A nanopore flow cell

Abstract

A flow cell device comprising an array of nanopore sensors, the flow cell device comprising; a main body comprising; a top surface, and a main surface, the main surface being arranged normal with respect to the plane of the top surface, and a sensor chamber comprising the array of nanopore sensors, the sensor chamber being arranged in the plane of the main surface; the top surface comprising a fluid inlet for introducing sample fluid into the flow cell device; wherein the flow cell device further comprises a lid connectable to main body such that when the lid is moved to an open position the fluid inlet is exposed.

Description

[0001] A NANOPORE FLOW CELL

[0002] FIELD OF THE APPLICATION

[0003] The present application relates to a flow cell for use as part of a sequencing device.

[0004] BACKGROUND

[0005] A flow cell sensing device is typically a microfluidic device that senses small volumes of liquid, typically on the scale of microlitres or smaller. Sample liquid may be introduced into the device via a fluid inlet and move along a flow channel to a sensor to be characterised. This is particularly advantageous in analysis of biological specimens, where smaller volumes can allow less intrusive sample acquisition from patients, and less use of expensive reagents. An example of such a device is Oxford Nanopore Technology’s MinlON sequencer, which performs DNA and RNA sequencing.

[0006] The flow cell is the part of the sequencing device which comprises a sensing chamber within which is located the sensor which senses the sample liquid. An example of a flow cell is Oxford Nanopore Technologies’ MinlON Flow Cell, which comprises a sensor comprising a nanopore array, and electronics for processing a signal produced by the sensor. The MinlON Flow Cell is for use as part of Oxford Nanopore Technology’s MinlON sequencer. The MinlON comprises additional electronics for performing further processing of the signal produced by the sensor. Flow cells are often consumable parts.

[0007] SUMMARY

[0008] A first aspect of the invention provides a flow cell device comprising an array of nanopore sensors, the flow cell device comprising; a main body comprising; a top surface, and a main surface, the main surface being arranged normal with respect to the plane of the top surface, and a sensor chamber comprising the array of nanopore sensors, the sensor chamber being arranged in the plane of the main surface; the top surface comprising a fluid inlet for introducing sample fluid into the flow cell device; wherein the flow cell device further comprises a lid connectable to main body such that when the lid is moved to an open position the fluid inlet is exposed.

[0009] Flow cells with an array of nanopore sensors enable the sensing of small molecules such as DNA. There is a desire to minimise the size or footprint of flow cells and associated devices such that they are compact and cost-effective. However, a key design consideration is that the nanopore array may be prone to becoming damaged when dried out. An example of a nanopore array is disclosed in WO2014064443, hereby incorporated by reference in its entirety.

[0010] The array of nanopore sensors comprises an array of nanopore sensing elements, each sensing element comprising a nanopore provided in a membrane supported on a support structure. The support structure defines a plurality of wells with a plurality of associated electrode, such that the membrane separates liquid in the wells from the liquid in the sensor chamber to provide a plurality of trans chambers, (i.e. the sensor chamber being referred to as a cis chamber).

[0011] The nanopore provides a fluid pathway through the membrane that fluidically connects the sample chamber to the respective trans chambers. The cis chamber and trans chambers may be provided with ionic liquids and the flow of ions through the nanopore may be measured in the presence of an analyte in order to characterise the analyte. Typical analytes that may be measured are polynucleotides such as DNA and RNA wherein measurements of ion flow may be carried out during translocation of polynucleotides through the array of nanopores.

[0012] Controlled translocation may be carried out under an applied potential difference across each membrane and measurements of a signal such as ionic current may be carried out in order to determine the nucleotide sequence.

[0013] The flow cell may be provided in a wet state, namely whereby ionic liquids are provided in the trans chambers and in the sample chamber. It is important to ensure that evaporation does not occur from the sample chamber and / or the trans chambers as this can affect the concentration of ions and therefore the ionic current. The solutions in the respective trans chambers and sample chamber are typically osmotically balanced in order to ensure that the ions in the respective chambers are maintained at the correct concentration.

[0014] Therefore, there is a need to maintain a wet environment for the array of nanopore sensors. Providing a lid attached to a flow cell allows for the flow cell to be fluidic sealed during transit, in use, or when placed in storage. In essence, a lid allows for the sealing of the device. The lid may take the form of a sealing film or closeable cap. The film may be retained by

[0015] By having a top surface with a fluid inlet (the top surface being arranged normal with respect to the flow cell) the overall size and volume of the device can be reduced. Most flow cell device have the fluid inlets arranged on the plane of the flow cell, meaning that flow cell and fluid inlets have to be arranged in the same plane. It has been found that a fluid inlet can be positioned on a different surface (and another plane) without impacting on the device function or causing undesired effects, such as the introduction of air bubbles into the flow cell fluid paths or array of nanopore sensors.

[0016] The top surface may comprise a fluid outlet. Typically the fluid outlet is used to extract sample liquid that has passed through the sensing chamber, although excess buffer solution or waste fluid could also be extracted. This allows for access to the fluidic inlet and outlet through one surface of the device. This also allows for a simplified and reduced sized flow cell since fluidic pathways can be routed to / from the same plane. An additional advantage is that the flow cell can be imbedded upright into an associated external device with respect to the plane of the main of the surface and a lab bench or workstation. This allows for efficient space saving since only the top surface with a fluid inlet and fluid outlet need to be accessed during use of the device. Additionally this configuration of the flow cell means that the associated external device can be easily arranged to house multiple flow cells adjacent one another without having to have a large horizontal footprint. In other words, the multiple flow cells can be arranged adjacent one another such that a large space is not required or taken up on a lab bench or within a workstation.

[0017] The lid may comprise a sealing material provided on a mating surface on the lid with respect to the top surface of the main body such that when the lid is in the closed position the lid fluidic seals the flow cell device. The mating surface is typically the face on the lid which is the opposite the top surface of flow cell. In some embodiments the seal ing material is an adhesive when the lid takes the form of a sealing film. It is envisaged that the adhesive would allow the sealing film to be resealed. In an alternative embodiment the sealing material takes the form of, for exampl e, an over-moul ded el astomer such as a Thermopl astic Elastomer (TPE) on the mating surface which aids in the formation of a fluidic seal between the two mating surfaces. As mentioned previously, it is desirable to maintain a wet environment in the sensing chamber. Allowing fluid to escape from the sensing chamber may be undesirable because it may result in the sensing chamber drying out or create an imbalance of concentration of ions. As mentioned above his may damage components located within the sensing chamber and / or affect the ionic current during use of the flow cell.

[0018] The lid may be pivotably connected to the main body by a hinge, the hinge being formed as part of the top surface of the flow cell device. Providing a pivotably connected lid rather than a peelable seal allows for a more robust closing mechanism. The adhesive on the seal may reduce in its ability to provide a seal whereas a pivotably connected lid can be reused for the lifetime of the flow cell. Additionally, the lid can be mounted and formed within the footprint of the device such that it does not add bulk or size to the overall product. In this connection the main body may further comprise hinge features for forming the pivotable connection between the main body and the lid to reduce the complexity and the overall size of the flow cell.

[0019] The lid may comprise a catch feature for locking the lid in the closed position. The catch feature may be configured to interlock with a part of the main body, such that the lid can be locked or retained in the closed position. This provides a better sealing arrangement for the flow cell so that it can be moved or transported and reduce the risk of liquid being spilled or leaking if the flow cell is dropped or mishandled.

[0020] The lid may further comprise an opening feature, the opening feature being configured such that force applied to the opening feature releases the catch. Optionally the opening feature may form part of the lid and is designed such that it can be opened by a single digit. Preferably the opening feature is designed such that it falls substantially within the footprint of the lid. This allows for the opening force applied to be aligned with the centre of gravity point of the device, thereby reducing the risk of tipping the fl ow cel 1 when an opening force is applied. Even more preferably, the opening feature forms a sprung or biased lever which, when a force is applied, moves the catch feature away from the interlocking feature of the main body of the flow cell. In other words, the catch is normally biased towards the interlocking feature of the main body and is moved away once force is applied to the opening feature on the lid. Additionally this means that the lid may be configured such that the force applied to the opening feature to open the lid is in the same direction as a force that is applied to close the lid.

[0021] In connection with the above statements this enables a simple sprung design for the opening feature which is within the footprint of the flow cell device. In addition the lid is designed in such a way that only one direction of force is required to open and close the lid which reduces the risk of a used tipping or mishandling the flow cell during use. Furthermore the simplicity of the form of the lid means that the flow cell can be operated by a robot for the automated addition of the fluid sample to the fluid inlet.

[0022] The flow cell device may further comprise; a locking member for retaining the sensor chamber against the main surface of the flow cell device; and a gasket for fluidic sealing the sensor chamber against main surface of the flow cell device. The flow cell is designed to be compact. This arrangement of a locking member allows for all of the necessary components to be held together and sealed without the need for screws or other mechanical means of retaining components in place. Optionally the locking member surrounds the sensor chamber but has a hole through which a thermal connection may be made to the sensor chamber. The thermal contact would allow a fluid, such as air, to flow over a face of the surface chamber and allow it to cool. Alternatively or additionally a thermal contact made be made with, for example, and electronic component such as the ASIC part of the flow cell.

[0023] The flow cell device may further comprise a cover which is attached to main surface of the flow cell device, the cover providing fluidic flow paths connecting the fluid inlet to the sensor chamber. The cover allows for a transparent seal such that the sensing chamber may be visible to a user of the flow cell. Additionally, by arranging the fluidic flow paths in the cover overall device complexity and parts for manufacture can be reduced. The flow cell device may further comprise an electronic component for electrically addressing the array of nanopore sensors. The electronic component may have retaining features which interact with features provided on both the locking member and the main body of the flow cell device. This arrangement of the locking member allows for all of the necessary components to be held together without the need for screws or other mechanical means of retaining components in place.

[0024] The electrical component may comprise an electrical connector for connecting to an external device. The electrical connector may be a USB-C interface. This allows for ease of interaction with an eternal device such as a docking station or an automated sampling handling device such as a robot. The external device may be an automated sampling handling device which is configured to interact with the lid of the flow cell device in order to introduce fluid sample into the device via the fluid inlet.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure l is a perspective view of a flow cell of the present invention;

[0027] Figure 2 is an exploded view of a flow cell of the present invention;

[0028] Figure 3 is a perspective view of the underside of a flow cell of the present invention;

[0029] Figures 4 - 6 are side views of a flow cell of the present invention with the lid held in various positions;

[0030] Figure 7 is a side view of a flow cell of the present invention with a film seal;

[0031] Figure 8 is a schematic diagram of a nanopore sensor element in the sensor chamber; and

[0032] Figure 9 is a flow cell of the present invention provided in an external device. DETAILED DESCRIPTION

[0033] Figure 1 shows a flow cell 1 comprising an array of nanopore sensors with a lid 3 and a main body 5. The main body 5 has a top surface 7 and a main surface 9. Figure 1 also shows the relative orientation between the flow cell 1, top surface 7 and a main surface 9 relative to a vertical axis 2, a length axis 4, a width axis 6, a horizontal plane 8, which extends along the length axis 4 and the width axis 6, and a vertical plane 10, which extends along the vertical axis 2 and the width axis 6. Typically in use, the flow cell is orientated as shown in Figure 1.

[0034] The flow cell 1 is shown in an exploded view in Figure 2 and comprises a main body 5 formed from, for example Cyclic olefin copolymer (COC), which together with a locking member 11, an electronic component 12, a gasket 13 and a cover 14 form a sensing chamber 15 (shown as 15 in Figure 2 which is the underside of the cover which forms an inner surface of the sensing chamber when these components (5, 11, 12, 13 and 14) are brought together along the dashed line A). As such, the gasket 13 is provided with an aperture 16 for forming a seal between the main body 5 and the electronic component 12 so that the flow cell 1 is fluidic sealed when constructed. The gasket 13 is formed from a pliable and elastic material such as Fluoroelastomer (FKM) which provides a fluidic seal and also deforms under pressure provided from the locking member 11 when the flow cell 1 is assembled.

[0035] The locking member 11 is formed out of a hard non-pliable material such as stainless steel and is provided with a set of retaining features 17 (one labelled to reduce complexity) which mate with a corresponding set of mating features 18 on the main body 5 such that the flow cell 1 is held together when assembled. The locking member further comprises a clip 19 which mates with an associated retainer 20 on the electronic component 12. Thus, the flow cell 1 is held together by the locking member 11 fastening to both the main body 5 and the electronic component 12. The locking member has an aperture 21 which reveals a part of the electronic component 12. This allows for access to the electronic component 12 through the back of the device if necessary (i.e. not through the sealed cover 14) and also allows for cooling or heating of the electronic component 12 as necessary during operation of the flow cell 1. The electronic component 12 comprises the array of sensing elements 133 which will be described later. The array of sensing elements 133 are formed on a substate 131 which overlies an ASIC 22 (underside shown in Figure 2). The substrate 131 forms part of the sensing chamber 15 with the gasket 13 and the cover 14. The electronic component 12 further comprises an electrical connector 23. The electrical connector 23 provides an interface between the flow cell 1 and an external device 24 which will be described later.

[0036] The electrical connector 23 may take the form of a traditional USB plug or socket so that power and data can be supplied / transferred to / from the flow cell 1. The electrical connector 23 is a USB-C style connector such that the footprint and size of the device can be minimised. Additionally, the electrical connector 23 takes the form of a USB-C plug so that the flow cell 1 cannot accidentally be inserted into a device such as a tablet or laptop computer by the user, since the flow cell 1 would be in the incorrect orientation for use. The main body 5 can be formed at least partly around the electrical connector 23 so as to offer protection and also ensure that the electrical connector 23 cannot be inserted into a device for which it is not intended. This is best shown in Figure 3.

[0037] The ASIC 22 is electrically connected to the sensor chamber 15 and the electrical connector 23. The ASIC 11 is for controlling the array of sensing elements 133 and a common electrode (not shown), and for processing signals output by the array of sensing elements 133 and providing the signals to the electrical connector 23.

[0038] The cover 14 is formed of a transparent material such as Cyclic olefin copolymer (COC) and is welded or adhered to a surface of the main body 5. The cover 14 comprises recesses 25 for forming chambers for holding fluid when the flow cell 1 is assembled. Additionally, the cover 14 comprises fluid flow paths 26 through the flow cell 1 once the flow cell is assembled.

[0039] The main body 5 additionally comprises an aperture 27 and a recess portion 28 for forming part of the sensor chamber 15 and a seal with the gasket 13, respectively. The top surface 7 of the main body 5 comprises a fluid inlet 29 and a fluid outlet 30. When assembled, the fl uid ini et 29 and fl id outl et 30 are connected fluidically by the fluid flow paths 26 provided in the cover 14. Sample or buffer fluid can enter the flow cell 1 via the fluid inlet 29 and flow via the fluid flow paths 26 into the sensing chamber 15. Fluid can be extracted from the flow cell 1 via the fluid outlet 30 which is fluidic connected to the sensing chamber 15 also via fluidic flow paths 26 provided by the cover 14.

[0040] The main body 5 also comprises a portion proximate the top surface 7 with a hinge cut-outs 31 and a bore 32 for accepting an axle 33 which together with the mating hinge feature 35 on the lid 3 for a pivotable connection between the lid 3 and the main body 5 when assembled. Also provided is a catch locking feature 34 which interacts with lid to hold the lid against the top surface 7 of the main body 5.

[0041] The lid 3 may be formed for example from a polycarbonate and comprises a matching mating hinge feature 35 which, when assembled allows the lid 3 and mating hinge features 35 to form flush with the main body 5 of the flow cell 1. The mating hinge features 35 comprise a matching bore 36 for housing the axle 33 such that the lid can pivot with respect to the axis B of the axle 33. The lid 3 additionally comprises a catch feature 37 for locking the lid 3 in the closed position against the main body 5 which will be described later. The catch feature 37 mates with the opposing catch locking feature 34 found on the main body 5. The lid 3 comprises an opening feature 38 which, when operated, releases the catch feature 37 from the opposing catch locking feature 34 found on the main body 5 and allows the lid 3 to move to the open position and reveal the fluid inlet 29 and the fluid outlet 30. Additionally, the underside (or side opposite the top surface 7 of the main body 5) may comprise a sealing material around its periphery to form a seal around the top surface 7 of the main body 5 when the lid 3 is in the closed position.

[0042] The opening and closing of the lid will now be described with reference to Figures 4 to 6.

[0043] Figure 4 shows the flow cell 1 where the lid 3 is in a closed position (i.e. held against the main body 5 and obscuring / sealing the fluid inlet 29 and the fluid outlet 30). The opening feature 38 takes the form of lever which is biased towards a position such that the catch feature 37 of the lid 3 is held against the opposing catch locking feature 34 found on the main body 5. Downward pressure on to the opening feature 38 releases the catch feature 37 of the lid 3 from the opposing catch locking feature 34 found on the main body 5. The lever of the opening feature 38 is designed such that the downward pressure also translates through the axial direction of the axl e 33 contributing to the opening of the l id 3 via a pivoting relationship with the main body 5 (as seen in Figure 5). The opening of the lid 3 as seen in Figure 6 reveals the fluid inlet 29 and the fluid outlet 30 on the top surface 7 of the main body 5.

[0044] In order to close the lid 3, the same downward pressure is applied against the other end of the lever forming part of the opening feature 38 (as shown in Figure 4 to 6 as the part of the lid above the catch locking feature 34. This design of the opening feature 38 also allows for the moment of force generated from the downward pressure applied by the user to be greater on closing or sealing the lid compared to the force required to open the lid 3 via the opening feature 38. Also the opening feature 38 can be designed or manufactured such that force from the user is absorbed by the flexing of the lever and / or from the flexibility of the material forming the opening feature 38.

[0045] This opening feature 38 as shown is shaped and particularly suited to automated sampling handling devices which are configured to interact with the lid 3 of the flow cell 1 in order to introduce fluid sample into the flow cell 1 via the fluid inlet 29. A simple digit or protruding member can be used to both open and close the lid 3 by, for example, a robot.

[0046] Figure 7 shows an embodiment where a sealing film 39 such as a biaxially oriented polypropylene (BOPP) is provided on the top surface 7 of the main body 5. The film 39 is adhered to the top surface 7 and may be reapplied to re-seal the fluid inlet 29 and / or the fluid outlet 30 during use. This may or may not be in addition to the seal provided by the sealing material around the periphery of opposing surface of the lid 3.

[0047] The sensing ability and design of the flow cell 1 will now be described. The sensor chamber 15 (shown schematically in Figure 8) has a substrate 131, and an array of nanopore sensing elements 133. The array of nanopore sensing elements 133 are located below the deep portion. The substrate 131 has an array of wells 135. The substrate 131 and the array of nanopore sensing elements 133 are 1 ocated in a bottom surface of the sensing chamber 27, in the portion of constant width 107

[0048] Each nanopore sensing element 134 of the array of nanopore sensing elements 133 has a membrane 139 and a sensor electrode 141. The membrane 139 is supported across one of the wells 135 of the array of wells 135 with a nanopore 143 inserted in the membrane 139. Each nanopore sensing element 134 of the array of nanopore sensing elements 133 supports a respective nanopore 143. Each nanopore 143 provides a nanopore channel 144 extending through it, a common chamber side 136 and a well side 138. The array of nanopore sensing elements 133 has a total surface area 153, when viewed in a plane which is parallel to the vertical plane 6. Each sensor electrode 141 is located in a respective well 135 such that each sensor electrode 141 is on the opposite side of their respective membrane 139 to the sensing chamber 15.

[0049] The sensing electrodes 141 output signals dependant on the interactions between the molecular entities and the nanopores 143. For example, translocation of a molecular entity through a nanopore 143 may alter an ion current flowing through the nanopore 143, which may be sensed by the sensing electrodes 141. The signals are output from an array of nanopore sensing elements 133 to the ASIC 22, which processes the signals. The signals are then sent to an external device 24 for further processing, and then sent to the external computing device where the signals are also processed, for example, to determine the DNA sequence of the molecular entities.

[0050] Figure 9 provides an example of an external device 24 which docks and retains the flow cell 1. The external device may dock multiple flow cells 1. In addition, the dock may comprise a data / power link 40 to connect the external device to a computer, lap top, phone or tablet (or any other device) to power and transfer data from the flow cell 1. It is also envisaged that the external device may be an automated sample preparation and polymer sequencing device with a dock or docks for a flow cell lor flow cells 1. Whil st particul ar exampl es have been described, it should be understood that these are illustrative examples only and that various modifications may be made without departing from the scope of the invention as defined by the claims.

Claims

CLAIMS1. A flow cell device comprising an array of nanopore sensors, the flow cell device comprising; a main body comprising; a top surface, and a main surface, the main surface being arranged normal with respect to the plane of the top surface, and a sensor chamber comprising the array of nanopore sensors, the sensor chamber being arranged in the plane of the main surface; the top surface comprising a fluid inlet for introducing sample fluid into the flow cell device; wherein the flow cell device further comprises a lid connectable to main body such that when the lid is moved to an open position the fluid inlet is exposed.

2. A flow cell device as claimed in claim 1, wherein the array of nanopore sensors comprises an array of nanopore sensing elements, each sensing element comprising a nanopore provided in a membrane supported on a support structure; wherein the support structure defines a plurality of wells, such that the membrane separates liquid in the wells from the liquid in the sensor chamber to form a plurality of trans chambers.

3. A flow cell device as claimed in claim 2 wherein the trans chambers comprise a sensing electrode and an ionic solution and wherein an ionic solution is provided in the sample chamber.

4. A flow cell device as claimed in any one of the preceding claims, wherein the top surface comprises a fluid outlet.

5. A flow cell device as claimed in any one of the preceding claims, wherein the lid comprises a sealing material provided on a mating surface with respect to the top surface of the main body such that when the lid is in the closed position the lid fluidic seals the flow cell device.

6. A flow cell device as claimed in any one of the preceding claims, wherein the lid ispivotably connected to the main body by a hinge, the hinge being formed as part of the top surface of the flow cell device.

7. A flow cell device as claimed in claim 6, wherein the main body comprises hinge features for forming a pivotable connection between the main body and the lid.

8. A flow cell device as claimed in any one of claims 6 or 7, wherein the lid comprises a catch feature for locking the lid in the closed position.

9. A flow cell device as claimed in claim 8, wherein the lid further comprises an opening feature, the opening feature being configured such that force applied to the opening feature releases the catch.

10. A flow cell device as claimed in claim 9, wherein the lid is configured such that the force applied to the opening feature to open the lid is in the same direction as a force that is applied to close the lid via the catch.

11. A flow cell device as claimed in any one of the preceding claims, wherein the flow cell device further comprises; a locking member for retaining the sensor chamber against the main surface of the flow cell device; and a gasket for fluidic sealing the sensor chamber against main surface of the flow cell device.

12. A flow cell device as claimed in any one of the preceding claims, wherein the flow cell device further comprises a cover which is attached to main surface of the flow cell device, the cover providing fluidic flow paths connecting the fluid inlet to the sensor chamber.

13. A flow cell device as claimed in any one of the preceding claims, wherein flow cell device further comprises an electronic component for electrically addressing the array of nanopore sensors.

14. A flow cell device as claimed in Claim 13, wherein the electronic component has retaining features which interact with features provided on both the locking member and the main body of the flow cell device.

15. A flow cell device as claimed in claim 13 or claim 14, wherein the electrical component comprises an electrical connector for connecting to an external device.

16. A flow cell device as claimed in claim 15, wherein the electrical connector is a USB- C interface.

17. A flow cell device as claimed in claims 15 or 16, wherein the external device is an automated sampling handling device which is configured to interact with the lid of the flow cell device in order to introduce fluid sample into the device via the fluid inlet.

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