System and method for preparing nucleotide solution
阅读说明:本技术 制备核苷酸溶液的系统和方法 (System and method for preparing nucleotide solution ) 是由 M.里德 Z.邢 D.马兰 E.泽吉贝尔 于 2019-08-16 设计创作,主要内容包括:本发明提供制备核苷酸溶液的系统和方法。该方法包括:使来自测序仪器的初始溶液储罐的水溶液连续流过流体耦合至测序仪器的容器,该容器包含核苷酸浓缩物;并将含有核苷酸的水溶液收集在存储容器中。(The present invention provides systems and methods for preparing nucleotide solutions. The method comprises the following steps: continuously flowing an aqueous solution from an initial solution reservoir of a sequencing instrument through a container fluidically coupled to the sequencing instrument, the container comprising a nucleotide concentrate; and collecting the aqueous solution containing the nucleotides in a storage container.)
1. A method for preparing a nucleotide solution, the method comprising:
in a system comprising a processing cartridge connected to a sequencing instrument, wherein the processing cartridge comprises a plurality of containers, each container in the plurality of containers having a frit in which a nucleotide concentrate is disposed;
flowing a quantity of an aqueous solution from an initial solution reservoir of a sequencing instrument continuously through one of a plurality of containers fluidly coupled to the sequencing instrument, flowing the aqueous solution through a frit to collect nucleotides from a nucleotide concentrate; and is
The aqueous solution containing the nucleotides is collected in a reagent storage container.
2. The method of claim 1, wherein the nucleotide concentrate is a concentrated solution.
3. The method of claim 1 or 2, wherein the nucleotide concentrate is lyophilized nucleotide.
4. A method according to any one of claims 1 to 3, wherein the vessel comprises a receptacle, a clip securing the seal to the receptacle, and a seal and receptacle surrounding the screen panel.
5. The method of claim 4, wherein the seal comprises a central aperture and a peripheral opening providing fluid access to the screen plate.
6. The method of claim 5, wherein the flowing comprises flowing into the container through a central aperture of the seal and out of the container through a peripheral opening.
7. The method of any one of claims 1-6, further comprising inserting the process cartridge into a sequencing instrument.
8. The method of claim 7, wherein the sequencing instrument comprises:
a docking station having:
a first stage including a second guide mechanism for receiving the process cartridge and complementary to the guide mechanism of the process cartridge; and
a second platform movable relative to the first platform and including a fluidic interface having a plurality of fluidic connectors, each fluidic connector including a tube for engaging an inlet of one of the plurality of receptacles of the process cartridge and including an outer ridge for closing an outlet of the receptacle; and is
Wherein said inserting the process cartridge comprises inserting the process cartridge into the docking station.
9. The method of claim 8, wherein each fluid connector includes a first port in fluid communication with a tube of the fluid connector and an inlet of the container, and includes a second port in fluid communication with an outlet of the container.
10. The method of claim 8, wherein the sequencing instrument comprises a plurality of reagent storage containers, each fluid connector being uniquely in fluid communication with one of the plurality of reagent storage containers.
11. The method of claim 8, wherein the docking station further comprises a drive mechanism for moving the second platform relative to the first platform, and wherein inserting the process cartridge into the sequencing instrument comprises moving the second platform closer to the first platform.
12. The method of claim 11, wherein the docking station includes a position sensor for determining a position of the second platform relative to the first platform, wherein moving the second platform closer to the first platform includes detecting the position of the second platform relative to the first platform.
13. The method of claim 11, wherein the docking station includes a process cartridge sensor for detecting the presence of a process cartridge, wherein moving the second platform closer to the first platform is in response to detecting the presence of a process cartridge.
14. A system for preparing a nucleotide solution, the system comprising:
a process cartridge having:
a housing having first and second major surfaces and defining a guide mechanism; and
a plurality of containers disposed in the housing, each container of the plurality of containers having a receptacle and a lid, the lid formed by a clip and a seal, a frit disposed in the receptacle within an enclosed space defined by the receptacle and the seal, the seal having an inlet and an outlet in fluid communication with the enclosed space, the frit comprising a nucleotide concentrate; and
a docking station having:
a first stage including a second guide mechanism for receiving the process cartridge and complementary to the guide mechanism of the process cartridge; and
a second platform movable relative to the first platform and including a fluidic interface having a plurality of fluidic connectors, each connector including a tube for engaging an inlet of one of the plurality of receptacles of the process cartridge and including an outer ridge for closing an outlet of the receptacle.
15. The system of claim 14, wherein each connector includes a first port in fluid communication with the tube of the connector and the inlet of the container, and includes a second port in fluid communication with the outlet of the container.
16. The system of claim 14 or 15, further comprising an initial solution storage container in fluid communication with the fluid interface.
17. The system of any one of claims 14-16, further comprising a plurality of reagent storage containers, each fluid connector being in fluid communication with only one reagent storage container.
18. The system of any one of claims 14-17, further comprising a drive mechanism for moving the second platform relative to the first platform.
19. The system of any one of claims 14-18, further comprising a position sensor for determining a position of the second platform relative to the first platform.
20. The system of any one of claims 14-19, further comprising a cartridge sensor for detecting the presence of a cartridge.
21. The system of any of claims 14-20, further comprising a lever disposed below the second platform for engaging with a notch in the process cartridge to ensure alignment of the plurality of containers with the fluid connectors of the fluidic interfaces.
22. The system of any one of claims 14-21, wherein the screen panel is fluid permeable.
23. The system of any one of claims 14-22, wherein the seal comprises a central bore and a peripheral opening providing fluid access to the screen deck.
24. The system of claim 23, wherein the seal comprises a protrusion for engaging with a protrusion of the screen deck.
25. A system according to claim 24 wherein the projection of the screening plate fits in a central bore of the seal.
26. The system of claim 25, wherein the screen plate includes a central aperture in fluid communication with a central aperture of a seal.
27. The system of claim 25 wherein a cavity is defined between the screen plate and the seal, the cavity being in fluid communication with the peripheral opening of the seal.
28. The system of any of claims 14-27, wherein the clip comprises a ridge for engaging a stem of a receiver.
29. The system of claim 28, wherein the seal engages the lip of the receiver when the ridge of the clip engages the stem of the receiver.
30. The system of any of claims 14-29, wherein the receiver may comprise a ridge for engaging a lip of the housing.
31. A process cartridge comprising:
a housing having first and second major surfaces and defining a guide mechanism; and
a plurality of containers disposed in the housing, each container of the plurality of containers having a receptacle and a lid formed by a clip securing the seal to the receptacle and a seal having an inlet and an outlet in fluid communication with an enclosed space defined by the receptacle and the seal, the seal comprising a nucleotide concentrate.
32. The cartridge of claim 31, wherein the nucleotide concentrate is a concentrated solution.
33. The cartridge of claim 31, wherein the nucleotide concentrate is a lyophilized nucleotide.
34. The process cartridge of any of claims 31-33, wherein said screen panel is fluid permeable.
35. The process cartridge of any of claims 31-34, wherein the seal comprises a central aperture and a peripheral opening providing fluid access to the screen plate.
36. A process cartridge according to claim 35, wherein said sealing member includes a projection for engaging with the projection of the screen plate.
37. The process cartridge as claimed in claim 36, wherein the projection of the screen plate is fitted in the center hole of the seal member.
38. The process cartridge of claim 36, wherein the screen plate includes a central aperture in fluid communication with the central aperture of the seal.
39. The process cartridge of claim 36, wherein a cavity is defined between the screen plate and the seal, the cavity being in fluid communication with the peripheral opening of the seal.
40. A cartridge according to any one of claims 31-39, wherein said clip includes a ridge for engaging with a stem of the receiver.
41. The process cartridge as claimed in claim 40, wherein the seal member engages with the lip of the receiver when the ridge of the clip engages with the stem of the receiver.
42. A cartridge according to any one of claims 31-41, wherein said receiver includes a ridge for engaging with a lip of the housing.
Technical Field
The present disclosure generally relates to a system and method for preparing a reagent solution.
Background
Biological and medical research is increasingly turning to sequencing for enhanced biological research and medicine. For example, biologists and zoologists are turning to sequencing to study the migration of animals, the evolution of species, and the origin of traits. Sequencing is being performed in the medical community to study the origin of the disease, sensitivity to drugs, and origin of infection. However, sequencing has historically been an expensive process, thus limiting its practice.
Disclosure of Invention
In a first embodiment, a method of preparing a nucleotide solution comprises: in a system comprising a processing cartridge connected to a sequencing instrument (wherein the processing cartridge comprises a plurality of containers, each container of the plurality of containers having a frit with a nucleotide concentrate disposed therein), continuously flowing a volume of an aqueous solution from an initial solution reservoir of the sequencing instrument through one of the plurality of containers fluidically coupled to the sequencing instrument, flowing the aqueous solution through the frit to collect nucleotides from the nucleotide concentrate; and collecting the aqueous solution containing the nucleotide in a reagent storage container.
In one example of the first embodiment, the nucleotide concentrate is a concentrated solution. For example, the nucleotide concentrate is lyophilized nucleotide.
In another example of the first embodiment and the above examples, the container includes a receptacle, a clip securing the seal to the receptacle, and a seal and the receptacle surrounding the screen panel. For example, the seal includes a central aperture and a peripheral opening that provide fluid access to the screen plate. In one example, the flow includes flowing into the container through a central aperture of the seal and out of the container through a peripheral opening.
In another example of the first embodiment and the above example, the method further comprises inserting the process cartridge into a sequencing instrument. In one example, the sequencing instrument includes a docking station having a first platform and a second platform, the first platform including a second guide mechanism for receiving and complementing the guide mechanism of the process cartridge, the second platform being movable relative to the first platform and including a fluidic interface having a plurality of fluidic connectors, each fluidic connector including a tube for engaging an inlet of one of a plurality of receptacles of the process cartridge and including an outer ridge for closing an outlet of the receptacle; wherein inserting the process cartridge includes inserting the process cartridge into the docking station. For example, each fluid connector includes a first port in fluid communication with the tube of the fluid connector and the inlet of the container, and includes a second port in fluid communication with the outlet of the container. In another example, the sequencing instrument includes a plurality of reagent storage containers, each fluid connector being uniquely in fluid communication with one of the plurality of reagent storage containers. In another example, the docking station further comprises a drive mechanism for moving the second platform relative to the first platform, and wherein inserting the process cartridge into the sequencing instrument comprises moving the second platform closer to the first platform. For example, the docking station includes a position sensor for determining a position of the second platform relative to the first platform, wherein moving the second platform closer to the first platform includes detecting the position of the second platform relative to the first platform. In another example, the docking station includes a cartridge sensor for detecting the presence of a cartridge, wherein moving the second platform closer to the first platform is in response to detecting the presence of a cartridge.
In a second embodiment, a system for preparing a nucleotide solution comprises: a process cartridge having a housing with first and second major surfaces and defining a guide mechanism; a plurality of containers disposed in the housing, each container of the plurality of containers having a receptacle and a lid, the lid formed by a clip and a seal, a frit disposed in the receptacle within an enclosed space defined by the receptacle and the seal, the seal having an inlet and an outlet in fluid communication with the enclosed space, the frit comprising a nucleotide concentrate. The system further includes a docking station having a first platform including a second guide mechanism for receiving the process cartridge and complementary to the guide mechanism of the process cartridge and a second platform movable relative to the first platform and including a fluid interface having a plurality of fluid connectors, each connector including a tube for engaging an inlet of one of the plurality of receptacles of the process cartridge and including an outer ridge for closing an outlet of the receptacle.
In one example of the second embodiment, each connector includes a first port in fluid communication with the tube of the connector and the inlet of the container, and includes a second port in fluid communication with the outlet of the container.
In another example of the second embodiment and the above examples, the system further comprises an initial solution storage container in fluid communication with the fluid interface.
In another example of the second embodiment and the above examples, the system further comprises a plurality of reagent storage containers, each fluid connector being in fluid communication with only one reagent storage container.
In another example of the second embodiment and the above example, a drive mechanism moves the second platform relative to the first platform.
In another example of the second embodiment and the above example, the system further comprises a position sensor for determining a position of the second platform relative to the first platform.
In another example of the second embodiment and the above examples, the system further includes a process cartridge sensor for detecting the presence of the process cartridge.
In another example of the second embodiment and the above example, the system further comprises a lever disposed below the second platform for engaging with a notch in the process cartridge to ensure alignment of the plurality of containers with the fluid connectors of the fluidic interface.
In another embodiment of the second embodiment and the above example, the screen panel is fluid permeable.
In another example of the second embodiment and the above examples, the seal comprises a central aperture and a peripheral opening providing fluid passage to the screen plate.
In another example of the second embodiment and the above examples, the seal comprises a protrusion for engaging with a protrusion of the screening deck. For example, the projection of the screen plate fits in the central hole of the seal. In one example, the screen plate includes a central bore in fluid communication with the central bore of the seal. In another example, a cavity is defined between the screen plate and the seal, the cavity being in fluid communication with the peripheral opening of the seal.
In another example of the second embodiment and the above examples, the clip includes a ridge for engaging with a stem of the receiver. For example, when the ridge of the clip engages the stem of the receiver, the seal engages the lip of the receiver.
In another example of the second embodiment and the above examples, the receiver may comprise a ridge for engaging with a lip of the housing.
In a third embodiment, a process cartridge includes: a housing having first and second major surfaces and defining a guide mechanism; and a plurality of containers disposed in the housing, each container of the plurality of containers having a receptacle and a lid formed by a clip for securing the seal to the receptacle and a seal in which a frit is disposed within an enclosed space defined by the receptacle and the seal, the seal having an inlet and an outlet in fluid communication with the enclosed space, the frit comprising a nucleotide concentrate.
In one example of the third embodiment, the nucleotide concentrate is a concentrated solution.
In another example of the third embodiment and the above examples, the nucleotide concentrate is a lyophilized nucleotide.
In another embodiment of the third embodiment and the above example, the screen panel is fluid permeable.
In another example of the third embodiment and the above examples, the seal comprises a central aperture and a peripheral opening providing fluid passage to the screen plate. For example, the seal comprises a projection for engaging with a projection of the screening deck. In one example, the projection of the screen plate fits in the central hole of the seal. In another example, the screen plate includes a central bore in fluid communication with the central bore of the seal. In another example, a cavity is defined between the screen plate and the seal, the cavity being in fluid communication with the peripheral opening of the seal.
In another example of the third embodiment and the above examples, the clip includes a ridge for engaging with a stem of the receiver. For example, when the ridge of the clip engages the stem of the receiver, the seal engages the lip of the receiver.
In another example of the third embodiment and the above examples, the receiver may comprise a ridge for engaging with a lip of the housing.
In a fourth embodiment, a system for preparing a nucleotide solution comprises: a process cartridge having a housing with first and second major surfaces and defining a guide mechanism; a plurality of containers disposed in the housing, each container of the plurality of containers having a receptacle and a lid formed by a clip and a seal, a screen disposed in the receptacle within an enclosed space defined by the receptacle and the seal, the seal having an inlet and an outlet in fluid communication with the enclosed space. The system further includes a docking station having a first platform including a second guide mechanism for receiving the process cartridge and complementary to the guide mechanism of the process cartridge and a second platform movable relative to the first platform and including a fluid interface having a plurality of fluid connectors, each connector including a tube for engaging an inlet of one of the plurality of receptacles of the process cartridge and including an outer ridge for closing an outlet of the receptacle.
In one example of the fourth embodiment, each connector includes a first port in fluid communication with the tube of the connector and the inlet of the container, and includes a second port in fluid communication with the outlet of the container.
In another example of the fourth embodiment and the above examples, the system further comprises an initial solution storage container in fluid communication with the fluid interface.
In another example of the fourth embodiment and the above examples, the system further comprises a plurality of reagent storage containers, each fluid connector being in fluid communication with only one reagent storage container.
In another example of the fourth embodiment and the above examples, a drive mechanism moves the second stage relative to the first stage.
In another example of the fourth embodiment and the above examples, the system further comprises a position sensor for determining a position of the second platform relative to the first platform.
In another example of the fourth embodiment and the above examples, the system further comprises a process cartridge sensor for detecting the presence of a process cartridge.
In another example of the fourth embodiment and the above examples, the system further comprises a lever disposed below the second platform for engaging with a notch in the process cartridge to ensure alignment of the plurality of containers with the fluid connectors of the fluidic interfaces.
In another embodiment of the fourth embodiment and the above examples, the screen panel is fluid permeable.
In another example of the fourth embodiment and the above examples, the seal comprises a central aperture and a peripheral opening providing fluid passage to the screen plate.
In another example of the fourth embodiment and the above examples, the seal comprises a protrusion for engaging with a protrusion of the screening deck. For example, the projection of the screen plate fits in the central hole of the seal. In one example, the screen plate includes a central bore in fluid communication with the central bore of the seal. In another example, a cavity is defined between the screen plate and the seal, the cavity being in fluid communication with the peripheral opening of the seal.
In another example of the fourth embodiment and the above examples, the clip includes a ridge for engaging with a stem of the receiver. For example, when the ridge of the clip engages the stem of the receiver, the seal engages the lip of the receiver.
In another example of the fourth embodiment and the above examples, the receiver may comprise a ridge for engaging with a lip of the housing.
In a fifth embodiment, a process cartridge includes: a housing having first and second major surfaces and defining a guide mechanism; and a plurality of receptacles disposed in the housing, each receptacle of the plurality of receptacles having a receptacle and a lid formed by a clip for securing the seal to the receptacle and a seal; a screen deck is disposed in the receptacle within an enclosed space defined by the receptacle and a seal having an inlet and an outlet in fluid communication with the enclosed space.
In one example of the fifth embodiment, the nucleotide concentrate is a concentrated solution.
In another example of the fifth embodiment and the above examples, the nucleotide concentrate is a lyophilized nucleotide.
In another embodiment of the fifth embodiment and the above examples, the screen panel is fluid permeable.
In another example of the fifth embodiment and the above examples, the seal comprises a central aperture and a peripheral opening providing fluid passage to the screen plate. For example, the seal comprises a projection for engaging with a projection of the screening deck. In one example, the projection of the screen plate fits in the central hole of the seal. In another example, the screen plate includes a central bore in fluid communication with the central bore of the seal. In another example, a cavity is defined between the screen plate and the seal, the cavity being in fluid communication with the peripheral opening of the seal.
In another example of the fifth embodiment and the above examples, the clip includes a ridge for engaging with a stem of the receiver. For example, when the ridge of the clip engages the stem of the receiver, the seal engages the lip of the receiver.
In another example of the fifth embodiment and the above examples, the receiver may comprise a ridge for engaging with a lip of the housing.
Drawings
The present disclosure may be better understood, and its numerous features and advantages made apparent to those skilled in the art by referencing the accompanying drawings.
FIG. 1 shows an exemplary sequencing system.
Figure 2 illustrates an exemplary sequencing apparatus.
Figure 3 illustrates an exemplary sequencing component.
Fig. 4 and 5 illustrate an exemplary method for preparing a sequencing device.
FIG. 6 illustrates an exemplary system for preparing reagents in a sequencing system.
Fig. 7, 8, and 9 illustrate an exemplary process cartridge system.
Fig. 10 shows an exemplary process cartridge.
Fig. 11, 12, 13, 14, 15, 16, 17, and 18 illustrate an exemplary container and its components used in conjunction with a process cartridge.
Fig. 19, 20, 21 and 22 show an exemplary docking station for receiving a process cartridge.
Fig. 23, 24, and 25 illustrate an example fluid coupling.
The use of the same reference symbols in different drawings indicates similar or identical items.
Detailed Description
In one embodiment, a system for preparing a reagent solution includes a process cartridge and a docking station. The process cartridge includes a container containing a reagent concentrate. The docking station may receive the process cartridges and be fluidly connected to each of the process cartridges. The initial solution may flow through each container and into a separate reagent storage container. The system has particular advantages when used to prepare nucleotide solutions for use in sequencing devices.
FIG. 1 schematically illustrates a system employing valves, for example, for performing pH-based nucleic acid sequencing. Each electronic sensor of the device generates an output signal that depends on the value of the reference voltage. The fluidic circuit allows for the delivery of multiple reagents to the reaction chamber.
In fig. 1, a
The
The device comprising a dielectric layer defining a pore formed by a first pathway and a second pathway and exposing a sensor pad is particularly suitable for detecting chemical reactions and by-products (e.g., detecting hydrogen ion release in response to nucleotide incorporation), and can be used for gene sequencing, as well as other applications. In one particular embodiment, a sequencing system includes a flow cell having a sensing array disposed therein, includes communication circuitry in electronic communication with the sensing array, and includes a container and a fluid control device in fluid communication with the flow cell. In one example, fig. 2 illustrates an enlarged cross-sectional view of the
Figure 3 shows an expanded view of
In one embodiment, the reaction performed in
In particular, the solid support may comprise copies of the polynucleotide. In the particular example shown in fig. 4, the polymer particles can be used as a carrier for polynucleotides in sequencing technology. For example, such hydrophilic particles can be used to immobilize polynucleotides for sequencing by fluorescence sequencing techniques. In another example, the hydrophilic particles can immobilize multiple copies of a polynucleotide for sequencing by ion sensing techniques. Alternatively, the treatment may improve the binding of the polymer matrix to the sensor array surface. The polymer matrix may capture analytes, such as polynucleotides for sequencing.
Typically, the polymer particles are treated to contain biomolecules, including nucleosides, nucleotides, nucleic acids (oligonucleotides and polynucleotides), polypeptides, sugars, polysaccharides, lipids, or derivatives or analogs thereof. For example, the polymer particles may be bound or attached to biomolecules. The ends or any internal portion of the biomolecule may be bound or attached to the polymer particle. The polymer particles may be bound or attached to the biomolecules by a linkage chemistry. The linkage chemistry includes covalent or non-covalent bonds, including ionic, hydrogen, affinity, dipole-dipole, van der waals, and hydrophobic bonds. Linkage chemistry includes affinity between binding partners, for example between: an avidin moiety and a biotin moiety; an epitope and an antibody or immunoreactive fragment thereof; antibodies and haptens; digoxin moieties and anti-digoxin antibodies; a fluorescein moiety and an anti-fluorescein antibody; an operator and a repressor protein; nucleases and nucleotides; lectins and polysaccharides; steroids and steroid binding proteins; active compounds and active compound receptors; hormones and hormone receptors; an enzyme and a substrate; immunoglobulins and protein a; or an oligonucleotide or polynucleotide and its corresponding complement.
As shown in fig. 4, a plurality of
For example, dispersed
In a particular embodiment, an enzyme (e.g., a polymerase) is present that binds to or is in close proximity to the hydrophilic particles or hydrogel particles of the dispersed phase droplets. In one example, a polymerase is present in the dispersed phase droplets to facilitate replication of the polynucleotide. A variety of nucleic acid polymerases can be used in the methods described herein. In one exemplary embodiment, the polymerase may include an enzyme, a fragment or subunit of an enzyme, which may catalyze the replication of a polynucleotide. In another embodiment, the polymerase may be a naturally occurring polymerase, a recombinant polymerase, a mutant polymerase, a variant polymerase, a fusion polymerase or other engineered polymerase, a chemically modified polymerase, a synthetic molecule, or analogs, derivatives, or fragments thereof.
Particles (e.g., particle 410) are formed after PCR or RPA, which may comprise
In embodiments of the invention, polymer particles resulting from the demulsification process may be collected and washed in preparation for sequencing. The collection can be performed by contacting a biotin moiety (e.g., linked to an amplified polynucleotide template attached to a polymer particle) with an avidin moiety and separating from the polymer particle lacking the biotinylated template. The collected polymer particles loaded with double stranded template polynucleotides may be denatured to produce single stranded template polynucleotides for sequencing. The denaturation step may comprise treatment with a base (e.g. NaOH), formamide or pyrrolidone.
In one exemplary embodiment, the
In one example, primers may be added to the
Sequencing can be performed by detecting the addition of nucleotides. The addition of nucleotides can be detected using methods such as fluorescence emission or ion detection. For example, a set of fluorescently labeled nucleotides can be provided to the
In another example, solutions containing a single nucleotide may be added sequentially. In response to nucleotide addition, the pH within the local environment of the
In particular, the sequencing system may include one or more apertures disposed on a sensor pad of an ion sensor, such as a Field Effect Transistor (FET). In an embodiment of the invention, a system includes a load toOne or more polymer particles disposed in one hole on a sensor pad of an ion sensor (e.g., a FET), or one or more polymer particles loaded into a plurality of holes disposed on a sensor pad of an ion sensor (e.g., a FET). In an embodiment of the present invention, the FET may be a chemFET or an ISFET. A "chemFET" (or chemical field effect transistor) includes a field effect transistor that is used as a chemical sensor. The structure of a chemFET is similar to that of a MOSFET transistor, in which the charge on the gate electrode is applied by a chemical process. "ISFET" (or ion sensitive field effect transistor) can be used to measure the concentration of ions in a solution; when ion concentration (e.g. H)+) When varied, the current through the transistor will vary accordingly.
In embodiments of the invention, one or more microfluidic structures may be fabricated over the FET sensor array to achieve confinement or confinement of biological or chemical reactions. For example, in one embodiment, the microfluidic structure may be configured as one or more wells (or microwells, reaction chambers, or reaction wells, the terms being used interchangeably herein) disposed above one or more sensors of the array such that the one or more sensors over which a given well is disposed can detect and measure the presence, level, or concentration of an analyte in the given well. In an embodiment of the invention, the FET sensor and reaction well may be 1: 1, in the same way.
Referring again to fig. 4, in another example, the
In another embodiment, the solid support (e.g., bead support) may comprise copies of the polynucleotide. In the particular example shown in fig. 5, the polymer particles can be used as a carrier for polynucleotides in sequencing technology. For example, such hydrophilic particles can be used to immobilize polynucleotides for sequencing using fluorescence sequencing techniques. In another example, the hydrophilic particles can immobilize multiple copies of a polynucleotide for sequencing using ion sensing techniques. Alternatively, the treatment may improve the binding of the polymer matrix to the sensor array surface. The polymer matrix may capture analytes, such as polynucleotides for sequencing.
As shown in FIG. 5, a plurality of
In a particular embodiment of seeding, the hydrophilic particles and the polynucleotide undergo Polymerase Chain Reaction (PCR) amplification or Recombinase Polymerase Amplification (RPA). In one example, the
In one example, a
The linker moiety attached to the polynucleotide and the linker moiety attached to the magnetic bead may be complementary and attached to each other. In one example, the linker moiety has an affinity and may comprise: an avidin moiety and a biotin moiety; an epitope and an antibody or immunoreactive fragment thereof; antibodies and haptens; a digoxin moiety and an anti-digoxin antibody; a fluorescein moiety and an anti-fluorescein antibody; an operator and a repressor protein; nucleases and nucleotides; lectins and polysaccharides; steroids and steroid binding proteins; active compounds and active compound receptors; hormones and hormone receptors; an enzyme and a substrate; immunoglobulins and protein a; or an oligonucleotide or polynucleotide and its corresponding complement. In one particular example, the linker moiety attached to the polynucleotide comprises biotin, and the linker moiety attached to the magnetic bead comprises streptavidin.
The
The
Optionally, the
In a particular embodiment, an enzyme (e.g., a polymerase) is present and the enzyme is bound to, or in close proximity to, the particle or bead. In one example, a polymerase is present in the solution or well to facilitate replication of the polynucleotide. A variety of nucleic acid polymerases can be used in the methods described herein. In one exemplary embodiment, the polymerase may include an enzyme, a fragment or subunit of an enzyme, which may catalyze the replication of a polynucleotide. In another embodiment, the polymerase may be a naturally occurring polymerase, a recombinant polymerase, a mutant polymerase, a variant polymerase, a fusion polymerase or other engineered polymerase, a chemically modified polymerase, a synthetic molecule, or analogs, derivatives, or fragments thereof.
Although the polynucleotide of the
In one exemplary embodiment, the
In one example, sequencing primers may be added to the
Sequencing can be performed by detecting the addition of nucleotides. The addition of nucleotides can be detected using methods such as fluorescence emission or ion detection. For example, a set of fluorescently labeled nucleotides can be provided to the
In another example, solutions containing a single nucleotide may be added sequentially. In response to nucleotide addition, the pH within the local environment of the
In an embodiment of the invention, the FET may be an array of FETs. An "array" as used herein is a planar arrangement of elements such as sensors or wells. The array may be one-dimensional or two-dimensional. A one-dimensional array may be an array having one column (or row) of elements in a first dimension and multiple columns (or rows) in a second dimension. The number of columns (or rows) in the first and second dimensions may be the same or different. The FET or array may include 102、103、104、105、106、107One or more FETs.
One exemplary system that involves sequencing by detecting ionic byproducts of nucleotide incorporation is the IonTorrent PGMTM、ProtonTMOr S5TMSequencer (Thermo Fisher Scientific), which is an ionic sequencing system, sequences nucleic acid templates by detecting hydrogen ions generated as a byproduct of nucleotide incorporation. Typically, hydrogen ions are released as a by-product of nucleotide incorporation that occurs during template-dependent nucleic acid synthesis by a polymerase. Ion Torrent PGMTM、ProtonTMOr S5TMThe sequencer detects nucleotide incorporation by detecting hydrogen ion by-products of nucleotide incorporation. Ion Torrent PGMTM、ProtonTMOr S5TMThe sequencer may comprise a plurality of template polynucleotides to be sequenced, each template being disposed in an array within a respective sequencing reaction well. The wells of the array may each be coupled to at least one ion sensor that can detect H as a byproduct of nucleotide incorporation+Release of ions or resulting change in pH of the solution. The ion sensor includes a Field Effect Transistor (FET) coupled to an ion sensitive detection layerLayer-sensible H+The presence of ions or a change in the pH of the solution. The ion sensor can provide an output signal indicative of nucleotide incorporation, which can be expressed as a magnitude and H in the corresponding well or reaction chamber+A voltage change associated with the ion concentration. The different types of nucleotides can flow continuously into the reaction chamber and can be incorporated into the extended primer (or polymerization site) by the polymerase in an order determined by the sequence of the template. Each nucleotide incorporation can be accompanied by H in the reaction well+Release of ions and corresponding change in local pH. H+The release of ions can be recorded by the FET of the sensor, which generates a signal indicating that nucleotide incorporation has occurred. Unincorporated nucleotides may not produce a signal during a particular nucleotide stream. The magnitude of the signal from the FET can also be correlated with the number of specific types of nucleotides incorporated into the extended nucleic acid molecule, allowing discrimination of homopolymer regions. Thus, during the operation of the sequencer, the instrument can simultaneously resolve the sequence of a large number of nucleic acid templates based on multiple nucleotide flows into the reaction chamber and incorporation monitoring across multiple wells or reaction chambers.
FIG. 6 illustrates an exemplary system for preparing reagent solutions in a sequencing device. In one example, the
The
The concentrated nucleotide may be mixed with the initial solution to produce a nucleotide solution that is stored in a separate container, such as
The
Fig. 7 shows an exemplary process cartridge system including a
The
Turning now to fig. 10, the
The
Fig. 11 illustrates an
As shown in the cross-sectional view of fig. 12, the
As shown in fig. 13 and 14, the
As shown in fig. 14, the
Fig. 15, 16, and 17 illustrate an
As shown in fig. 18, the
The docking station is now reviewed and is shown in fig. 19, 20, 21 and 22. The docking station may also include sensors, such as
A sensor 2054 may also be disposed on the
Fig. 21 shows a bottom view of
For example, as shown in fig. 22, when the
In one particular example, fluid may flow into the
Fig. 23, 24, and 25 illustrate an
In a first embodiment, a method of preparing a nucleotide solution comprises: in a system comprising a processing cartridge connected to a sequencing instrument (wherein the processing cartridge comprises a plurality of containers, each container of the plurality of containers having a frit with a nucleotide concentrate disposed therein), continuously flowing a volume of an aqueous solution from an initial solution reservoir of the sequencing instrument through one of the plurality of containers fluidically coupled to the sequencing instrument, flowing the aqueous solution through the frit to collect nucleotides from the nucleotide concentrate; and collecting the aqueous solution containing the nucleotide in a reagent storage container.
In one example of the first embodiment, the nucleotide concentrate is a concentrated solution. For example, the nucleotide concentrate is lyophilized nucleotide.
In another example of the first embodiment and the above examples, the container includes a receptacle, a clip securing the seal to the receptacle, and a seal and the receptacle surrounding the screen panel. For example, the seal includes a central aperture and a peripheral opening that provide fluid access to the screen plate. In one example, the flow includes flowing into the container through a central aperture of the seal and out of the container through a peripheral opening.
In another example of the first embodiment and the above example, the method further comprises inserting the process cartridge into a sequencing instrument. In one example, the sequencing instrument includes a docking station having a first platform and a second platform, the first platform including a second guide mechanism for receiving and complementing the guide mechanism of the process cartridge, the second platform being movable relative to the first platform and including a fluidic interface having a plurality of fluidic connectors, each fluidic connector including a tube for engaging an inlet of one of a plurality of receptacles of the process cartridge and including an outer ridge for closing an outlet of the receptacle; wherein inserting the process cartridge includes inserting the process cartridge into the docking station. For example, each fluid connector includes a first port in fluid communication with the tube of the fluid connector and the inlet of the container, and includes a second port in fluid communication with the outlet of the container. In another example, the sequencing instrument includes a plurality of reagent storage containers, each fluid connector being uniquely in fluid communication with one of the plurality of reagent storage containers. In another example, the docking station further comprises a drive mechanism for moving the second platform relative to the first platform, and wherein inserting the process cartridge into the sequencing instrument comprises moving the second platform closer to the first platform. For example, the docking station includes a position sensor for determining a position of the second platform relative to the first platform, wherein moving the second platform closer to the first platform includes detecting the position of the second platform relative to the first platform. In another example, the docking station includes a cartridge sensor for detecting the presence of a cartridge, wherein moving the second platform closer to the first platform is in response to detecting the presence of a cartridge.
In a second embodiment, a system for preparing a nucleotide solution comprises: a process cartridge having a housing with first and second major surfaces and defining a guide mechanism; a plurality of containers disposed in the housing, each container of the plurality of containers having a receptacle and a lid, the lid formed by a clip and a seal, a frit disposed in the receptacle within an enclosed space defined by the receptacle and the seal, the seal having an inlet and an outlet in fluid communication with the enclosed space, the frit comprising a nucleotide concentrate. The system further includes a docking station having a first platform including a second guide mechanism for receiving the process cartridge and complementary to the guide mechanism of the process cartridge and a second platform movable relative to the first platform and including a fluid interface having a plurality of fluid connectors, each connector including a tube for engaging an inlet of one of the plurality of receptacles of the process cartridge and including an outer ridge for closing an outlet of the receptacle.
In one example of the second embodiment, each connector includes a first port in fluid communication with the tube of the connector and the inlet of the container, and includes a second port in fluid communication with the outlet of the container.
In another example of the second embodiment and the above examples, the system further comprises an initial solution storage container in fluid communication with the fluid interface.
In another example of the second embodiment and the above examples, the system further comprises a plurality of reagent storage containers, each fluid connector being in fluid communication with only one reagent storage container.
In another example of the second embodiment and the above example, a drive mechanism moves the second platform relative to the first platform.
In another example of the second embodiment and the above example, the system further comprises a position sensor for determining a position of the second platform relative to the first platform.
In another example of the second embodiment and the above examples, the system further includes a process cartridge sensor for detecting the presence of the process cartridge.
In another example of the second embodiment and the above example, the system further comprises a lever disposed below the second platform for engaging with a notch in the process cartridge to ensure alignment of the plurality of containers with the fluid connectors of the fluidic interface.
In another embodiment of the second embodiment and the above example, the screen panel is fluid permeable.
In another example of the second embodiment and the above examples, the seal comprises a central aperture and a peripheral opening providing fluid passage to the screen plate.
In another example of the second embodiment and the above examples, the seal comprises a protrusion for engaging with a protrusion of the screening deck. For example, the projection of the screen plate fits in the central hole of the seal. In one example, the screen plate includes a central bore in fluid communication with the central bore of the seal. In another example, a cavity is defined between the screen plate and the seal, the cavity being in fluid communication with the peripheral opening of the seal.
In another example of the second embodiment and the above examples, the clip includes a ridge for engaging with a stem of the receiver. For example, when the ridge of the clip engages the stem of the receiver, the seal engages the lip of the receiver.
In another example of the second embodiment and the above examples, the receiver may comprise a ridge for engaging with a lip of the housing.
In a third embodiment, a process cartridge includes: a housing having first and second major surfaces and defining a guide mechanism; and a plurality of containers disposed in the housing, each container of the plurality of containers having a receptacle and a lid formed by a clip for securing the seal to the receptacle and a seal in which a frit is disposed within an enclosed space defined by the receptacle and the seal, the seal having an inlet and an outlet in fluid communication with the enclosed space, the frit comprising a nucleotide concentrate.
In one example of the third embodiment, the nucleotide concentrate is a concentrated solution.
In another example of the third embodiment and the above examples, the nucleotide concentrate is a lyophilized nucleotide.
In another embodiment of the third embodiment and the above example, the screen panel is fluid permeable.
In another example of the third embodiment and the above examples, the seal comprises a central aperture and a peripheral opening providing fluid passage to the screen plate. For example, the seal comprises a projection for engaging with a projection of the screening deck. In one example, the projection of the screen plate fits in the central hole of the seal. In another example, the screen plate includes a central bore in fluid communication with the central bore of the seal. In another example, a cavity is defined between the screen plate and the seal, the cavity being in fluid communication with the peripheral opening of the seal.
In another example of the third embodiment and the above examples, the clip includes a ridge for engaging with a stem of the receiver. For example, when the ridge of the clip engages the stem of the receiver, the seal engages the lip of the receiver.
In another example of the third embodiment and the above examples, the receiver may comprise a ridge for engaging with a lip of the housing.
In a fourth embodiment, a system for preparing a nucleotide solution comprises: a process cartridge having a housing with first and second major surfaces and defining a guide mechanism; a plurality of containers disposed in the housing, each container of the plurality of containers having a receptacle and a lid formed by a clip and a seal, a screen disposed in the receptacle within an enclosed space defined by the receptacle and the seal, the seal having an inlet and an outlet in fluid communication with the enclosed space. The system further includes a docking station having a first platform including a second guide mechanism for receiving the process cartridge and complementary to the guide mechanism of the process cartridge and a second platform movable relative to the first platform and including a fluid interface having a plurality of fluid connectors, each connector including a tube for engaging an inlet of one of the plurality of receptacles of the process cartridge and including an outer ridge for closing an outlet of the receptacle.
In one example of the fourth embodiment, each connector includes a first port in fluid communication with the tube of the connector and the inlet of the container, and includes a second port in fluid communication with the outlet of the container.
In another example of the fourth embodiment and the above examples, the system further comprises an initial solution storage container in fluid communication with the fluid interface.
In another example of the fourth embodiment and the above examples, the system further comprises a plurality of reagent storage containers, each fluid connector being in fluid communication with only one reagent storage container.
In another example of the fourth embodiment and the above examples, a drive mechanism moves the second stage relative to the first stage.
In another example of the fourth embodiment and the above examples, the system further comprises a position sensor for determining a position of the second platform relative to the first platform.
In another example of the fourth embodiment and the above examples, the system further comprises a process cartridge sensor for detecting the presence of a process cartridge.
In another example of the fourth embodiment and the above examples, the system further comprises a lever disposed below the second platform for engaging with a notch in the process cartridge to ensure alignment of the plurality of containers with the fluid connectors of the fluidic interfaces.
In another embodiment of the fourth embodiment and the above examples, the screen panel is fluid permeable.
In another example of the fourth embodiment and the above examples, the seal comprises a central aperture and a peripheral opening providing fluid passage to the screen plate.
In another example of the fourth embodiment and the above examples, the seal comprises a protrusion for engaging with a protrusion of the screening deck. For example, the projection of the screen plate fits in the central hole of the seal. In one example, the screen plate includes a central bore in fluid communication with the central bore of the seal. In another example, a cavity is defined between the screen plate and the seal, the cavity being in fluid communication with the peripheral opening of the seal.
In another example of the fourth embodiment and the above examples, the clip includes a ridge for engaging with a stem of the receiver. For example, when the ridge of the clip engages the stem of the receiver, the seal engages the lip of the receiver.
In another example of the fourth embodiment and the above examples, the receiver may comprise a ridge for engaging with a lip of the housing.
In a fifth embodiment, a process cartridge includes: a housing having first and second major surfaces and defining a guide mechanism; and a plurality of receptacles disposed in the housing, each receptacle of the plurality of receptacles having a receptacle and a lid formed by a clip for securing the seal to the receptacle and a seal; a screen deck is disposed in the receptacle within an enclosed space defined by the receptacle and a seal having an inlet and an outlet in fluid communication with the enclosed space.
In one example of the fifth embodiment, the nucleotide concentrate is a concentrated solution.
In another example of the fifth embodiment and the above examples, the nucleotide concentrate is a lyophilized nucleotide.
In another embodiment of the fifth embodiment and the above examples, the screen panel is fluid permeable.
In another example of the fifth embodiment and the above examples, the seal comprises a central aperture and a peripheral opening providing fluid passage to the screen plate. For example, the seal comprises a projection for engaging with a projection of the screening deck. In one example, the projection of the screen plate fits in the central hole of the seal. In another example, the screen plate includes a central bore in fluid communication with the central bore of the seal. In another example, a cavity is defined between the screen plate and the seal, the cavity being in fluid communication with the peripheral opening of the seal.
In another example of the fifth embodiment and the above examples, the clip includes a ridge for engaging with a stem of the receiver. For example, when the ridge of the clip engages the stem of the receiver, the seal engages the lip of the receiver.
In another example of the fifth embodiment and the above examples, the receiver may comprise a ridge for engaging with a lip of the housing.
It should be noted that all of the activities described in the general description or the examples above are not necessarily required, that a portion of a specific activity may not be required, and that one or more other activities may be performed in addition to the activities described. Further, the order of enumerated activities is not necessarily the order in which they are necessarily performed.
The inventive concept has been described in the foregoing specification with reference to specific embodiments. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the present invention as set forth in the claims below. Accordingly, the specification and figures are to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of present invention.
As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having" or any variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited to only those features but may include other features not expressly listed or inherent to such process, method, article, or apparatus. Furthermore, unless expressly stated otherwise, "or" means an inclusive rather than exclusive or. For example, either of the following satisfies condition a or B: a is true (or present), B is false (or not present), a is false (or not present) and B is true (or present), and both a and B are true (or present).
In addition, the terms "a" or "an" are used to describe elements and components described herein. This is done merely for convenience and to give a general sense of the scope of the invention. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.
Benefits, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any feature(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or element of any or all the claims.
After reading this specification, skilled artisans will appreciate that certain features are described herein for clarity in the context of separate embodiments, but such features may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. Further, reference to a value given as a range includes each value within that range.
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