirable to maintain the heater at a constant temperature. Illustratively, this can be used to reduce run time by eliminating the time required to transition the heater temperature over the time required to transition the sample temperature. Furthermore, this arrangement can improve the electrical efficiency of the system because only a sample and sample vessel with a small thermal cycle are required, rather than requiring a much larger (greater thermal mass) peltier device. For example, the instrument may include multiple heaters (i.e., two or more) positioned relative to the bag to complete a thermal cycle, the heaters being at temperatures set for annealing, elongation, denaturation, for example. For many applications, two heaters may be sufficient. In various embodiments, the heater may be moved, the bag may be moved, or the fluid may be moved relative to the heater to complete the thermal cycle. Illustratively, the heaters may be arranged linearly, in a circular arrangement, or the like. Suitable heater types have been discussed above with reference to first stage PCR.

When fluorescence detection is desired, an optical array 890 may be provided. As shown in fig. 2, optical array 890 includes a light source 898 and a camera 896, light source 898 illustratively being a filtered LED light source, filtered white light, or laser illumination. The camera 896 illustratively has a plurality of photodetectors, each photodetector corresponding to a second-stage aperture 582 in the pouch 510. Alternatively, the camera 896 may capture an image containing all of the second stage apertures 582, and the image may be divided into separate fields corresponding to each second stage aperture 582. Depending on the configuration, the optical array 890 may be stationary, or the optical array 890 may be placed on a mover attached to one or more motors and moved to obtain a signal from each individual second-stage aperture 582. It should be understood that other arrangements are possible. The embodiment of the second stage heater shown in fig. 18 provides a heater on the opposite side of the bag 510 from that shown in fig. 2. This orientation is merely illustrative and may be determined by spatial constraints within the instrument. The photodetectors and heaters may be on either side of array 581, provided that second stage reaction zone 580 is disposed in an optically transparent material.

As shown, the computer 894 controls the valve 899 of the compressed air source 895 and, thus, all of the pneumatics of the instrument 800. Additionally, in other embodiments, many of the pneumatic systems in the instrument may be replaced with mechanical actuators, pressure applying devices, and the like. Computer 894 also controls heaters 886 and 888 and optical array 890. Each of these components is electrically connected via a cable 891, although other physical or wireless connections are within the scope of the invention. It should be understood that the computer 894 may be housed within the instrument 800 or may be external to the instrument 800. In addition, computer 894 may include a built-in circuit board that controls some or all of the components, and may also include an external computer, such as a desktop or laptop PC, to receive and display data from the optical array. An interface may be provided, such as a keyboard interface, which includes keys for entering information and variables such as temperature, cycle time, etc. Illustratively, a display 892 is also provided. For example, the display 892 may be an LED, LCD, or other such display.

Other prior art instruments teach PCR in sealed flexible containers. See, for example, U.S. patent nos. 6,645,758, 6,780,617, and 9,586,208, which are incorporated herein by reference. However, the inclusion of cell lysis within a sealed PCR vessel may improve ease and safety of use, particularly when the sample to be tested may contain biological hazards. In the embodiments shown herein, waste from cell lysis and waste from all other steps remain within the sealed bag. Nevertheless, it should be understood that the bag contents may be removed for further testing.

As discussed above, fig. 2 shows an illustrative instrument 800 that may be used with bag 510. The instrument 800 includes a support member 802, and the support member 802 may form a wall of the housing or be mounted within the housing. The instrument 800 may further include a second support member (not shown) that is optionally movable relative to the support member 802 to allow insertion and withdrawal of the bag 510. Illustratively, once the bag 510 has been inserted into the instrument 800, a cover may cover the bag 510. In another embodiment, the two support members may be fixed, with the bag 510 held in place by other mechanical means or pneumatic pressure.

In the illustrative example, heaters 886 and 888 are mounted on support members 802. However, it should be understood that this arrangement is merely illustrative and that other arrangements are possible. Illustrative heaters include peltier and other block heaters, resistive heaters, electromagnetic heaters, and thin film heaters as are known in the art to thermally cycle the contents of the bubble cap 864 and second stage reaction zone 580. A bladder plate 810 having bladders 822, 844, 846, 848, 864, 866, hard seals 838, 843, 852, 853, and seals 871, 872, 873, 874 forms a bladder assembly 808, the bladder assembly 808 illustratively being mounted on a movable support structure that is movable toward the bag 510 such that the pneumatic actuator is placed in contact with the bag 510. When the bag 510 is inserted into the instrument 800 and the movable support member is moved toward the support member 802, the various blisters of the bag 510 are in position adjacent to the various pockets of the pocket assembly 810 and the various seals of the assembly 808, such that activation of the pneumatic actuator can force liquid out of one or more blisters of the bag 510 or can form a pinch valve with one or more channels of the bag 510. The relationship between the blisters and channels of bag 510 and the pockets and seals of assembly 808 is shown in more detail in fig. 3.

While the pressure transducer 880 (e.g., a window pouch) discussed above with respect to fig. 3 is one example of a device that may be capable of at least partially sealing the fluid in the reaction of the aperture 582 or the high density reaction zone 580 during the reaction, it may be desirable in some cases to form a permanent or semi-permanent seal that is capable of maintaining the integrity of the fluid contents of the reaction aperture after the reaction is complete — e.g., hours, days, or weeks after the reaction vessel is removed from the instrument. Note that forming a durable, more durable seal after use of the reaction vessel may also have the effect of better sealing the fluid contents in the reaction well during the reaction. The present invention provides reaction vessels, methods and systems for sealing in situ respective reaction wells in a closed reaction vessel to form a seal using conditions already present in normal reactions. For example, the heat and pressure present in some thermal cycling reactions may be used to deform a sealing material to form a seal in situ, thereby sealing one or more reaction wells in a reaction vessel and forming a seal that effectively seals the wells during the reaction and remains after the thermal cycling is completed and the reaction vessel is removed from the instrument. Furthermore, the illustrative sealable reaction vessels, methods, and systems do not risk premature adhesion and sealing prior to reaction. Also, because the conditions required to form the seal are already present in normal reaction conditions, the containers, methods, and systems described herein do not require any additional steps or processes to form the seal. Reaction wells sealed according to the methods and systems described herein can be stored and readied on the same or different instruments. Such reaction wells may be used to measure well-to-well variability or instrument-to-instrument variability. In addition, reaction wells sealed according to the methods and systems described herein can be used to make standards (e.g., fluorescence standards), which can be used to calibrate an instrument. Because the sealing material is contained within the reaction vessel and there is little risk of premature formation of a seal, the use of a sealable reaction vessel and the methods and systems described herein do not require any special handling or sample preparation on the part of the user.

Turning now to fig. 5A and 5B, illustrated are cross-sectional views of embodiments of a reaction vessel 5000 for performing multiple reactions on a fluid sample in a closed system. Although the reaction vessel 5000 shows several parallel reaction wells 5035, this is merely illustrative. The in situ sealing systems described herein may be used to seal in situ any portion of a reaction vessel, such as, but not limited to, a reaction well or multiple parallel reaction wells, reaction chambers (e.g., reaction blisters), fluid flow channels, and the like. As shown in fig. 5A, the reaction vessel 5000 is shown in an initial undeformed/unsealed state 5000 a. Fig. 5B illustrates the reaction vessel 5000 in a deformed/sealed state 5000B.

The reaction vessel 5000 includes a first outer layer 5010, a second outer layer 5020, an array layer 5030, and a plurality of reaction holes 5035 formed as a series of voids or holes formed in the array layer 5030. In embodiments employing pressure, the material used to form one or more layers of the reaction vessel 5000 is illustratively sufficiently flexible to allow the pressure to have a desired effect. However, even in embodiments employing pneumatic pressure, only certain regions of the reaction vessel 5000 need be flexible. In addition, only one side of the reaction vessel 5000 needs to be flexible as long as the selected portion (e.g., on at least one side of the array layer 5030) can be easily deformed. Other areas of the reaction vessel 5000 may be made of or reinforced with rigid materials. Thus, it will be understood that when the terms "flexible bag" or "flexible reaction vessel" or the like are used, only portions of the bag or reaction vessel need be flexible. The materials used to fabricate the first outer layer 5010, the second outer layer 5020 and the array layer 5030 are discussed in detail above with reference to the pouch 510 and the array 581. Non-limiting examples of materials that may be used include, but are not limited to, polyester, polyethylene terephthalate (PET), polycarbonate, polypropylene (PP), or polymethyl methacrylate. In the illustrated embodiment, a flexible outer layer 5020 is bonded to one end 5053 of the array layer 5030 to seal one end of the aperture 5035. The second outer layer 5020 can be bonded directly to the array layer 5030 (e.g., by thermal or ultrasonic welding) or the layer 5020 can comprise an adhesive layer (e.g., a pressure sensitive adhesive or a heat activated adhesive (not shown)) that can bond the layer 5020 to the array layer 5030.

In the illustrated embodiment, reaction vessel 5000 includes a sealing layer 5040, where 5040a refers to layer 5040 prior to deformation and sealing, and 5040b refers to layer 5040 after deformation and sealing. The sealing layer 5040 is coupled to the inner surface 5047 of the first outer layer 5010 such that the sealing layer 5040 is positioned adjacent to the open ends of the array apertures 5035. In the initial, undeformed/unsealed state 5000a of the reaction vessel 5000, the first flexible outer layer 5010 and the sealing layer 5040a are spaced apart from the array layer 5035 and fluid can flow into (or out of) the open ends 5055 of the plurality of apertures 5035. Once the fluid sample has filled the apertures 5035, pressure can be applied to the outer surfaces 5049 of the layer 5010 to press the layers 5010 and 5040 into contact with the second ends 5051 of the array layer 5030 to form a temporary seal over the open ends 5055 of the plurality of apertures (not shown).

Fig. 5B indicates what may occur under reaction conditions (e.g., during a thermal cycling reaction) (e.g., when one or both of heat and pressure may be applied). In the illustrated embodiment, the reaction conditions result in the formation of a seal to seal the open end 5055 of the aperture 5035. By pressing the layers 5010 and 5040 against the array layer 5030, for example, heat can be applied to the reaction vessel 5000 adjacent to the layer 5020 to facilitate reactions (e.g., nucleic acid amplification reactions) in the plurality of wells 5035 while pressure is applied at the surface 5049 adjacent to the layer 5010. In other embodiments, heat and pressure may be applied to the same side of the reaction vessel 5000. Illustratively, the heat and pressure provided to facilitate the reaction can cause the sealing layer 5040 to deform (as illustratively represented by 5040 b) to form an in situ seal without the need for additional heat and pressure. The deformed sealing layer 5040b may be deformed about the second end 5051 of the array layer 5030 (example deformations are illustratively shown at 5042 and 5044) and pressed into the bore opening 5055 to create a sealing plug (e.g., shown at 5044) that enters the open end 5055 of the bore 5035 such that the fluid contents of the bore cannot flow out and mix during or after the reaction. When the reaction is complete and heat and pressure are removed, the seal (e.g., a permanent or semi-permanent seal) sealing the open end 5055 of each aperture 5035 remains along the array layer 5030, second end 5051 at the interface between second end 5051 at 5042/5044 and sealing layer 5040.

In one embodiment, the sealing layer 5040 may be applied directly to the inner surface 5047 of the outer layer 5010, or the sealing material 5040 may be included as a layer or part of a separate film layer that is bonded to the inner surface 5047 of the outer layer 5010 adjacent to the second end of the array layer 5030. For example, a sealing layer 5040 (which illustratively may include an adhesive, a swelling material that swells in an aqueous environment, a wax, etc.) may be applied directly to the inner surface 5047 of the outer layer 5010 as a continuous layer, a spray coating, or the like. In another embodiment, the sealing material 5040 may be coated onto or may be part of another film layer that may be bonded to the inner surface 5047 of the outer layer 5010 adjacent to the second end 5051 of the array layer 5030. The film layer may include a backing layer (e.g., a PET layer) and a sealing material applied to the backing. In one embodiment, such a film layer can be bonded (e.g., by thermal welding, laser welding, etc.) directly to the upper flexible layer 5010. In another embodiment, such a film layer may include a second adhesive layer (e.g., a pressure sensitive adhesive) that is also applied to the backing layer that adheres the film layer to the upper flexible layer.

Examples of suitable heat-activated and pressure-activated adhesives include, but are not limited to, Ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA), Ethylene Methyl Acetate (EMA), ethylene n-butyl acrylate (EnBA), Ethylene Acrylic Acid (EAA), Thermoplastic Polyurethane (TPU), polycaprolactone, silicone rubber, thermoplastic elastomers, waxes (e.g., microcrystalline wax), polyethylene, polypropylene, low density polyethylene, copolymers thereof, and combinations thereof. Suitable heat and pressure activated adhesives, waxes, and the like can soften or partially or completely melt under thermal cycling conditions to deform into the reaction holes 5035 of the sealed array layer 5030 and substantially seal the reaction holes 5035 of the array layer 5030. The melting temperature of the adhesive should be below the highest temperature of the reaction and above ambient temperature. In one embodiment, an adhesive having a melting point in the range of about 60 ℃ to about 100 ℃ (e.g., about 65-95 ℃, about 70-90 ℃, about 75-85 ℃, or about 80-85 ℃) is used. However, it will be understood that there is an interaction between pressure and heat, and that the temperature ranges listed are merely illustrative. For example, if the pressure is relatively increased, less heat may be required to deform the adhesive to form the seal, or, on the other hand, if the pressure is relatively decreased, more heat may be required to form the seal. When heat and pressure are removed from the reaction vessel 5000, the adhesive will re-cure to form a seal that seals the respective aperture 5035.

Heat and pressure are not the only in situ reaction parameters or processes that can be used for pore sealing. Other in situ processes that can create a permanent seal include, but are not limited to: a liquid-sensitive adhesive layer sealing the hole when the reaction liquid is supplied to the hole; the pores may be provided with an adhesive catalyst, solvent or reagent that reacts with the adhesive layer upon pore filling; a moisture absorbent material may be provided around the microporous openings, which may expand and block the openings in the presence of water; or a hygroscopic material may be provided within the pores and may be used to absorb the sample (e.g., like a sponge) as it enters, thereby preventing sample components from leaving.

Referring now to fig. 6A and 6B, cross-sectional views of another embodiment of a high density reaction zone 6000 configured for in situ sealing are shown. The embodiment of fig. 6A and 6B is similar to that shown in fig. 5A and 5B except that an in-situ seal material 6040 is disposed on an end 6051 of the high density array layer 6030 adjacent to an open end 6055 of a bore 6035. As in the previous examples, 6040 generally refers to the sealing material, 6040a refers to the sealing material in an initial, undeformed/unsealed state, and 6040b refers to the sealing material in a deformed/sealed state. As shown in fig. 6A, the reaction vessel 6000 is shown in an initial undeformed/unsealed state 6000 a. Fig. 6B illustrates the reaction vessel 6000 in a deformed/sealed state 6000B.

The reaction vessel 6000 includes a first outer layer 6010, a second outer layer 6020, an array layer 6030, and a plurality of reaction wells 6035 formed as a series of voids or holes formed in the array layer 6030. The materials used to make the first outer layer 6010, the second outer layer 6020, and the array layer 6030 are discussed in detail elsewhere herein. In the illustrated embodiment, the second outer layer 6020 is bonded to the first end 6053 of the array layer 6030 to seal the first end of the bore 6035. The second outer layer 6020 may be bonded directly to the second end 6053 of the array layer 6030 (e.g., by thermal or ultrasonic welding) or the layer 6020 may include an adhesive layer (e.g., a pressure sensitive adhesive or a heat activated adhesive (not shown)) that may bond the layer 6020 to the array layer 6030.

In the illustrated embodiment, the reaction vessel 6000 includes a sealing material 6040 disposed on a second end 6051 of the array layer 6030 opposite the first end 6053. In the initial undeformed/unsealed state 6000a of the reaction vessel 6000, the sealing material 6040 is in the unsealed state 6040a, and the first flexible outer layer 6010 is separated from the sealing material 6040 so that fluid may flow into (or out of) the open ends 6055 of the plurality of apertures 6035. Once the fluid sample has filled hole 6035, pressure may be applied to the exterior of layer 6010 at surface 6049 to press layer 6010 into contact with sealing material 6040, thereby creating a temporary seal between interior surface 6047 of layer 6010 and sealing material 6040, which sealing material 6040 covers open end 6055 of hole 6035.

With the layer 6010 pressed onto the sealing material 6040, for example, heat may be applied to the reaction vessel 6000 adjacent to the layer 6020 to facilitate reactions (e.g., nucleic acid amplification reactions) in the plurality of wells 6035. As shown in fig. 6B, the heat and pressure provided to facilitate the reaction can cause the sealing material 6040 to change from its initial state 6040a to a deformed/sealed state 6040B to form an in-situ seal such that the fluid contents of the bore 6035 cannot flow out of the open end 6055 of the bore 6035 and mix during or after the reaction. In one illustrative example, the sealing material 6040 may be a thermoset polymer or a thermoplastic polymer. When the reaction is complete and the heat and pressure are removed, the seal (e.g., permanent or semi-permanent seal) sealing each hole 6035 remains along the interface between the layer 6010 and the deformed sealing material 6040 b.

In one embodiment, the sealing material 6040 may be applied directly to the second end 6051 of the array layer 6030. For example, sealing material 6040, which may be an adhesive, a swelling agent that swells in an aqueous environment, a wax, or the like, is applied directly to second end 6051 of array layer 6030 such that it is disposed adjacent to inner surface 6047 of outer layer 6010. For example, as discussed in detail above, the array layer may be made of a relatively thick card material having apertures formed therein to form an array of sample apertures. For example, the array layer material has a thickness of about 0.3 to about 1 millimeter (e.g., about 0.4 millimeters) as compared to the thickness of the outer layer of about 0.02 to about 0.1 millimeters. In an example embodiment, the sealing material (e.g., a heat sensitive adhesive) may be applied to the card layer as a continuous coating in the form of droplets, grid lines, or the like. Holes may then be formed in the card layer, leaving behind the array layer, with the holes being bounded by the encapsulant material. In another embodiment, the sealing material may be applied after the array layer and the holes are formed.

In yet another embodiment, the sealing material 6040 may comprise a membrane material that may be bonded to the array layer 6030. The film material may include a backing layer (e.g., a PET layer) and a sealing material applied to the backing layer as disclosed herein. In one embodiment, such a film material may be bonded (e.g., by thermal welding, laser welding, etc.) directly to the second end 6051 of the array layer 6030. In another embodiment, such a film layer may include a second adhesive layer (e.g., a pressure sensitive adhesive) that may adhere the film layer to the second end 6051 of the array layer 6030. Holes may be formed in array layer 6030 before or after applying the membrane material to array layer 6030. If the membrane material is applied to the array prior to forming the holes in the array, the holes may be formed through the array card, the membrane and the in situ seal adhesive. If the sealing material is applied to the array as a film carrying an adhesive layer after formation of the array holes, corresponding holes may be formed in the film/adhesive before attaching the film to the array.

Examples of suitable heat-activated and pressure-activated adhesives (e.g., Ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA)) are discussed above with reference to fig. 5A and 5B. Suitable heat and pressure activated adhesives, waxes, and the like may at least partially melt under reaction conditions (e.g., thermal cycling conditions) to substantially seal the reaction wells 6035 of the array layer 6030. In one embodiment, the heat and pressure activated adhesive has a melting point in the range of about 60 ℃ to about 100 ℃. When the heat and pressure are removed from the reaction vessel 6000, the heat and pressure activated adhesive will re-cure to form a seal that seals the respective holes 6035 along the interface between the interior surface 6047 of the layer 6010 and the sealing material 6040.

Referring now to fig. 7A and 7B, there is shown a cross-sectional view of yet another embodiment of a reaction vessel 7000 configured for in situ sealing. The embodiment of fig. 7A and 7B is similar to the high density reaction zone of the reaction vessel shown in the previous examples. As in the previous examples, 7040 generally refers to the sealing material, 7040a refers to the sealing material in an initial, undeformed/unsealed state, and 7040b refers to the sealing material in a deformed/sealed state. As shown in fig. 7A, reaction vessel 7000 is shown in an initial undeformed/unsealed state 7000 a. Fig. 7B illustrates the reaction vessel 7000 in the deformed/sealed state 7000B.

The reaction vessel 7000 includes a first outer layer 7010, a second outer layer 7020, an array layer 7030, and a plurality of reaction wells 7035 formed as a series of voids or holes formed in the array layer 7030. Materials used to fabricate the first outer layer 7010, the second outer layer 7020, and the array layer 7030 are discussed in detail herein. In the illustrated embodiment, the second outer layer 7020 is bonded to the first end 7053 of the array layer 7030 to seal the first end of the aperture 7035. The second outer layer 7020 can be bonded directly to the first end 7053 of the array layer 7030 (e.g., by thermal or ultrasonic welding) or the layer 7020 can include an adhesive layer (e.g., a pressure sensitive adhesive or a heat activated adhesive) (not shown) that can bond the layer 7020 to the array layer 7030. In the illustrated embodiment, the reaction vessel 7000 includes a sealing layer 7040 coupled to the inner surface 7047 of the first outer layer 7010. The sealing layer 7040 is similar to sealing layer 5040 shown in fig. 5A and 5B.

Fig. 7A and 7B show an illustrative embodiment of a reaction vessel 7000 that includes a physical barrier over the opening of array well 7035. Sandwiched between the first outer layer 7010, the sealing layer 7040, and the second outer layer 7020 of the reaction vessel 7000 is an array layer 7030 having apertures 7035. Disposed on the second end 7051 of the array layer 7030 is a perforated layer 7050, the perforated layer 7050 being provided to act as a physical barrier, wherein the perforations 7055 allow a fluid sample to flow into the apertures 7035 (e.g., a partial vacuum in the apertures 7035) in the presence of a force, but impede backflow from the apertures in the absence of a force. Illustratively, the perforated layer 7050 is a plastic film layer (illustratively, by heat sealing) that has been sealed to the second end 7051 of the array layer 7030, although it is understood that other securing methods may be employed. It should also be understood that the materials used for the array layer 7030 and the materials used for the perforated layer 7050 and the second outer layer 7020 should be compatible with each other, with the sealing method, and with the chemicals used.

In the initial undeformed/unsealed state 7000a (fig. 7A), the first outer layer 7010 and the sealing layer 7040 are separated from the perforated layer 7050 and the array layer 7035, and as a result, fluid can flow into (or out of) the plurality of apertures 7035 via the openings 7055. An illustrative way of filling high density arrays (e.g., array wells 7035) in a closed system without cross-contamination is discussed in U.S. patent No. 8,895,295, which has been incorporated herein by reference. In the illustrative embodiment shown in fig. 7A and 7B, a perforated layer 7050 is provided, similar to the perforated layer 7050 of U.S. patent No. 8,895,295. The perforated layer 7050 allows fluid to flow into each of the apertures 7035 in the presence of a force, but the perforations are small enough to substantially prevent fluid from flowing into or out of the apertures in the absence of a force. For example, a predetermined amount of vacuum in the pores 7035 can be sufficient to draw fluid through the openings 7055 of the perforated layer 7050 and into the pores; once a predetermined vacuum is "consumed" in filling the aperture, fluid will generally not readily flow into or out of the aperture 7035 through the opening 7055. After filling the array apertures 7035, the apertures 7035 in the array layer 7030 can be temporarily sealed by applying pressure to the first outer layer 7010 adjacent to the surface 7049, as discussed, for example, in U.S. patent No. 8,895,295, to press the first outer layer 7010 and the sealing layer 7040 against the upper surface 7052 of the perforated layer 7050.

Where the layer 7040 is pressed against the upper surface 7052 of the perforated layer 7050 to form a temporary seal by applying pressure adjacent to the layer 7010, heat can be applied to the reaction vessel 7000 (e.g., adjacent to the layer 7020) to facilitate a reaction (e.g., a nucleic acid amplification reaction) in the plurality of apertures 7035. As shown in fig. 7B, the heat and pressure provided to facilitate the reaction can cause the sealing layer 7040 in an initial, undeformed/unsealed state 7040a to deform, as shown at 7040B, to create a seal such that the fluid contents of the aperture 7035 cannot flow out through the opening 7055 and mix during or after the reaction. In the illustrated embodiment, the sealing layer 7040 can be deformed in the sealed state 7040b to at least partially fill the perforated layer hole 7055 to form a sealing plug 7044. The sealing layer 7040 may be further sealed as shown, for example, at 7042 at the interface between the upper surface 7052 of the perforated layer 7050 and the sealing layer 7040 b. When the reaction is complete and heat and pressure are removed, the seal (e.g., permanent or semi-permanent seal) sealing the respective apertures 7035 is left along the interface between the perforated layer 7050, the opening 7055, and the deformed sealing material 7040 b.

As described in detail with reference to fig. 5A and 5B, the sealing layer 7040 can be applied directly to the inner surface 7047 of the outer layer 7010, or the sealing material 7040 can comprise a separate film layer bonded to the inner surface 7047 of the outer layer 7010 such that the sealing material 7040 is disposed adjacent to the perforated layer 7050. The sealing layer 7040 applied directly to the inner surface of the outer layer 7010 can be, for example, sprayed or coated onto the inner surface of the outer layer 7010. The film layer carrying the sealing material 7040 can be bonded (e.g., by thermal welding, laser welding, etc.) directly to the interior surface 7047 of the outer layer 7010, or such film layer can comprise a second adhesive layer (e.g., a pressure sensitive adhesive) that adheres a backing layer adjacent to the layer 7010 to the adhesive layer 7040 adjacent to the perforated layer 7050.

In various embodiments, the sealing layer 7040 can include a binder, a swelling material that swells in an aqueous environment, a wax (e.g., a microcrystalline wax), the like, and combinations thereof. Typical swelling agents include hydrophilic crosslinked polymers that swell to 10 to 1000 times their own weight in aqueous media. Examples of suitable heat-activated and pressure-activated adhesives (e.g., Ethylene Vinyl Acetate (EVA), Ethylene Ethyl Acetate (EEA)) are discussed above with reference to fig. 5A and 5B. Suitable heat and pressure activated adhesives, waxes, and the like at least partially soften or melt under reactive conditions (e.g., thermal cycling conditions) to adhere to the perforated layer 7050 and, preferably, deform into the perforated layer holes 7055 to substantially seal the reactive pores 7035 of the array layer 7030. In one embodiment, the heat and/or pressure activated adhesive has a melting point in the range of about 60 ℃ to about 100 ℃.

The embodiment of fig. 8A and 8B is similar to the embodiment of fig. 6A and 6B and 7A and 7B, except that the in-situ sealing material 8040 is disposed on the perforation layer 8050 between the holes 8055 rather than directly on the array layer (see, e.g., the sealing material 6040 of fig. 6A disposed on the end 6051). As in the previous examples, 8040 generally refers to the sealing material, 8040a refers to the sealing material in an initial, undeformed/unsealed state, and 8040b refers to the sealing material in a deformed/sealed state. As shown in fig. 8A, the reaction vessel 8000 is shown in an initial undeformed/unsealed state 8000 a. Fig. 8B illustrates the reaction vessel 8000 in a deformed/sealed state 8000B.

The reaction vessel 8000 includes a first outer layer 8010, a second outer layer 8020, an array layer 8030, a plurality of reaction wells 8035 formed as a series of voids or holes in the array layer 8030, and a perforated layer 8050. Materials used to fabricate the first outer layer 8010, the second outer layer 8020, the perforated layer 8050, and the array layer 8030 are discussed in detail elsewhere herein. In the illustrated embodiment, the second outer layer 8020 is bonded to the first end 8053 of the array layer 8030 to seal the first end of the aperture 8035. The second outer layer 8020 can be bonded directly to the first end 8053 of the array layer 8030 (e.g., by thermal or ultrasonic welding) or the layer 8020 can include an adhesive layer (e.g., a pressure sensitive adhesive or a heat activated adhesive) (not shown) that can bond the layer 8020 to the first end 8053 of the array layer 8030. Similarly, the perforated layer 8050 can be coupled to a second end 8051 of the array layer 8030 opposite the first end 8053 to partially seal the second end of the aperture 8035. The perforated layer 8050 may be formed from a film layer that may be directly bonded to the second end 8051 of the array layer 8030 (e.g., by thermal or ultrasonic welding) or the perforated layer 8050 may be formed from a film layer that includes an adhesive layer (e.g., a pressure sensitive adhesive or a heat activated adhesive) (not shown) that may bond the perforated layer 8050 to the second end 8051 of the array layer 8030.

In the illustrated embodiment, the reaction vessel 8000 includes a sealing material 8040 disposed on an upper surface 8052 of the perforated layer 8050 such that the sealing material 8040 is adjacent to an inner surface 8047 of the outer layer 8010. In the illustrated embodiment, the sealing material 8040 appears as discrete droplets or beads of sealing material applied to the perforated layer 8050 adjacent to the holes 8055, but this is merely illustrative. The sealing material 8040 may be applied as a continuous layer on top of the perforated layer 8050, or, as will be discussed in more detail with reference to fig. 9, the sealing material 8040 may be part of the film material applied to the perforated layer 8050, or, alternatively, the perforated layer 8050 may be made of a film having an in-situ sealing material on one side. With the sealing material 8040 in the initial, undeformed/unsealed state 8040a shown in fig. 8A, the first outer layer 8010 is separated from the sealing material 8040, and fluid can flow into the plurality of apertures 8035 through the holes 8055 of the perforated layer 8050. Once the fluid sample has filled the aperture 8035, pressure can be applied adjacent the outer surface 8049 of the outer layer 8010 to press the layer 8010 into contact with the sealing material 8040, creating a temporary seal. When heat and/or pressure is applied (e.g., in a thermal cycling reaction), the sealing material can deform and adhere the inner surface 8047 of the outer layer 8010 to the sealing material 8040 in the sealed state 8040b to form a more permanent seal.

In one embodiment, the sealing material 8040 may be applied directly to the upper surface 8052 of the perforated layer 8050. For example, the sealing material 8040 can be an adhesive, swelling agent, wax, or the like, or combinations thereof, that is applied directly to the upper surface 8052 of the perforated layer 8050 such that the sealing material is adjacent to the inner surface of the outer layer 8010. In an example embodiment, the sealing material (e.g., heat sensitive adhesive) may be applied as a continuous coating, droplets, grid lines, etc. onto the perforated layer material, and perforations may then be formed, leaving a perforated layer 8050 with holes 8055 bounded by the sealing material 8040. In another embodiment, the sealing material 8040 (e.g., droplets or grid lines) can be applied after bonding the perforated layer 8050 to the array layer 8030. In yet another embodiment, the sealing material 8040 may be part of a film layer applied to the perforated layer 8050. In such embodiments, the film layer comprising the sealing material may include apertures that are approximately the same size and substantially correspond to the holes 8055 in the perforated layer 8050, or alternatively, the layer of sealing material may include holes that are substantially larger than the holes 8055 in the perforated layer 8050. Such a film layer can be directly bonded (e.g., by thermal welding, laser welding, etc.) to the perforated layer 8050. In another embodiment, such a film layer may include a second adhesive layer (e.g., a pressure sensitive adhesive) that may adhere the film layer carrying the sealing material to the perforated layer 8050.

In another example, the perforated layer in the embodiment of fig. 8A and 8B may be made of a film substrate material that includes a sealing layer. One example of such a film-based material 9000 is schematically shown in fig. 9. The film 9000 includes a backing layer 9002 (e.g., a PET layer) and first and second adhesive layers 9004, 9006. A perforated layer similar to 8050 can be prepared by making perforations similar to perforations 8055 in film 9000 and then adhering the perforated film to an array such as array 8030. In various embodiments, the first adhesive layer 9004 and the second adhesive layer 9006 can be the same adhesive, or they can be different adhesives. For example, the first adhesive layer 9004 can be an adhesive (e.g., a pressure sensitive adhesive, a radiation activated adhesive (e.g., a uv catalyzed epoxy)), a conventional epoxy, a surface activated silicone, a cyanoacrylate, a ketone, a latex, an anaerobic adhesive, or an acrylate adhesive) selected to bond the film 9000 to an array, preferably without heating, and the second adhesive layer 9006 can be a sealing layer (e.g., a heat sensitive adhesive) that can form an in situ seal under reaction conditions (e.g., heat and pressure) to form a permanent or semi-permanent seal between the adhesive layer 9006 of the perforated layer and the inner surface of the reaction vessel outer layer. In one embodiment, the adhesive used for the second adhesive layer 9006 may be selected from the group including, but not limited to: heat-activated and/or pressure-activated adhesives, swelling materials that swell in an aqueous environment, waxes, water-activated adhesives, and combinations thereof. In one embodiment, the film material 9000 may be bonded directly (e.g., by thermal welding, laser welding, etc.) to the array layer. In another embodiment, such a film layer may include a second adhesive layer (e.g., a pressure sensitive adhesive or a heat sensitive adhesive) that may adhere the film 9000 to the array.

It will also be appreciated that in the embodiments shown in fig. 5A, 5B, 7A and 7B, a film such as film material 9000 may be used to make the sealing material applied to the outer layer. For example, the first adhesive layer 9004 can be an adhesive (e.g., a pressure sensitive adhesive) selected to bond the film 9000 to an outer layer (e.g., to the surface 7047 of the outer layer 7010 of fig. 7A and 7B), preferably without heating, and the second adhesive layer 9006 can be a sealing layer (e.g., a heat sensitive adhesive) that can form an in situ seal under selected reaction conditions (e.g., heat and/or pressure). Depending on the embodiment, the first adhesive layer 9004 may be selected to bond the film 9000 to an inner surface of the first layer adjacent to the array layer or the perforated layer, and the second adhesive layer 9006 may be selected to form a permanent or semi-permanent seal between the adhesive layer 9006 and the second end of the array layer (fig. 5A and 5B) or between the adhesive layer 9006 and the perforated layer (fig. 7A and 7B) under reactive conditions.

Referring now to fig. 10A-10C, cross-sectional views of system 10000 are illustrated. Fig. 10A-10C illustrate examples of how an in-situ seal may be formed in an instrument 10005 having a reaction vessel that includes a high-density reaction zone and an in-situ sealing feature. Figure 10D illustrates the high density reaction zone after an in situ seal has been formed in the instrument of figures 10A-10C, similar to that shown in figures 7A and 7B. Although the reaction vessels shown in system 10000 are the reaction vessels shown in fig. 7A and 7B, it will be understood that this is for illustrative purposes only, and that any of the reaction vessels shown herein may be received in the instrument 10005.

The instrument 10005 with system 10000 is shown to include an opening between the heater 10010 and the pressure transducer 10020 that is configured to receive a reaction vessel that includes a high density reaction zone and in-situ sealing features. The instrument 10005 shown in fig. 10A-10C is only a portion of an instrument, and it is to be understood that the heater 10010 and pressure transducer 10020 can be included in an instrument that performs several functions, such as the instrument 800 of fig. 2, or the heater 10010 and pressure transducer 10020 can be part of a separate instrument configured to apply pressure and heat to a reaction vessel (e.g., to thermal cycling for nucleic acid amplification).

The reaction vessel 7000 includes a first outer layer 7010, a second outer layer 7020, an array layer 7030, and a plurality of reaction wells 7035 formed as a series of voids or holes formed in the array layer 7030. In the illustrated embodiment, the second outer layer 7020 is bonded to the first end 7053 of the array layer 7030 to seal the first end of the aperture 7035. A second, opposite end 7051 of the array layer includes a perforated layer 7050 located over the openings of the array apertures 7035 to act as a physical barrier, the perforations 7055 allowing fluid sample to flow into the apertures 7035, but can help prevent backflow of fluid from the apertures. The reaction vessel 7000 also includes a sealing layer 7040 coupled to the inner surface 7047 of the first outer layer 7010. In the illustrated embodiment, the sealing layer 7040 can deform in response to heat and pressure to form a seal (e.g., a semi-permanent seal) that seals the opening of the reaction well during the reaction and remains after the heat and pressure are removed. In the illustrated embodiment, 7040 refers generally to the sealing layer, 7040a refers to the sealing layer in an undeformed/unsealed state, and 7040b refers to the sealing layer in a deformed/sealed state.

In an initial step shown in fig. 10A, the reaction vessel 7000 may be disposed between the heater 10010 and the pressure transducer 10020. In an initial step, the heater 10010 and the pressure transducer 10020 may not have been activated, and the sealing layer 7040a and the first outer layer 7010 may not have been pressed into contact with the perforated layer 7050, which allows the aperture 7035 to be filled with a fluid. Suitable examples of heaters for heater 10010 can include, but are not limited to, peltier heaters and other block heaters, resistive heaters, electromagnetic heaters, and thin film heaters as are known in the art. The pressure transducer 10020 can be mechanically or pneumatically actuated, as described in detail above with reference to the pressure transducer 880 of fig. 3. When it is desired to fluorescently excite and detect fluorescence from the contents of the well 7035, the pressure transducer may be a transparent plastic balloon or the like, which may be inflated on the reaction vessel after the well 7035 is filled with the reaction mixture.

In fig. 10B, the pressure transducer 10020 and the heater 10010 are activated. In the illustrated embodiment, actuation of the pressure transducer 10020 has the effect of pressing the second outer layer 7020 of the reaction vessel 7000 against the heater 10010 to facilitate the transfer of heat from the heater 10010 to the fluid in the reaction well 7035. Likewise, actuation of the pressure transducer 10020 may press the layers 7010 and 7040 against the perforated layer 7050 to seal closed the aperture 7035 and purge excess fluid from the high-density reaction zone. In the illustrated embodiment, actuation of the heater 10010 and/or the pressure transducer 10020 has the effect of transforming the layer of sealing material 7040 to form a seal capable of sealing the reaction aperture.

Such a seal is shown in fig. 10. In this caseUnder heat and/or pressure, the sealing layer 7040 deforms from the initial state 7040a to the sealed state 7040b to adhere to the perforated layer 7050 at 7042 and to plug the holes 7055 in the perforated layer 7050 at 7044. The reaction vessel 7000 can be subjected to a first temperature (T) at the interface indicated at 10030 between the heater 10010 and the second outer layer 7020₀) A second intermediate temperature (T) indicated at 10032_i) And a third temperature (T) indicated at 10034_s). In one illustrative example, T₀Can be about 95-105 deg.C (e.g., about 96 deg.C), T_iCan be about 95-100 deg.C (e.g., about 95 deg.C), and T_sCan be in the range of about 60 deg.C to about 100 deg.C (e.g., about 65-95 deg.C, about 70-90 deg.C, about 75-85 deg.C, or about 80-85 deg.C). In one embodiment, the heater 10010 can be configured for an isothermal reaction, and under reaction conditions, T₀、T_iAnd T_sThe temperature present may be substantially static. In another embodiment, the heater 10010 can be configured for thermal cycling, and T₀、T_iAnd T_sThe temperatures present may not be static, but may be highest when the heater 10010 is in a high temperature portion of the thermal cycle (e.g., denaturation) and lower when the heater 10010 is in a lower temperature portion of the thermal cycle (e.g., annealing). In one embodiment, the sealing material of the sealing layer 7040 can be selected such that it is at T_sUnder heat and/or pressure to form a seal that seals the reaction aperture 7035. For example, the sealing material may be a heat and pressure activated adhesive having a softening or melting point in the range of about 60 ℃ to about 100 ℃ (e.g., about 65-95 ℃, about 70-90 ℃, about 75-85 ℃, or about 80-85 ℃). However, the sealing material can be a swelling agent that swells in an aqueous environment, a wax, or the like that is activated by heat and/or pressure (e.g., by water vapor) to form a seal that seals the reaction pores 7035.

As shown in fig. 10D, when heat and pressure are removed from the reaction vessel 7000 (e.g., when the reaction vessel is removed from the instrument 10005), the sealing material 7040b will re-solidify to form a seal that seals the respective aperture 7035. Reaction wells sealed according to the methods and systems described herein may be retained for a period of time for subsequent validation of results and/or further analysis or re-reading on a different instrument. Such reaction wells may be used to measure well-to-well variability or instrument variability from instrument to instrument. In addition, reaction wells sealed according to the methods and systems described herein can be used to make standards (e.g., fluorescence standards), which can be used to calibrate an instrument. Because the sealing material forms a seal in situ, the seal may enhance the effectiveness of the perforated layer to further prevent fluids from flowing into or out of the wells while the reaction is in progress, thereby substantially preventing the contents of each reaction well from intermixing with the contents of other reaction wells.

Examples of the invention

The following examples are intended to illustrate embodiments of the invention and are not intended to limit the scope of the specification or the appended claims.

FIG. 11 illustrates a time course experiment of several time points (1 week, 3 weeks in the process) where the fluorescent material was retained in the wells of the high density reaction zone with and without the in situ sealing material. Fig. 11 illustrates the effectiveness of in-situ sealing during and after the reaction process to substantially isolate individual wells. In the illustrated example, a pattern of fluorescent dye is spotted in an array of microwells with and without an array of in situ sealing layers. An example of an array of holes with associated materials for forming an in situ seal is illustrated in fig. 5A-8B (e.g., fig. 7A and 7B). Both arrays showed sufficient temporary sealing during the reaction phase (in-process column). However, when the array was examined at a later point in time (after 3 hours and after 1 week), the array without the in-situ sealing layer showed significant mixing of the fluorescent dye from the original well to the adjacent wells. In contrast, the array with the in-situ sealing layer showed good sealing, with the fluorescent dye substantially retained in the original wells and little evidence of dye leakage to adjacent wells.

In this embodiment, a film material having a layer of Ethylene Vinyl Acetate (EVA) in situ sealant material applied thereto is placed on the inner surface of the outer layer adjacent to the open ends of the array apertures in an arrangement similar to the embodiment shown in FIGS. 7A and 7B. Although EVA is used as the in-situ sealing material in this example, other materials may also be used, such as, but not limited to, Ethylene Ethyl Acetate (EEA), Ethylene Methyl Acetate (EMA), ethylene n-butyl acrylate (EnBA), Ethylene Acrylic Acid (EAA), Thermoplastic Polyurethane (TPU), polycaprolactone, silicone rubber, thermoplastic elastomers, waxes (e.g., microcrystalline wax), polyethylene, polypropylene, low density polyethylene, copolymers thereof, and combinations thereof. Suitable heat-and pressure-activated adhesives, waxes, and the like typically have a softening or melting point in the range of about 60 ℃ to about 100 ℃ (e.g., about 65-95 ℃, about 70-90 ℃, about 75-85 ℃, or about 80-85 ℃). As shown with reference to fig. 10C, the temperature range experienced by the in situ sealing material is typically within this range during the reaction (e.g., thermal cycling reaction).

Heat and pressure are not the only in situ reactive components that can be used for pore sealing. Other in situ processes that can create a permanent seal include, but are not limited to: the liquid filling the hole may activate the liquid sensitive adhesive layer to seal the hole; the micropores may be filled with an adhesive catalyst, solvent, or reagent that reacts with the adhesive layer when filling the pores; or the hygroscopic material around the openings of the pores may spread and block the openings in the presence of water.

The limitations set forth in the claims are to be interpreted broadly based on the language employed in the claims and not limited to specific examples described in the foregoing detailed description, which examples are to be construed as non-exclusive and non-exhaustive. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

It should also be understood that various features of certain embodiments may be compatible with, combined with, included in, and/or incorporated into other embodiments of the present disclosure. For example, systems, methods, and/or articles of manufacture consistent with certain embodiments of the present disclosure may include, incorporate, or otherwise include features described in other embodiments disclosed and/or described herein. Thus, the disclosure of certain features in connection with specific embodiments of the disclosure should not be construed as limiting the application or inclusion of such features to specific embodiments. Moreover, unless a feature is described as being required in a particular embodiment, features described in various embodiments may be optional and may not be included in other embodiments of the disclosure. Moreover, any feature disclosed herein may be combined with any other feature of the same or different embodiments disclosed herein, unless the feature is described as requiring another feature in combination therewith.