Methods to control dissolved gas

A method of sensing nucleotide reactions includes flowing at least one nucleotide solution from a container of at least two containers of a sensor system. The sensor system includes a sensor sensitive to a byproduct of nucleotide incorporation. Each container of the at least two containers includes a different nucleotide solution. The sensor system enters an idle mode after flowing. The method further includes cycling the at least two containers through at least two cycles. Each cycle includes depressurizing the at least two containers for a first period and pressurizing the at least two containers for a second period. The method also includes pressurizing the at least two containers when the sensor system enters an active mode.

FIELD OF THE DISCLOSURE

This disclosure, in general, relates to systems and methods for reducing dissolved gas in a sensor system.

BACKGROUND

Sensors for measuring a chemical or biological response are increasingly relying on measurement of ion concentration or pH. More recently, sensor systems for detecting reactions in molecular biology use sensors for measuring ionic concentration or pH. In particular, sensor systems measuring ionic concentration or pH have been used for quantitative polymer chain reaction (PCR) and genetic sequencing.

SUMMARY

In a first aspect, a method of sensing nucleotide reactions includes flowing at least one nucleotide solution from a container of at least two containers of a sensor system. The sensor system includes a sensor sensitive to a byproduct of nucleotide incorporation. Each container of the at least two containers includes a different nucleotide solution. The sensor system enters an idle mode after flowing. The method further includes cycling the at least two containers through at least two cycles. Each cycle includes depressurizing the at least two containers for a first period and pressurizing the at least two containers for a second period. The method also includes pressurizing the at least two containers when the sensor system enters an active mode.

In a second aspect, an apparatus includes at least two containers. Each container of the at least two containers includes a different nucleotide solution. The apparatus further includes a degasser in fluid communication with a container of the at least two containers. The degasser is to receive a nucleotide solution from the container of the at least two containers and is to separate dissolved gas from the nucleotide solution received from the container of the at least two containers. The apparatus further includes a flow chamber in fluid communication with the degasser. A sensor is disposed within the flow chamber. The sensor is sensitive to a byproduct of nucleotide incorporation.

DETAILED DESCRIPTION

In an exemplary embodiment, a plurality of containers including reagent solutions is connected to a flow control device that selectively provides a reagent solution from a container to a sensor device. The sensor device includes a flow chamber. The sensor device can include an array of sensors and an array of reaction chambers or wells in fluid communication with the flow chamber. The reaction chambers or wells of the array of reaction chambers or wells are operatively coupled to sensors of the sensor array. In particular, the sensors of the sensor array are sensitive to ionic concentration or pH. For example, a sensor of the sensor array can include a field effect transistor (FET), such as a chemically sensitive FET (chemFET), for example, an ion sensitive field effect transistor (ISFET). The reagent solutions can flow out of the flow chamber to a waste port or waste container. The system can include one or more degassers fluidically coupled between containers of the plurality of containers and the flow control device. In another example, a degasser can be fluidically coupled between the flow control device and the flow chamber of the sensor device. Optionally, the system can include a vacuum device operatively coupled to an outer chamber of the one or more degassers.

In a further example, a method for detecting a chemical or biological reaction includes flowing a reagent solution from a container through a flow control device to a flow chamber of a sensor device. The sensor device can include a plurality of reaction chambers in fluid communication with the flow chamber. Sensors of the sensor device can be operatively coupled to the plurality of reaction chambers and can detect products or byproducts of chemical or biological reactions. Following flowing, the system can enter into an idle mode. The method can further include cycling a pressure within the reagent solution container when the system is in idle mode. Cycling can include depressurizing the reagent solution container for a first period and re-pressurizing the container using an inert gas for a second period. When the sensor system is again activated or placed in an active node in preparation for flowing the reagent solution or detecting a chemical or biological reaction, a reagent solution container can be re-pressurized using the inert gas. In an example, the first period is at least twice the second period.

In a particular example, the systems and methods find use in measuring or detecting chemical or biological reactions that produce very small changes in ionic concentration or pH. Exemplary systems utilize small reaction chambers having volumes in a range of 0.01 fL to 10 pL. The sensor can measure ionic concentration or pH. For example, the sensor can detect a change in pH of 0.01 to 0.2 or have resolution in a range of 0.001 to 0.02 pH units. It has been discovered that such sensitive measurement of ionic concentration or pH can be influenced by even small amounts of dissolved carbon dioxide either dissolved as a result of the preparation of reagent solutions or diffusion through container walls when the sensor system sits idle, particularly at a near neutral pH, e.g., a pH in a range of 6 to 8. Such a problem increases noise within the system and increases errors associated with detecting the incorporation of nucleotides. The proposed systems and methods mitigate this discovered problem and improve signal-to-noise ratio and accuracy of the sensor measurement.

FIG. 1includes an illustration of an exemplary sensor system100that includes one or more reagent containers102,104,106,108, and110. The reagent containers (102,104,106,108, and110) are fluidically connected to a flow control device122. The flow control device122selectively provides reagent solutions from the reagent containers (102,104,106,108, and110) to a sensor device126. The sensor device126includes a flow chamber128in fluid communication with a plurality of reaction chambers134associated with a sensor array130. In an example, the array of sensors130includes field effect transistors, such as chemFETs, e.g., ion sensitive field effect transistors (ISFET). In particular, the array of sensors130can be sensitive to ionic concentration or, for example, pH. In an example, the reaction chamber134has a volume in a range of 0.01 fL to 10 pL, such as a range of 0.01 fL to 1 pL, a range of 0.05 fL to 0.5 pL, a range of 0.1 fL to 100 fL, or a range of 0.1 fL to 10 fL. In an embodiment, such sensors can be used to detect nucleotide incorporation or can be used for quantitative analysis of gene expression (e.g., qPCR).

The reagent containers (102,104,106,108, and110) can include reagent or wash solutions useful for facilitating a reaction to be detected in the sensor device126. In a particular example, the containers can include nucleotide solutions, each including an individual nucleotide species (e.g., T, A, C, or G) and one or more wash solutions.

One or more degassers can be placed in fluid communication between the containers and the flow control device122or between the flow control device122and the sensor device126. For example, a degasser112can be in fluid communication between the container102and the flow control device122. In another example, a degasser114is in fluid communication between the container104and the flow control device122. In a further example, a degasser116is in fluid communication between the container106and the flow control device122, a degasser118is in fluid communication between the container108and the flow control device122, or a degasser120is in fluid communication between the container110and the fluid control device for flow control device122. In an additional example, a degasser124is in fluid communication between the flow control device122and the sensor device126. Each degasser can include a membrane defining a flow path and a chamber defined outside of the membrane and inside of a housing of the degasser.

The system100can further include a vacuum device136. The vacuum device136can be connected to one or more of the degassers (112,114,116,118,120, or124). Alternatively, the degassers (112,114,116,118,120, or124) can be open to the atmosphere or can include a check valve that permits one-way flow from within the degasser to outside of the degasser. The vacuum system134can be in fluid communication with the chamber inside the degasser. In a further example, the degassers can be purged with an inert gas, such as helium or nitrogen.

Each of the containers102,104,106,108, or110can be connected to an inert gas system132. In an example, the inert gas system132pressurizes each of the containers (102,104,106,108, or110) to provide a driving force to drive fluid from the containers to the flow control device122. In addition, when the system is idle, the inert gas system132depressurizes the containers (102,104,106,108, or110) and periodically re-pressurizes the containers with an inert gas. For example, the inert gas can include a noble gas, such as helium, or an inert diatomic gas, such as nitrogen.

When in an active mode, the sensor system100pressurizes the containers102,104,106,108, or110and the flow control device122selectively permits flow from one or more of the containers to the sensor device126. In the example of a sequencing system, solutions including individual nucleotides species can be sequentially provided to the sensor device126by the flow control device122. A wash solution can be provided by the flow control device122intermediately between the flow of each reagent solution including a nucleotides species. The sensor device126can include within one or more reaction chambers134template nucleic acids to which a nucleotide within a reagent solution can hybridize. A byproduct of such a reaction can influence the pH of the microenvironment within the reaction chamber, which can in turn be sensed by a sensor associated with the reaction chamber134.

Depending on the nature of the reaction taking place within the reaction chamber134, the ion concentration or pH change in the microenvironment defined by the reaction chamber134is small. For example, the sensor can be configured to measure a pH change within a range of less than 0.5 pH units, such as less than 0.3 pH units, or less than 0.2 pH units at a pH in a range of 6 to 8. In particular, the pH sensor may have a resolution for measuring pH within a range of 0.005 to 0.05, such as a range of 0.005 to 0.02, or even a range of 0.01 to 0.02 pH units.

Such a system is sensitive to small fluctuations in pH, particularly those resulting from changes in the reagent solutions or wash solution. As such, cycling pressure when the system is idle, degassing reagent solutions prior to entry into the sensor device, or a combination thereof, reduces measurement errors by the sensor device126associated with nucleotide incorporation or primer extension.

In particular, the pressure within the containers can be cycled when the system is in an idle mode. In an example illustrated inFIG. 2, a method200includes activating a sensor system, as illustrated at202. Activating the sensor system can include supplying a sensor device for the sensor system or performing calibration functions to prepare the system for performing a chemical or biological reaction. In an example of a genetic sequencing system, a chip device including a flow cell can be applied within the system and fluidically connected to a flow control device that can selectively flow nucleotide solutions and a wash solution from a set of containers to the flow cell.

The containers including reagent solutions, such as nucleotide solutions or a wash solution, can be pressurized using an inert gas, as illustrated at204. In an example, the pressure within the containers is used to drive reagent solution flow that is controlled by the flow control device.

The flow control device can permit a solution to flow from one of the containers and selectively provide the solution to a flow chamber of the sensor device, as illustrated at206. In a genetic sequencing device, the flow control device can selectively permit a nucleotide solution including a species of nucleotide to flow through the flow chamber of the sensor device followed by sequential flow of another nucleotide solution. Optionally, a wash solution can be provided to the flow chamber of the sensor device between flows of nucleotide solution.

Once a test or run is complete, the sensor system can be placed in idle mode, as illustrated at208. In idle mode, reagent solutions are not drawn from the one or more of the containers. Optionally, the sensor device can be removed, particularly in a chip-based sequencing device.

Once the system is in an idle mode, the inert gas system can cycle pressure within the containers that include reagent solutions or wash solutions, as illustrated at210. In particular, the inert gas system can cycle the pressure of the containers at least twice within an idle period, such as at least 5 times, at least 10 times, at least 20 times, or even at least 100 times. To perform another test, the system can be activated again, as illustrated at202.

To cycle pressures within the containers, a method300illustrated inFIG. 3includes depressurizing a container, as illustrated at302. In an example, the container can be depressurized to atmospheric pressure, such as 0 PSIG. In another example, the container can be depressurize to a pressure not more than 50% of the pressure of the container during an active run of the system, such as not more than 35% of the active pressure or even not more than 20% of the active pressure.

A container can be held at the pressure, as illustrated at304, for a first period. In an example, the first period can be in a range of 1 min. to 60 min, such as a range of 1 min to 30 min, or a range of 5 min to 20 min.

Following the first period, the inert gas system can re-pressurize a container, as illustrated at306. For example, the inert gas system can re-pressurize a container to a pressure in a range of 60% to 150% of the pressure utilized during an active run, such as a range of 75% to 115% of the active run pressure, or range of 85% to 105% of the active run pressure, or approximately 100% of the active run pressure.

Pressure within the container can be held for a second period, as illustrated at308. In an example, the second period can be in a range of 5 seconds to 20 min such as a range of 5 seconds to 10 min, a range of 5 seconds to 5 min, a range of 5 seconds to 1 min., or a range of 10 seconds to 30 seconds. In particular, a ratio of the first period to the second period can be in a range of 10:1 to 100:1, such as a range of 15:1 to 75:1, a range of 20:1 to 50:1, or even a range of 25:1 to 45:1, or approximately 30:1 (first period: second period).

Following the second period, the container can be again depressurized, as illustrated at302and the method repeated, each repeat of the method constituting a cycle.

In particular, as illustrated inFIG. 6, a method600includes installing wash reagent containers, as illustrated at602. The wash reagent containers can include a wash reagent prepared as part of configuring the sequencing device. The wash reagent in the containers can be installed within the system and purged, as illustrated604, with an inert gas, such as helium or diatomic nitrogen.

Optionally, the pH of reagents in the wash containers can be measured. In a particular example, the pH can be adjusted, as illustrated at606. Such adjustment can be made using a pH adjusting solution.

The method600further includes installing nucleotide containers including nucleotide reagents, such as concentrated nucleotide reagents, as illustrated608. For example, four different nucleotide solutions, each including a different nucleotide (e.g., A, T, G, or C) can be coupled to the sequencing system. The containers can be purged with an inert gas, such as helium or diatomic nitrogen, as illustrated610. Optionally, the nucleotide containers can be filled using a buffered solution, such as the reagent solution, as illustrated at612.

Following the initiation of the sequencing system, the system can cycle the pressure while waiting to run, as illustrated614. For example, the system can use a pressure cycle as described above. At the direction of a user or based on a time or other automatic system, a run can be initiated, as illustrated616. The run generally includes sequentially drawing the nucleotide solutions with intermediate draws of wash reagent and applying the solutions and reagents over a sequencing device.

Once the run is complete, the system can determine whether an additional run is going to take place, as illustrated at616. If the additional run is to be performed, the system can pressure cycle while waiting to run, as illustrated614. If the system has completed the desired runs, the instrument can undergo a cleaning process, as illustrated620. Following the cleaning process, the system can be started again with the installation of wash reagent containers, as illustrated602.

The system can include degassers in fluid communication between containers and a flow control device or between a flow control device and a sensor device. In an example illustrated inFIG. 4, a degasser400includes a membrane402within a chamber408defined by a housing420. The membrane402defines a flow path for fluid flowing from inlet404to outlet406.

In particular, the membrane402includes a gas permeable tube coiled within the chamber408. An exemplary membrane402can be formed of a gas permeable polymeric material, such as a fluoropolymer including a perflourinated polymer or a perflourinated copolymer, polyphenol sulfide (PPS), polyether ether ketone (PEEK) or derivatives thereof, or any combination thereof. An exemplary fluoropolymer includes polytetrafluoroethylene (PTFE), fluorinated ethylene propylene (FEP) copolymer, a copolymer of tetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride (THV), a copolymer of tetrafluoroethylene and perfluoro methylvinylether (PFA or MFA), a fluoropolymer having a fluorinated oxolane in its backbone, perfluoroether, or any combination thereof.

The chamber408can include one or more ports, such as ports410or412. In an example, the port412is an effluent port and can be connected to a vacuum system. As illustrated, the port412may include a check valve414preventing flow from entering the chamber through the port412. The port410can be connected to a vacuum pressure controller or purged with an inert gas, such as helium or nitrogen. Alternatively, the port410can be left open to atmosphere or can be capped, as illustrated with cap416.

In particular, it was found that a method that includes cycling pressure of the containers using an inert gas reduced pH changes, including pH drift, and provides improved buffering over an extended period. The use of the degassing further stabilizes and reduces fluctuations within the solution, leading to reduced nucleotide incorporation measurement errors. A combination of such aspects provides both long-term and short-term stability of the pH and dissolved gas leading to improved sequencing, particularly for systems operating at a pH in a range of 6 to 8 or systems including a heated sequencing device. In fact, the proposed system provides the ability to tune pH by varying the cyclical purge profile. In particular, the pH influences enzyme activity and thus influences signal in the system. Low pH, in particular, can reduce enzyme activity.

EXAMPLE

Five reagent solutions are treated by cycling pressures over a 20 hour period. The reagent solutions include four nucleotide solutions and a wash solution. The respective containers are depressurized to atmospheric pressure for 10 minutes and are re-pressurized with nitrogen for 20 seconds. This cycle is repeated over a 20 hour period. A similar set of solutions is held at atmospheric pressure for comparison.

As illustrated inFIG. 5, the cycled solutions (upper graphs) exhibit a lower change in pH over the 20 hour period than the solutions held at atmospheric pressure (lower graphs). In particular, the nucleotide solutions that undergo cycling change in pH by less than 0.4 units, whereas the nucleotide solutions that are held at atmospheric pressure change by 0.8 pH units. The wash solution that is cycled maintains a near constant pH, whereas the wash solution that is held at atmospheric pressure changes by approximately 0.2 pH units.

In a first aspect, a method of sensing nucleotide reactions includes flowing at least one nucleotide solution from a container of at least two containers of a sensor system. The sensor system includes a sensor sensitive to a byproduct of nucleotide incorporation. Each container of the at least two containers includes a different nucleotide solution. The sensor system enters an idle mode after flowing. The method further includes cycling the at least two containers through at least two cycles. Each cycle includes depressurizing the at least two containers for a first period and pressurizing the at least two containers for a second period. The method also includes pressurizing the at least two containers when the sensor system enters an active mode.

In an example of the first aspect, the first period is at least twice the second period. In another example of the first aspect and the above example, a ratio of the first period to the second period is in a range of 10:1 to 100:1. For example, the ratio is in a range of 15:1 to 75:1.

In a further example of the first aspect and the above example, the sensor system further includes a flow chamber in fluid communication with the at least two containers and further includes a reaction chamber disposed within the flow cell and operatively coupled to the sensor. For example, the reaction chamber has a volume in a range of 0.01 fL to 10 pL.

In an additional example of the first aspect and the above example, the sensor system further includes a template nucleic acid disposed proximal to the sensor.

In another example of the first aspect and the above example, the sensor includes an ion sensitive field effect transistor (ISFET). In a further example of the first aspect and the above example, the byproduct influences pH.

In an additional example of the first aspect and the above example, flowing the at least one nucleotide solution includes flowing through a degasser.

In a second aspect, an apparatus includes at least two containers. Each container of the at least two containers includes a different nucleotide solution. The apparatus further includes a degasser in fluid communication with a container of the at least two containers. The degasser is to receive a nucleotide solution from the container of the at least two containers and is to separate dissolved gas from the nucleotide solution received from the container of the at least two containers. The apparatus further includes a flow chamber in fluid communication with the degasser. A sensor is disposed within the flow chamber. The sensor is sensitive to a byproduct of nucleotide incorporation.

In an example of the second aspect, the sensor includes an ISFET sensor. For example, the ISFET sensor can be disposed within a reaction chamber disposed within the flow chamber. In a further example, the reaction chamber has a volume in a range of 0.01 fL to 10 pL.

In another example of the second aspect and the above examples, the degasser includes a gas permeable tubing in fluid communication with the at least two containers. In an example, the gas permeable tube includes a fluoropolymer.

In a further example of the second aspect and the above examples, the byproduct influences pH.

In an additional example of the second aspect and the above examples, the apparatus further includes a flow control device in fluid communication between the degasser and the flow chamber.

In another example of the second aspect and the above examples, the apparatus further includes a flow control device. The degasser is in fluid communication between the flow control device and the flow chamber.

In a further example of the second aspect and the above examples, the apparatus further includes a vacuum device operatively coupled to the degasser.

In an additional example of the second aspect and the above examples, the apparatus further includes an inert gas system in fluid communication with the at least two containers.