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WO2025238376A1 - Cuve à circulation à nanopores et procédé - Google Patents

Cuve à circulation à nanopores et procédé

Info

Publication number
WO2025238376A1
WO2025238376A1 PCT/GB2025/051068 GB2025051068W WO2025238376A1 WO 2025238376 A1 WO2025238376 A1 WO 2025238376A1 GB 2025051068 W GB2025051068 W GB 2025051068W WO 2025238376 A1 WO2025238376 A1 WO 2025238376A1
Authority
WO
WIPO (PCT)
Prior art keywords
valve
flow cell
sensing chamber
liquid
nanopore
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/GB2025/051068
Other languages
English (en)
Inventor
Paul MACKETT
Steven Ireland
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oxford Nanopore Technologies PLC
Original Assignee
Oxford Nanopore Technologies PLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oxford Nanopore Technologies PLC filed Critical Oxford Nanopore Technologies PLC
Publication of WO2025238376A1 publication Critical patent/WO2025238376A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores

Definitions

  • the present application relates to a flow cell for use as part of a nanopore array device and a method.
  • Nanopore array devices have been developed that comprise an array of nanopore sensing elements, thereby increasing data collection by allowing plural nanopore sensing elements to sense interactions in parallel, typically from the same sample.
  • Nanopore sensing elements may typically employ an electrical signal across a nanopore channel to generate a measurement signal that is interpreted to sense and/or characterise molecular entities as they interact with the nanopore channel.
  • an electrical signal is applied as a potential difference or current across the nanopore channel that will provide a meaningful measurement signal to be interpreted.
  • the measurement can include, for example, one of ionic current flow, electrical resistance, or voltage.
  • An example of such a device is Oxford Nanopore Technology’s MinlON, which performs DNA and RNA sequencing.
  • a first aspect provides a flow cell for use as part of a nanopore array device, the flow cell comprising: a sensing chamber comprising an array of nanopore sensing elements for sensing a liquid; a sensing chamber inlet for allowing the liquid to enter the sensing chamber; a sensing chamber outlet for allowing the liquid to exit the sensing chamber; and a valve moveable between: a first position in which the valve allows the liquid to flow through the sensing chamber inlet and the liquid to flow through the sample chamber outlet; and a second position in which the valve inhibits the liquid from flowing through the sensing chamber inlet and the liquid from flowing through the sample chamber outlet.
  • a user of the flow cell only needs to operate one device (the valve) to inhibit flow through both the sensing chamber inlet and the sensing chamber outlet. This may make the flow cell simpler to use and/or reduce the likelihood of the user forgetting to inhibit flow through one of the sensing chamber inlet or the sensing chamber outlet when compared with a flow cell which has a respective separate valve for each of the inlet and the outlet.
  • the user may move the valve between the two positions to best suit the different stages of operation of the flow cell.
  • the user may place the valve in the first position when it is desirable for liquid to be able to enter and exit the sensing chamber, such as when adding a sample liquid to the sensing chamber. This may result in waste liquid already in the sensing chamber (such as a transport liquid) being displaced by the sample liquid and flowing out of the sensing chamber via the sensing chamber outlet.
  • the user may place the valve in the second position when it is desirable for liquid to be inhibited from flowing into or out of the sensing chamber, for example, after the sensing liquid has been added to the sensing chamber.
  • the nanopore array device is a sequencing instrument.
  • the flow cell comprises a waste collection channel located downstream of the sensing chamber outlet; the waste collection channel is fluidically connectable to the sensing chamber outlet to collect the liquid.
  • the liquid may be collected in a controlled manner in the collection chamber rather than leaking out of the flow cell, which may result in the liquid damaging other components of the flow cell.
  • a total volume of the waste collection channel is no less than a total volume of the sensing chamber. This may provide sufficient volume for all the liquid stored in the sensing chamber during transport to be displaced out of the sensing chamber and into the waste collection channel prior to operation of the flow cell. This may be desirable to enable two different liquids, one with properties suited for transportation, and one with properties suited for running an assay, to be used.
  • the total volume of the waste collection channel is no less than 1.5, 2, 2.5, 3, or 5 times greater than the total volume of the sensing chamber.
  • the total volume of the collection chamber is no less than 1ml.
  • the waste collection channel has a serpentine shape. This may make the waste collection channel more compact and may thereby provide a more compact flow cell, for a given length of waste collection channel. Additionally, the serpentine shape may make liquid collected in the waste collection channel easier to control than if the waste collection channel had a non-serpentine shape.
  • the valve comprises a transfer channel configured to fluidically connect the sensing chamber outlet to the waste collection channel; when the valve is in the first position, the transfer channel is fluidically connected to the sensing chamber outlet and the waste collection channel to allow the liquid to flow from the sensing chamber outlet to the waste collection channel; and when the valve is in the second position, the transfer channel is fluidically disconnected from at least one of the sensing chamber outlet and the waste collection channel in order to inhibit the liquid from flowing from the sensing chamber outlet to the waste collection channel.
  • a transfer channel which is fluidically connected/disconnected from at least one of the sensing chamber outlet and waste collection channel may provide a simple and robust means of inhibiting the flow of the liquid from the sensing chamber outlet to the waste collection channel. Additionally or alternatively, a transfer channel may provide a convenient means for providing the capability to redirect a liquid flow direction as oppose to merely blocking it.
  • the flow cell comprises a waste port for allowing the liquid to exit the flow cell; the waste port is located downstream of the sensing chamber outlet; and the waste port is fluidically connectable to the waste collection channel.
  • the valve By providing the valve moveable between the two positions, the user may place the valve in the second position when emptying the waste collection channel via the waste port to reduce the likelihood of the sensing chamber also being emptied when the waste collection channel is emptied.
  • Comprising a waste port may enable the user to empty the waste collection channel and reuse the flow cell, rather than disposing of the flow cell once the waste collection channel is full.
  • the waste port has a minimum width of no less than 1mm. This may be sufficient width to enable a large enough pipette to be inserted into the waste port to empty the waste collection channel in a single operation rather than using a smaller pipette, which may take multiple operations to empty the waste collection channel.
  • the valve when the valve is in the first position, the valve inhibits the liquid from flowing from the waste collection channel to the waste port; and when the valve is in the second position, the valve allows the liquid to flow from the waste collection channel to the waste port.
  • the waste collection channel may only be emptied via the waste port when liquid flow between the sensing chamber and the waste collection channel is inhibited. Thereby the likelihood of the user accidentally draining the sensing chamber when emptying the waste collection channel may be reduced.
  • the valve comprises a transport channel configured to fluidically connect the waste collection channel to the waste port; when the valve is in the first position, the transport channel is fluidically disconnected from at least one of the waste collection channel and the waste port to inhibit the liquid from flowing from the collection chamber the waste port; and when the valve is in the second position, the transport channel is fluidically connected to the waste collection channel and the waste port to allow the liquid to flow from the waste collection channel to the waste port.
  • a transport channel which is fluidically connected/disconnected from at least one of the waste collection channel and the waste port may provide a simple and robust means of inhibiting the flow of the.
  • the valve comprises a common channel configured to fluidically connect one of the sensing chamber outlet to the waste collection channel, and the waste collection channel to the waste port; when the valve is in the first position, the common channel is: fluidically connected to the sensing chamber outlet and the waste collection channel to allow the liquid to flow from the sensing chamber outlet to the waste collection channel; and fluidically disconnected from at least one of the waste collection channel and the waste port to inhibit the liquid from flowing from the collection chamber the waste port; and when the valve is in the second position, the common channel is: fluidically disconnected from at least one of the sensing chamber outlet and the waste collection channel in order to inhibit the liquid from flowing from the sensing chamber outlet to the waste collection channel; and fluidically connected to the waste collection channel and the waste port to allow the liquid to flow from the waste collection channel to the waste port.
  • the common channel performs the functions of the transport channel and the transfer channel is a single channel. This may provide a simpler design for the valve than if the transport channel and the transfer channel were separate channels.
  • the waste collection channel comprises: a waste collection channel inlet for allowing the liquid to enter the waste collection channel; and an air vent for allowing air to exit the waste collection channel.
  • the air vent may allow air located in the waste collection channel to exit the waste collection channel when the liquid is being collected in the waste collection channel. This may enable the waste collection channel to collect more liquid than if the air vent were omitted.
  • the waste collection channel comprises a fluid trap and a main portion located downstream of the fluid trap.
  • the fluid trap may inhibit liquid collected in the waste collection channel from exiting the main portion in the event that the flow cell is held vertically or moved around.
  • the fluid trap has a serpentine shape.
  • the waste collection channel inlet is located at an opposite end of the waste collection channel to the air vent. Due to the behaviour of the liquid at microfluidic scales, air may be unable to pass through or around the liquid entering the waste collection channel.
  • the air vent were located at the same end of the waste collection channel as the waste collection channel inlet, air located at the opposite end of the waste collection channel to the waste collection channel inlet may not be able to pass through or around the liquid and thereby may become trapped by the liquid. This may thereby inhibit the filling of the waste collection channel by the liquid. Therefore, locating the waste collection channel inlet at an opposite end of the waste collection channel to the air vent may enable the waste collection channel to collect more liquid than if the air vent were located at the same end of the waste collection channel as the waste collection channel inlet.
  • the waste collection channel inlet is for allowing the liquid to flow from the waste collection channel to the waste port.
  • the air vent may prevent a vacuum being created within the waste collection channel which may inhibit the removal of the liquid from the waste port.
  • the air vent has a non-circular cross section.
  • a pipette tip may not seal with the air vent, which may reduce the likelihood of the user accidentally attempting to draw liquid out of the air vent, which could result in liquid being inadvertently drawn out of the sample chamber and drying out the sample chamber.
  • the air vent has a square cross section.
  • the valve comprises an input port for allowing the liquid to be inserted directly into the sensing chamber inlet from outside the microfluid device.
  • the liquid can be inserted directly into the sensing chamber inlet, rather than having to travel via an additional channel intermediate the input port and the sensing chamber inlet. This may remove a requirement to, upon commencing use of the flow cell, draw back a liquid contained within the sensing chamber into the additional channel, which may be required to reduce the likelihood of air bubbles being introduced into the sensing chamber. Removing this additional drawing back stage may simplify the operation of the flow cell.
  • the input port has a minimum width of no less than 0.5mmmm. This may be sufficient width to enable a pipette to be inserted into the input port in order to insert the liquid.
  • the input port has a circular cross section. This may improve the seal between the pipette, which generally have a circular cross section, and the structure defining the input port. This may improve the ability of the pipette to insert the liquid without introducing air bubbles.
  • the input port has a conical shape. A conical shape may allow for a range of pipette tips to be accommodated in the input port.
  • the valve is configured to rotate between the first position and the second position.
  • Rotating (rather than, for example, sliding) may reduce the space required on the flow cell for the valve and thereby provide a more compact flow cell. Space for the valve on the flow cell may be limited, so providing a more compact valve design may be desirable. Additionally, rotation may also allow a simple lever to be used to provide a mechanical advantage.
  • the flow cell comprises: a main body defining a sensing chamber, the main body comprising a surface through which light can pass into the sensing chamber; and a light shield for inhibiting passage of light through the surface; the light shield is moveable relative to the sensing chamber to vary an amount of light that can reach the nanopore array; and the light shield is coupled to the valve such that movement of the valve causes movement of the light shield relative to the sensing chamber.
  • Providing a light shield may enable a user to vary the amount of light which can reaches the nanopore based on the different requirements of the stages of using the flow cell.
  • the nanopore array when loading a liquid into the sensing chamber, it may be desirable to allow light to reach the nanopore array so that the user can check if there are air bubbles present in the nanopore array. The presence of air bubbles may degrade the performance of the nanopore array.
  • Coupling the valve to the light shield may reduce the number of operations the user is required to perform and thereby make the flow cell easier to use, relative to the light shield and valve not being coupled to one another, because the user may only need to move the valve rather than having to move both the valve and the light shield during operation of the flow cell.
  • the light shield is coupled to the valve such that a movement of the valve from the closed position to the open position causes the light shield to move from the covered position to the uncovered position.
  • the light shield is coupled to the valve such that a movement of the valve from the open position to the closed position causes the light shield to move from the uncovered position to the covered position.
  • the light shield and the valve are separate components.
  • Providing the valve and light shield as separate components may enable greater design flexibility when compared to the light shield and valve being a single, integrally formed, component. This greater design flexibility may enable the light shield and the valve to be more easily accommodated within the space constraints on a flow cell, which may typically be tight.
  • one of the light shield and the valve comprises a projection; the other of the light shield and the valve comprises a slot configured to receive the projection and couple the light shield to the valve such that the movement of the valve causes the movement of the light shield relative to the sensing chamber to vary the amount of light which can reach the nanopore array.
  • a slot and a projection to couple the valve to the light shield may provide a simple to manufacture and robust mechanism for coupling the valve to the light shield.
  • one of the light shield and the valve is configured to move linearly; and the other of the light shield and the valve is configured to rotate.
  • the different rotational and linear motions of the light shield and the valve may be dictated by the space constraints and location of other components of the flow cell.
  • the slot and projection may provide a convenient means for coupling the light shield to the valve whilst accommodating the different rotational and linear motions.
  • the light shield is moveable between: an uncovered position in which a first amount of light can reach the array of nanopore sensing elements; and a covered position in which a second amount of light can reach the array of nanopore sensing elements; and the second amount of light is no greater than 50% of the first amount of light.
  • the performance of the array of nanopore sensing elements may improve as the amount of light which can reach the nanopore, when the light shield is in the covered position, decreases. Therefore, by the second amount of light being no greater than 50% of the first amount of light, the performance of the array of nanopore sensing elements may be improved to a greater extent than if the second amount of light were greater than 50% of the first amount of light.
  • the second amount of light is no greater than 40%, 30%. 20%, 10%, 5%, 1%, or 0.1% of the first amount of light.
  • no light can reach the array of nanopore sensing elements.
  • the light shield when the light shield is in the uncovered position, the light shield overlies no greater than 20% of a total surface area of the array of nanopore sensing elements when viewed in a horizontal plane. This may make air bubbles easier to detect than if the light shield overlies a greater amount of the array of nanopore sensing elements when in the uncovered position, and thereby improve the functionality of the flow cell.
  • the light shield when the light shield is in the uncovered position, the light shield overlies no greater than 10%, 5%, 1%, or 0.1% of the total surface area of the array of nanopore sensing elements when viewed in the horizontal plane.
  • the light shield when in the uncovered position, does not overlie any part of the array of nanopore sensing elements when viewed in the horizontal plane.
  • the horizontal plane is horizontal when the flow cell is placed on a horizontal surface in an intended use configuration.
  • the light shield when the light shield is in the covered position, the light shield overlies no less than 80% of a total surface area of the array of nanopore sensing elements when viewed in a horizontal plane.
  • the performance of the array of nanopore sensing elements may be improved to a greater extent than if the light shield overlies a lesser amount of the total surface area of the array of nanopore sensing elements.
  • the light shield when the light shield is in the covered position, the light shield overlies no less than 85%, 90%, 95% , 99%, or 99.9% of the total surface area of the array of nanopore sensing elements when viewed in the horizontal plane.
  • the light shield when the light shield is in the uncovered position, the light shield overlies all of the total surface area of the array of nanopore sensing elements when viewed in the horizontal plane.
  • the array of nanopore sensing elements has a total surface area measured in a horizontal plane; and the light shield has a total surface area measured in the horizontal plane which is no greater than 200% of the total surface area of the array of nanopore sensing elements.
  • a more compact light shield may be more easily integrated onto the flow cell, which are typically subject to tight space constraints, and may be easier for the user to use.
  • the valve is configured to rotate by no greater than 90° between the first position and the second position. This may make the valve easier to use because the user does not need to rotate the valve as much than if a greater rotation were required to move the valve between the two positions.
  • the valve is configured to rotate by not greater than 80°, 70°, 60°, 50°, or 45° between the first position and the second position.
  • the flow cell is configured such that the valve remains within the footprint of the flow cell during rotation between the first position and the second position. This may enable the valve to be operated without interfering with other structures surrounding the flow cell.
  • the nanopore array sensing device comprises a first stop and a second stop; the first stop and the second stop are configured to inhibit rotation of the valve by greater than 90° between the first position and the second position; when the valve is in the first position, the valve abuts the first stop; and when the valve is in the second position, the valve abuts the second stop. Abutting the valve with one of the stops provides physical feedback to the user that the valve is in the first position or the second position. This physical feedback may make the valve easier to use than if the stops were omitted and, for example, the user had to visually identify when the valve was in the desired position.
  • the valve is configured to rotate by no less than 10° between the first position and the second position. If the rotation is too small, it may be relatively easy for the user to accidentally rotate the valve into the incorrect position. Therefore, by providing a rotation of no less than 10° between the first position and the second position, the likelihood of the user accidentally rotating the valve into the incorrect position may be reduced compared to if a smaller rotation were provided.
  • the valve is configured to rotate by not less than 20°, 30°, 40°, or 45°, between the first position and the second position.
  • the sensing chamber comprises an upstream portion which is located between the sensing chamber inlet and the array of nanopore sensing elements; the upstream portion has a volume of no less than lOpL; and a width of the sensing chamber increases between the sensing chamber inlet and the array of nanopore sensing elements.
  • a “bubble trap” may be provided. If a bubble enters the sensing chamber via the sensing chamber inlet, it may stick to a wall which defines the sensing chamber due to surface tension. Due to the increasing width of the sensing chamber, the bubble may then travel along the wall until the width of the sensing chamber has increased sufficiently for a portion of the liquid inserted behind the bubble to flow around and bypass the bubble.
  • the portion of the liquid inserted behind the bubble may no longer be applying a force to the bubble to move the bubble towards the array of nanopore sensing elements, and the bubble may remain in its location and stop progressing towards the array of nanopore sensing elements.
  • This may be desirable because if a bubble were to impinge on the array of nanopore sensing elements, the bubble could inhibit the operation of the array of nanopore sensing elements or damage the array of nanopore sensing elements. Therefore, by providing the bubble trap, the flow cell may be able to accommodate bubbles being inserted into the sensing chamber inlet without the bubbles inhibiting the operation of or damaging the array of nanopore sensing elements.
  • the volume of the upstream portion is no greater than lOOpL.
  • the sensing chamber may thereby be more easily accommodated in the tight space constraints of a flow cell than if the volume of the upstream portion were greater.
  • the upstream portion may need to be filled with a sample liquid before the sample liquid can reach the array of nanopore sensing elements to be analysed.
  • the upstream portion having a volume of no greater than lOOpL may reduce the volume of sample liquid that is required to be prepared than if the volume of the upstream portion were greater than lOOpL.
  • the width of the sensing chamber measured at the array of nanopore sensing elements is no less than 2mm.
  • the ability of the flow cell to accommodate bubbles being inserted into the sensing chamber inlet without the bubbles inhibiting the operation of or damaging the array of nanopore sensing elements may be increased relative to if the width at the array of nanopore sensing elements were less than 2mm
  • width of the sensing chamber immediately adjacent to the sensing chamber inlet is no greater than 1.5 times a width of the sensing chamber inlet.
  • the width of the sensing chamber immediately adjacent to the sensing chamber inlet is no greater than 1.4, 1.3, 1.2, or 1.1 times the width of the sensing chamber inlet.
  • the width of the sensing chamber is measured in a horizontal plane, the horizontal plane being horizontal when the flow cell is in a normal operational orientation.
  • the benefits of the bubble trap may be achieved without increasing the vertical height of the sensing chamber. This may enable the bubble trap to be incorporated into flow cells with vertical space constraints.
  • the flow cell comprises: a nanopore sensing element configured to support a nanopore, the nanopore sensing element comprising a first electrode; and the flow cell comprises a second electrode; the nanopore sensing element is configured to interact with the second electrode to output an electrical signal at the first electrode that is dependent on an interaction of the liquid with the nanopore; the nanopore sensing element is located within the sensing chamber; and the second electrode is located further from the sensing chamber inlet than the nanopore sensing element.
  • the second electrode is located further from any air bubbles entering the sensing chamber from the sensing chamber inlet.
  • the operation of the second electrode may be less likely to be affected by air bubbles entering the sensing chamber via the sensing chamber inlet than if the second electrode were located closer to the sensing chamber inlet than the nanopore sensing element.
  • the flow cell comprises a plurality of nanopore sensing elements; each nanopore sensing element of the plurality of nanopore sensing element comprises a respective nanopore and a respective first electrode; and each nanopore sensing element of the plurality of nanopore sensing elements is configured to interact with the second electrode to output a respective electrical signal at their respective first electrode that is dependent on an interaction of the liquid with their respective nanopore.
  • a flow cell for use as part of a nanopore array device, the flow cell comprising: a sensing chamber comprising an array of nanopore sensing elements for sensing a liquid; a sensing chamber inlet for allowing the liquid to enter the sensing chamber; and an input port for allowing the liquid to be inserted directly into the sensing chamber inlet from outside the microfluid device, wherein: there is a minimum clearance of no less than 1mm between the sensing chamber inlet and the array of nanopore sensing elements; and a width of the sensing chamber increases between the sensing chamber inlet and the array of nanopore sensing elements.
  • a method comprising: providing a flow cell according to the first aspect of the present invention; and moving the valve between: a first position in which the valve allows the liquid to flow through the sensing chamber inlet and the liquid to flow through the sample chamber outlet; and a second position in which the valve inhibits the liquid from flowing through the sensing chamber inlet and the liquid from flowing through the sample chamber outlet.
  • the array of nanopore sensing elements comprises an array of nanopores provided in a corresponding array of membranes.
  • Each membrane may have a single nanopore inserted into it.
  • the nanopore may typically be a protein nanopore provided in an amphipathic membrane wherein the nanopore provides a channel through the membrane extending from one side to the other.
  • the membrane is typically supported on a support structure separating a cis chamber from a trans chamber.
  • An array of support structures for supporting the array of membranes may have a common cis chamber and an array of trans chambers.
  • the flow cell may be provided with an ionic liquid in the cis chamber covering the nanopores and ionic liquids in the trans chambers.
  • the ionic liquid in the cis chamber may be removed or displaced by a liquid containing the analyte to be sensed by the nanopore array.
  • the cis and trans chambers may contain respective electrodes and ionic flow through the membrane may be measured over time during translocation of the analyte through a nanopore under an applied potential difference across the nanopore.
  • the ionic liquid may comprise an alkali metal halide such as potassium or rubidium chloride.
  • the ionic liquid may further comprise a buffer such as HEPES or Tris-HCl buffer.
  • the pH may vary from 4.0 to 12.0 and is preferably about 7.5.
  • the analyte may for example comprise a polynucleotide, a polypeptide, a protein or a polysaccharide.
  • the polynucleotide may be chosen for example from DNA or RNA.
  • the polynucleotide may be made up of deoxyribonucleotide bases or ribonucleotide bases.
  • Nucleic acids may also include synthetic nucleic acids (XNA), such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA) and peptide nucleic acid (PNA).
  • HNA hexitol nucleic acid
  • CeNA cyclohexene nucleic acid
  • TAA threose nucleic acid
  • GNA glycerol nucleic acid
  • LNA locked
  • the translocation of the analyte through the nanopore may be controlled, for example by a binding protein.
  • Suitable polynucleotide binding proteins for controlling translocation of polynucleotides for use in the flow cell are known in the art and include polymerases, exonucleases, helicases and topoisomerases.
  • a preferred enzyme is a helicase which may be or be derived from a Hel308 helicase, a RecD helicase, such as Tral helicase or a TrwC helicase, a XPD helicase or a Dda helicase.
  • the helicase may be any of the helicases, modified helicases or helicase constructs disclosed in WO2013/057495, WO 2013/098562, WO2013098561, WO 2014/013259; WO 2014/013262 and WO 2014013260.
  • Binding proteins such as unfoldases are also known in the art and may be used for controlling the translocation of polypeptides through a nanopore.
  • the flow-cell may comprise, during use, the binding protein and/or the analyte.
  • the protein nanopore may be selected for example from a number of known pores such as those derived from P-barrel pores or a-helix bundle pores, such as a-hemolysin, anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin (Msp).
  • the transmembrane protein pore may be derived from derived from Spl or haemolytic protein fragaceatoxin C (FraC).
  • the pore may be derived from CsgG, examples of which are disclosed in WO 2016/034591.
  • the amphipathic membrane may be chosen for example from a lipid bilayer or polymer.
  • the polymer may be a di or triblock copolymer such as disclosed in WO2014064444 and US6723814.
  • Figure 1 is a perspective view of an example sequencing instrument
  • Figure 2 is a perspective view of a flow cell of the example sequencing instrument
  • Figure 3 is a perspective view of a bottom surface of the flow cell
  • Figure 4 is a perspective view of a main body of the flow cell
  • Figure 5 is a top down view of a sensing chamber, a connection channel, a waste collection channel, a waste channel, and a sensor of the flow cell;
  • Figure 6 is a magnified view of part of the top surface of the flow cell
  • Figure 7 is enlarged top down view of a contact surface of the top surface of the main body of the flow cell
  • Figure 8 is a sectioned view of the main body of the flow cell along a plane which is parallel to a vertical plane;
  • Figure 9 is an enlarged perspective view of a guide recess of the top surface of the main body of the flow cell
  • Figure 10 is a schematic diagram of the sensor
  • Figure 11 is a perspective view of a valve of the flow cell
  • Figure 12 is a bottom plan view of a bottom surface of the valve
  • Figure 13 is a perspective view of the valve
  • Figure 14 is a perspective view of a light shield of the flow cell
  • Figure 15 is a top down view of the light shield
  • Figure 16 is a perspective view of a base of the example sequencing instrument
  • Figure 17 is a top down view of the flow cell in a first configuration
  • Figure 18 is an enlarged sectioned view of the flow cell along a vertical plane
  • Figure 19 is a top down view of the flow cell with the valve located in a closed position and the light shield in a covered position;
  • Figure 20 is a sectioned view of the flow cell along a plane which is parallel to the vertical plane of the flow cell;
  • Figure 21 is a top down view of the flow cell with the valve located in an open position and the light shield in an uncovered position;
  • Figure 22 is a top down view of some of the components of the main body of the flow cell and a sectioned view of the valve, when the valve is in the open position;
  • Figure 23 is a top down view of some of the components of the main body of the flow cell and a sectioned view of the valve, when the valve is in the closed position;
  • Figure 25 is a flow chart showing steps of an example method for manufacturing the main body of the flow cell
  • Figure 26 is a perspective view of an upper part of the main body of the flow cell
  • Figure 29 is a flow chart showing steps of a second method.
  • Figure 30 is a flow chart showing steps of a third method.
  • Figure 1 shows a sequencing instrument 1 (which is an example of a nanopore array device) having a flow cell 3 and a base 5.
  • Figure 1 also shows the relative orientation between the sequencing instrument 1 and 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.
  • the flow cell 3 (shown in isolation in Figure 2 and Figure 3) has a main body 7, a sensor 9 (shown in Figure 5), an Application Specific Integrated Circuit (ASIC) 11 (shown in Figure 10), a common electrode 13 (shown in Figure 5), a bubble sensor 15 (shown in Figure 5), a valve 17, a light shield 19, and an electrical connector 21.
  • ASIC Application Specific Integrated Circuit
  • the main body 7 of the flow cell 3 (shown in isolation in Figure 4 and Figure 5) has a top surface 23, a bottom surface 25, a sensing chamber 27, a connection channel 29, a waste collection channel 31, and a waste channel 33.
  • the sensing chamber 27 and the channels 27,29,31,33 are shown in isolation along with the sensor 9, the bubble sensor 15, and the common electrode 13 in Figure 5.
  • the main body 7 of the flow cell 3 has a maximum width 35 of 28.3mm and a maximum length 37 of 93.5mm, measured in a plane parallel to the horizontal plane 8, and a maximum height 39 of 11.5mm, measured in a plane parallel to the vertical plane 10.
  • a maximum width 35 of no greater than 40mm, a maximum length 37 of no greater than 120mm, and a maximum height 39 of no greater than 30 is also envisaged.
  • the top surface 23 of the main body 7 of the flow cell 3 has a contact surface 41, a first overhang 43, a second overhang 45, a first stop 47, a second stop 49, a guide recess 51, a fin 53, and a clip recess 55.
  • the top surface 23 of the main body 7 of the flow cell 3 is formed from transparent Cyclic Olefin Copolymer (COC).
  • the contact surface 41 (enlarged views of which is shown in Figure 6 and Figure 7) is defined on the top surface 23 of the main body 7 of the flow cell 3.
  • the contact surface 41 has a periphery 57 which is circular in shape and a ridge 59 (which may be referred to as an additional projection).
  • the ridge 59 projects upwards from the contact surface 41 and is concentric with the periphery 57 of the contact surface 41.
  • the ridge 59 has an annular shape.
  • the ridge 59 is sized and dimensioned to be received within a valve recess 173 of the valve 17 (as will be discussed in more detail below).
  • the first and second overhangs 43,45 are integrally formed with the main body 7 of the flow cell 3, specifically by being injection moulded in a single operation.
  • the first overhang 43 has a vertical portion 65, a horizontal portion 67, and a mould split line 69.
  • the vertical portion 65 of the first overhang 43 projects upwards from the top surface 23 of the main body 7 of the flow cell 3.
  • the horizontal portion 67 of the first overhang 43 projects parallel to, and over, the contact surface 41 such that the horizontal portion 67 of the first overhang 43 overlies the contact surface 41.
  • a first recess 71 is defined between the vertical portion 65 of the first overhang 43, the horizontal portion 67 of the first overhang 43, and the contact surface 41.
  • the first recess 71 has a width 75, measured radially outwards from the periphery 57 of the contact surface 41 and in a plane which is parallel to the horizontal plane 8, of 1.1mm.
  • the first recess 71 is sized and dimensioned to receive a first projection 165 of the valve (which will be discussed below in more detail).
  • the first overhang 43 When viewed from the side (as shown in Figure 8), the first overhang 43 has an inverted L shape. When viewed from above (as shown in Figure 7), the first overhang 43 has an arcuate shape.
  • the vertical portion 65 of the second overhang 45 projects upwards from the top surface 23 of the main body 7 of the flow cell 3 on an opposite side of the contact surface 41 to the first overhang 43.
  • the second overhang 45 is otherwise identical to the first overhang 43 such that the second overhang 45 defines a second recess 77 which is identical to the first recess 71.
  • the second overhang 45 is identical to the first overhang 43 except that the second overhang 45 has a different length B to the length A of the first overhang 43 (discussed below).
  • the first gap 79 is located on an opposite side of the contact surface 41 to the second gap 81.
  • the first overhang 43 subtends a central angle 83 of 79.2°, measured in a plane which is parallel to the horizontal plane 8.
  • the second overhang 45 subtends a central angle 87 of 66.7°, measured in the plane which is parallel to the horizontal plane 8.
  • the first gap 79 subtends a central angle 85 of 112.8°.
  • the second gap 81 subtends a central angle 86 of 101.4°.
  • the first overhang 43 extends around the periphery 57 of the contact surface 41 for a length A of 15.4mm, such that the first recess 71 extends around the periphery 57 of the contact surface 41 for the length A of 15.4mm.
  • the second overhang 45 extends around the periphery 57 of the contact surface 41 for a length B of 13.3mm, such that the second recess 77 extends around the periphery 57 of the contact surface 41 for the length B of 13.3mm.
  • the mould split line 69 of the first overhang 43 i s located on a surface of the horizontal portion 67 of the first overhang 43 which faces away from the contact surface 41. Thereby, the mould split line 69 is spaced from a surface of the first overhang 43 which defines the first recess 71.
  • the mould split line 69 of the second overhang 45 is correspondingly located on a surface of the horizontal portion 67 of the second overhang 45 which faces away from the contact surface 41.
  • the first and second stops 47,49 are each cuboidal in shape.
  • the first stop 47 projects upwards from the top surface 23 of the main body 7 of the flow cell 3.
  • the first stop 47 has a first end 89 which is physically connected to the first overhang 43 and a second end 91 which is spaced along the top surface 23 of the main body 7 of the flow cell 3 from the contact surface 41.
  • the second stop 49 is identical to the first stop 47, except that the second stop 49 is physically connected to the second overhang 43.
  • the guide recess 51 (an enlarged view of which is shown in Figure 9) is defined by the top surface 23 of the main body 7 of the flow cell 3.
  • the guide recess 51 has a deep portion 91, a shallow portion 93, a base 95, and a wall 97.
  • the deep portion 91 has a greater depth than the depth of the shallow portion 93.
  • the deep portion 91 is adjacent to the shallow portion 93.
  • the deep portion 91 and the shallow portion 93 are each sized and dimensioned to receive a main body portion 201 of the light shield 19 (discussed below in more detail).
  • the deep portion 91 is vertically above the sensing chamber 27.
  • the shallow portion 93 is located towards a periphery of the top surface 23 of the main body 7 of the flow cell 3.
  • the wall 97 projects upwards from the base 95 of the guide recess 51.
  • the fin 53 projects upwards from the base 95 of the guide recess 51 into the guide recess 51.
  • the fin 53 extends in a width wise direction (parallel to the width axis 6) to span the entire guide recess 51.
  • the fin 53 is sized and dimensioned to be received within a groove 203 of the light shield 19 (discussed below in more detail).
  • the clip recess 55 (shown in the enlarged view of Figure 9) is located towards one end 99 of the top surface 23 of the main body 7 of the flow cell 3.
  • the clip recess 55 is sized to receive a clip 227 which is part of the base 5 (discussed in more detail below) to mechanically connect the flow cell 3 to the base 5.
  • the sensing chamber 27 comprising a sensing chamber inlet 101 and a sensing chamber outlet 103.
  • the sensing chamber 27 has a volume of 130pl.
  • the sensing chamber inlet 101 has a circular cross section and a minimum width 105, measured in a plane which is parallel to the horizontal plane, of 1.2mm.
  • the sensing chamber inlet 101 is located in the contact surface 41.
  • the sensing chamber inlet 101 is located at an opposite end of the sensing chamber 27 to the sensing chamber outlet 103.
  • a width 109 of the sensing chamber 27 immediately adjacent (measured within 1mm) to the sensing chamber inlet 101, measured parallel to the width axis 6, is 1.2mm, which is equal to the minimum width 105 of the sensing chamber inlet 101.
  • the width 109 of the sensing chamber 27 immediately adjacent to the sensing chamber inlet 101 being no greater than 1.5 times the minimum width 105 of the sensing chamber inlet 101 is also envisaged.
  • the width 111 of the portion of constant width 107, measured parallel to the width axis 6, is 8.4mm.
  • the width 111 of the portion of constant width 107 being no less than 2mm is also envisaged.
  • the portion of constant width 107 is located vertically below (in a direction parallel to the vertical axis 2) the deep portion 91.
  • the sensor 9 is located in the portion of constant width 107.
  • a volume of an upstream portion 102 of the sensing chamber 27 which is located between the sensing chamber inlet 101 and the sensor 9 is 34pL.
  • the upstream portion 102 have a volume of between lOpL and lOOpL is also envisaged.
  • connection channel 29 has a connection channel inlet 113 and a connection channel outlet 115.
  • the connection channel inlet 113 is located at an opposite end of the connection channel 29 to the connection channel outlet 115.
  • the connection channel inlet 113 is physically connected to the sensing chamber outlet 103 such that the sensing chamber 27 is fluidically connected to the connection channel 29.
  • the connection channel outlet 115 is located in the contact surface 41.
  • the waste collection channel 31 has a waste collection channel inlet 117, an air vent 119, a main portion 122 and a fluid trap 124.
  • the waste collection channel 31 has a volume of 2.1ml, which is approximately 16 times greater than the volume of the sensing chamber 27.
  • the waste collection channel 31 having other volumes, which are no less than the volume of the sensing chamber 27, is also envisaged.
  • the waste collection channel 31 has a serpentine shape.
  • the waste collection channel inlet 117 is located in the contact surface 41 and is spaced from the connection channel outlet 115 by a distance 114 of 1.5mm along the contact surface 41.
  • the waste collection channel inlet 117 is located at an opposite end of the waste collection channel 31 to the air vent 119.
  • the air vent 119 has a square cross section.
  • the air vent having a different, non-circular cross section is also envisaged.
  • the air vent 119 is located in the top surface 23 of the main body 7 of the flow cell 3 and is spaced from the contact surface 41.
  • the main portion 122 has a greater volume than the fluid trap 122.
  • the fluid trap 122 is located upstream of the main portion 122 and towards the waste collection channel inlet 117.
  • the fluid trap 122 has a serpentine shape.
  • the waste channel 33 has a waste channel inlet 121 and a waste port 123.
  • the waste port 123 has a circular cross section.
  • the waste channel inlet 121 is located in the contact surface 41.
  • the waste channel inlet 121 is spaced from the waste collection channel inlet 117 by a distance 125 of 1.4mm along the contact surface 41, which is equal to the distance 114 by which the waste collection channel inlet 117 is spaced from the connection channel outlet 115.
  • the waste channel inlet 121 is located on an opposite side of the waste collection channel inlet 117 to the connection channel outlet 115.
  • the waste channel inlet 121 is located at an opposite end of the waste channel 33 to the waste port 123.
  • the waste port 123 has a minimum width 127 of 1.2mm.
  • the waste port 123 having a minimum width 127 of no less than 1mm is also envisaged.
  • the waste port 123 is located in the top surface 23 of the main body 7 of the flow cell 3 and is spaced from the contact surface 41.
  • the sensor 9 (shown schematically in Figure 10) has a substrate 131, and an array of nanopore sensing elements 133.
  • the sensor 9 is located vertically 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 located in a bottom surface of the sensing chamber 27, in the portion of constant width 107
  • 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 horizontal plane 8, of 72mm 2 .
  • 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 27.
  • the ASIC 11 is electrically connected to the sensor 9 and the electrical connector 21.
  • the ASIC 11 is for controlling the sensor 9 and common electrode 13, and for processing signals output by the sensor 9 and providing the signals to the electrical connector 21.
  • the common electrode 13 is located in the connection channel 29 and is thereby downstream of the sensing chamber outlet 103 and the sensor 9 which has the array of nanopore sensing elements 133.
  • the common electrode 13 is for providing a common reference potential to each of the nanopore sensing elements 133.
  • the bubble sensor 15 is for detecting a bubble in the sensing chamber 27.
  • the bubble sensor 15 is an optical bubble sensor having a light-emitting diode and a phototransistor receiver located opposite one another. If a clear liquid passes between the light-emitting diode and the phototransistor receiver, a maximum current reading is detected, whereas, a dark liquid produces a minimum current reading. A bubble is detected, as it passes between the light-emitting diode and the phototransistor receiver, by a fluctuation in the output current signal, which is intermediate to that of the maximum and minimum current outputs.
  • Other types of bubble sensors are envisaged including ultrasonic bubble sensors.
  • the valve 17 (shown in isolation in Figures 11 to 13) has a hub 161, a valve handle 163, a first projection 165, a second projection 167, a top surface 162, and a bottom surface 164.
  • the valve 17 is formed from Polyurethane, which is resiliently deformable.
  • the hub 161, the valve handle 163, and the first and second projections 165,167 are integrally formed with one another, specifically by being injection moulded in a single operation.
  • the hub 161 has a common channel 169, an input port 171, and a valve recess 173.
  • the hub 161 is disc shaped such that a periphery 181 of the hub 161 is circular.
  • the common channel 169 has a common channel inlet 183 and a common channel outlet 185.
  • the common channel inlet 183 and the common channel outlet 185 are located on the bottom surface 162 of the valve 17.
  • the common channel outlet 185 is spaced from the common channel inlet 183 along the bottom surface 164 of the valve 17 by a distance 187 of 3mm, which is equal to the distance 114 between the connection channel outlet 115 and the waste collection channel inlet 117, and the distance 125 between the waste collection channel inlet 117 and the waste channel inlet 121.
  • the common channel 169 When viewed from below (as shown in Figure 12) the common channel 169 has an arcuate shape.
  • the input port 171 extends between the top surface 162 of the valve 17 and the bottom surface 164 of the valve 17.
  • the input port 171 has a conical shape.
  • the input port 171 has a circular cross section, a minimum width 189 of 1.8mm and a maximum width of 1.8mm.
  • the input port 171 having a minimum width 189 of no less than 0.5mm is also envisaged.
  • the valve recess 173 is defined in a bottom side of the hub 161 and has an annular shape.
  • the valve recess 173 is sized and dimensioned to receive the ridge 59.
  • the valve handle 163 has a slot 191 and is elongate in shape.
  • the valve handle 163 projects outwards from a portion of the periphery 181 of the hub 161 which is located between the first projection 165 and the second projection 167.
  • the slot 191 is defined in the bottom surface 164 of the valve 163 and is sized and dimensioned to receive a light shield projection 207 of the light shield 19 (discussed below in more detail).
  • the first projection 165 has an arcuate shape when viewed from below (as shown in Figure 12).
  • the first projection 165 projects horizontally outwards from the periphery 181 of the hub 161 and extends around the periphery 181 of the hub 161 for a length 193 of 16.9mm, which is 110% of the length A which the first and second overhangs 43,45 extend discontinuously around the periphery 57 of the contact surface 41.
  • the first projection 165 extending around the periphery 181 of the hub 161 for a length 193 of no less than 50% of the length A which the first and second overhangs 43,45 extend around the periphery 57 of the contact surface 41 is also envisaged.
  • the first projection 165 subtends a central angle 195, measured in a plane which is parallel to the horizontal plane 8, of 95°, which is less than the central angles 85 subtended by the first gap 79. Thereby, the first projection 165 is sized to fit through the first gap 79.
  • the first projection 165 is sized and dimensioned to be received within the first recess 71.
  • the first projection 165 has a width 197, measured in a plane which is parallel to the horizontal plane 8, of 0.7mm, which is 63% of the width 75 of the first recess 71.
  • the first projection 165 having a width 197 which is no less than 20% of the width 75 of the first recess 71 is also envisaged.
  • a lubricant 198 is located between the first overhang 43 and the first projection 165, and between the contact surface 41 and the first projection 165.
  • the second projection 167 is identical to the first projection 165, except that the second project projects from an opposite side of the periphery 181 of hub 161 to the first projection 165.
  • the light shield 19 (shown in isolation in Figure 14 and Figure 15) has a main body 201, a groove 203, a light shield handle 205, and a light shield projection 207.
  • the light shield 19 is formed from opaque Polycarbonate.
  • the light shield 19 has a total surface area 209, measured in a plane which is parallel to the horizontal plane 8, of 91mm 2 , which is 126% of the total surface area 153 of the array of nanopore sensing elements 133.
  • the light shield 19 having a total surface area 209 of no less than 100%, and no greater than 200% of the total surface area 153 of the array of nanopore sensing elements 133 is also envisaged.
  • the main body 201 of the light shield 19 has a top surface 208 and a bottom surface.
  • the main body 201 of the light shield 19 has a planar shape and is formed of opaque Polycarbonate.
  • the main body 201 of the light shield 19 has a length and a width which are sized to be received in either of the shallow portion 93, and the deep portion 91.
  • the groove 203 extends between the top surface 208 and the bottom surface of the main body 201 of the light shield 19 and extends along a whole width 211 of the main body 201 of the light shield 19 (extends parallel to the width axis 6) such that the groove 203 divides the main body 201 of the light shield 19 into first and second parts 213,215.
  • the groove 203 is sized and dimensioned to receive the fin 53.
  • the light shield handle 205 extends from the top surface 208 of the main body 201 of the light shield 19 and extends across the groove 203 to join the first and second parts 213,215 of the main body 201 of the light shield 19 together.
  • the light shield projection 207 projects upwards from the main body 201 of the light shield 19 and is located on an opposite side of the main body 201 of the light shield 19 to the light shield handle 205.
  • the light shield projection 207 is sized and dimensioned to be received within the slot 191 of the valve handle 163.
  • the electrical connector 21 is located on the bottom surface 25 of the main body 7 of the flow cell 3 and is for connecting to a corresponding electrical connector on the base 5 (discussed in more detail below) to electrically connect the flow cell 3 to the base 5.
  • the base 5 (shown in isolation in Figure 16) has a main body 221, an electrical connector 223, a processor 225, a clip 227, a lid 229, and a cable 231.
  • the main body 221 of the base 5 has a top surface 233.
  • the main body 221 of the base 5 has a cuboidal shape.
  • a base recess 235 is defined in the top surface 233 of the main body 221 of the base 5.
  • the base recess 235 is sized and dimensioned to receive the flow cell 3.
  • the electrical connector 223 is located in the base recess 235 and, as described previously, is for connecting to the electrical connector 21 on the flow cell 3 to electrically connect the flow cell 3 to the base 5.
  • the processor 225 is located within the main body 221 of the base 5 and is electrically connected to the electrical connector 223 of the base 5 such that signals received from the flow cell 3 via the electrical connectors 21,213 can be received and processed by the processor 225.
  • the clip 227 is located at one end of the base recess 235. As described above, the clip 227 is sized so that a portion of the clip 227 can be received within the clip recess 55 to mechanically connect the flow cell 3 to the base 5.
  • the lid 229 has a lid recess 237 and a pivot 239.
  • the lid recess 237 is sized and dimensioned to receive the flow cell 3.
  • the base recess 235 and the lid recess 237 are sized and dimensioned such that when the flow cell 3 is located in the base recess 235 and the lid recess 237, the base 5 completely encloses the flow cell 3.
  • the pivot 239 pivotably connects the lid 229 to the main body 221 of the base 5 such that the lid 229 can be opened and closed.
  • the cable 231 extends from the main body 221 of the base 5 and is electrically connected to the processor 225.
  • the cable 231 is for electrically connecting the processor 225 to an external computing device, such as a tablet computer, such that signals and power can be transferred between the external computing device and the processor 225.
  • valve 17 To connect the valve 17 to the main body 7 of the flow cell 3, the valve 17 is located above the top surface 23 of the main body 7 of the flow cell 3 with the first projection 165 vertically aligned with the first gap 79, the second projection 167 vertically aligned with the second gap 81 (as shown in Figure 17), and the ridge 59 vertically aligned with the valve recess 173.
  • the valve 17 may be referred to as being in a first configuration.
  • valve 17 is then moved down towards the top surface 23 of the main body 7 of the flow cell 3 such that the first projection 165 passes through the first gap 79 and the second projection 167 passes through the second gap 81.
  • the valve 17 is continued to be moved down until the bottom surface 164 of the valve 17 contacts the contact surface 41 and the ridge 59 is received within the valve recess 173.
  • a minimum clearance 241 shown in the enlarged section view of Figure 18, measured parallel to the vertical axis 2, between the ridge 59 and the portion of the bottom surface 164 of the valve 17 which defines the valve recess 173 is 0.15mm.
  • valve 17 is then rotated in a plane which is parallel to the horizontal plane 8 by approximately 90° in a clockwise direction, which moves the valve handle 163 towards the stops 47,49 (shown in Figure 19).
  • a minimum clearance 243 shown in Figure 20
  • a minimum clearance 243 measured in a plane parallel to the horizontal plane 8, between each of the first and second projections 165,167 and the surface of each overhang 43,45 defining their corresponding recess 71,77 is 0.1mm.
  • the contact surface 41 and the horizontal portion 67 of each of the first and second overhangs 43,45 block the movement of the projections 165,167 in a direction 245 parallel to the vertical axis 2, thereby connecting the valve 17 to the main body 7 of the flow cell.
  • the first and second projections 165,167 and the first and second overhangs 43,45 operate in a similar manner to a bayonet fitting.
  • valve handle 163 During the rotation of the valve 17, the valve handle 163 is bent upwards, in the direction 245 parallel to the vertical axis 2, away from the top surface 23 of the main body 7 of the flow cell 3 so that the valve handle 163 can pass over one of the first and second stops 47,49. If this was not performed, the valve handle 163 would abut one of the first and second stops 47,49 and prevent the valve 17 from being rotated further to complete the connection of the valve 17 to the main body 7 of the flow cell 3. Once the valve handle 163 has passed over the stop, the valve handle 163 is released and, due to the valve 17 being resiliently deformable, the valve handle 163 moves back towards the top surface 23 of the main body 7 of the flow cell 3.
  • valve 17 may be referred to as being in a second configuration.
  • the valve handle 163 which can be resiliently deformed over the first and second stops 47,49, the user can select when the first and second stops 47,49 inhibit or allow movement of the valve 17 between the first configuration (shown in Figure 17) and the second configuration (shown in Figure 19 and Figure 21).
  • the rotation of the valve 17 is constrained to be a maximum of approximately 45° between an open position (shown in Figure 19) and a closed position (shown in Figure 21) by the valve handle 163 abutting either one of the first and second stops 47,49 at either end of this 45° rotation. It is envisaged that the stops 47,49 may be differently located such that the rotation between the open position and the closed position is no greater than 90° and no less than 10 °. The first and second stops 47,49 prevent the valve handle 163 from projecting outside a footprint of the flow cell 3 during rotation between the open position and the closed position.
  • the lid 229 is opened, and the flow cell 3 is inserted into the base 5 and the clip 227 moved such that the portion of the clip 227 is received in the clip recess 55 to mechanically connect the flow cell 3 to the base 5.
  • the electrical connector 21 of the flow cell 3 overlies, and is thereby connected to, the electrical connector of the base 5 such that the flow cell 3 is electrically connected to the base 5.
  • a conditioning ionic liquid 242 (which may be a buffer liquid) is located within the sensing chamber 27 (which may be referred to as a cis chamber) and is in contact with the common chamber side 136 of the nanopore 143.
  • a trans ionic liquid 244 is located within each of the wells 135 and in contact with the well side 138 of each of the nanopores 143. The nanopores 143 separate the trans ionic liquid 244 from the conditioning ionic liquid 242.
  • the user puts the valve 17 into the open position (shown in Figure 21 and Figure 22) by moving the valve handle 163 such that it abuts the second stop 49.
  • the input port 171 is vertically aligned with the sensing chamber inlet 101.
  • the common channel 169 is positioned such that the common channel inlet 183 is connected to the connection channel outlet 115, and the common channel outlet 185 is connected to the waste collection channel inlet 117.
  • the valve 17 blocks the waste channel inlet 121 to fluidically disconnect the waste channel 33 from the waste collection channel 31.
  • the light shield projection 207 is received in the slot 191 of the valve handle 163, and the valve handle 163 abuts the light shield handle 205.
  • the main body 201 of the light shield 19 is received in the shallow portion 93 such that no part of the main body 201 of the light shield 19 is located in the deep portion 91.
  • the light shield 19 does not overlie any part of the array of nanopore sensing elements 133, so the light shield 19 does not block any of the light from outside the flow cell 3 from reaching the array of nanopore sensing elements 133.
  • the light shield 19 is in an uncovered position, in that the array of nanopore sensing elements 133 is not covered by the light shield 19.
  • the fin 53 is located in the groove 203 in the light shield 19.
  • the user inserts a sample liquid containing molecular entities (which may be referred to as an analyte) which are to be sensed directly into the sensing chamber inlet 101 via the input port 171.
  • the sample liquid then passes through the sensing chamber inlet 101 into the sensing chamber 27 and travels to the senor 9.
  • the insertion of the sample liquid causes the conditioning ionic liquid 242 (which is then referred to as a waste liquid) to be displaced out of the sensing chamber 27 and into the connection channel 29.
  • the waste liquid flows through the connection channel 29, through the common channel 169, and into the waste collection channel 31, where it is collected.
  • air already present in the waste collection channel 31 is displaced and exits the waste collection channel 31 via the air vent 119.
  • the input port 171 When the valve 17 is in the closed position, the input port 171 is vertically misaligned with the sensing chamber inlet 101 and the hub 161 blocks the sensing chamber inlet 101.
  • the common channel 169 is positioned such that the common channel inlet 183 is connected to the waste collection channel inlet 117, and the common channel outlet 185 is connected to the waste channel inlet 121.
  • the valve 17 blocks the connection channel outlet 115 such that the waste collection channel 31 is fluidically disconnected from the connection channel 29.
  • the valve handle 163 is coupled to the light shield 19 such that as the valve handle 163 moves into the closed position, the light shield 19 also moves.
  • the main body 201 of the light shield 19 slides linearly in a width direction 251 parallel to the width axis 6 until the main body 201 of the light shield 19 is located in the deep portion 91.
  • sliding of the light shield 19 along a length direction 253 parallel to the length axis 4 is restricted by the interaction of the fin 53 and the groove 203, and the main body 201 of the light shield 19 and the top surface 23 of the main body 7 of the flow cell 3 which defines the guide recess 51.
  • the fin 53 and the groove 203, and the main body 201 of the light shield 19 and the top surface 23 of the main body 7 of the flow cell 3 which defines the guide recess 51 may be considered as a guide which guides the sliding of the light shield 19.
  • the main body 201 of the light shield 19 completely overlies the total surface area 153 of the array of nanopore sensing elements 133 when viewed in a plane which is parallel to the horizontal plane 8 such that substantially no light from outside the flow cell 3 can reach the array of nanopore sensing elements 133.
  • the light shield 19 is in a covered position.
  • the ASIC 11 controls the common electrode 13 and the sensor electrodes 141 such that an electrical potential is created between the common electrode 13 and the sensor electrodes 141. This electrical potential causes the molecular entities to translocate trough the nanopores 143 and an ion current to flow through the nanopores 143.
  • 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 the sensor 9 to the ASIC 11, which processes the signals.
  • the signals are then sent to the processor 225 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.
  • the user can empty the waste collection channel 31 by inserting a pipette into the waste port 123, and applying suction using the pipette which draws the waste liquid out of the waste collection channel 31, through the common channel 169, through the waste channel 33, and out of the waste port 123.
  • the user can close the lid 229 to completely enclose the flow cell 3 within the base 5.
  • the user can then move the valve 17 to the open position to start another assay. This is performed by rotating the valve 17 by 45° in a clockwise direction until the valve handle 163 abuts the first stop 47. During this rotation, the valve handle 163 will also abuts the light shield handle 205 and thereby couple the valve handle 163 to the light shield handle 205 such that as the valve handle 163 moves into the open position, the light shield 19 also slides.
  • the main body 201 of the light shield 19 slides linearly along the direction 251 which is parallel to the width axis 6 of the flow cell 3 until the main body 201 of the light shield 19 is located in the shallow portion 93.
  • the light shield 19 is formed from an opaque material.
  • An alternative sequencing instrument comprises an alternative light shield and an alternative base. The other features of the alternative sequencing instrument are identical to those of the sequencing instrument 1 described above.
  • a main body of the alternative light shield has a light shield polariser 303 instead of being formed from an opaque material.
  • the light shield polariser 303 has a polarisation axis 305.
  • the main body of the alternative light shield remains stationary in the deep portion 91 during operation rather than moving between the deep portion 91 and the shallow portion 93. Thereby, the light shield polariser 303 remains fixed in a position which overlies the total surface area 153 of the array of nanopore sensing elements 133 when viewed in a plane which is parallel to the horizontal plane 8.
  • a lid of the alternative base has a lid polariser 307.
  • the lid polariser 307 has a polarisation axis 309.
  • the lid polariser 307 is vertically above the light shield polariser 303 such that light which passes through the lid polariser 307 is attenuated by the light shield polariser 303.
  • the polarisation axis 309 of the lid polariser 307 and the polarisation axis 305 of the light shield polariser 303 are angularly offset from each other when viewed in two parallel reference planes 311,313 (shown in Figure 24). Specifically, the polarisation axes 305,309 are orthogonal to one another. Thereby, the light shield polariser 303 and the lid polariser 307 are crossed such that substantially all of the light is attenuated. Thereby, the user can open and close the lid of the alternative base, which causes the lid polariser 307 to move relative to the light shield polariser 303, to vary the amount of light which can reach the array of nanopore sensing elements 133.
  • the light shield polariser 303 and the lid polariser 307 could be a fixed distance from one another, and the polarisation axes 305,309 moved relative to one another to vary the amount of light which can reach the array of nanopore sensing elements 133.
  • the valve 17 has a single common channel 169.
  • the single common channel 169 may be replaced by two separate channels: a transfer channel and a transport channel.
  • the transfer channel is fluidically connected to the connection channel outlet 115 and the waste collection channel inlet 117 when the valve is in the open position.
  • the transfer channel is fluidically disconnected from the connection channel inlet 113 and the waste collection channel inlet 117 when the valve is in the closed position.
  • the transport channel is fluidically connected to the waste collection channel inlet 117 and the waste channel inlet 121 when the valve is in the closed position.
  • the transport channel is fluidically disconnected from the waste collection channel inlet 117 and the waste channel inlet 121 when the valve is in the closed position.
  • the light shield when the valve 17 is moved from the closed position to the open position, the light shield is coupled to the valve 17 by the valve handle 163 abutting the light shield handle 205.
  • the light shield is coupled to the valve 17 during the movement from the closed to the open position by the light shield projection 207 being received in the slot 191 of the valve handle 163.
  • the light shield handle 205 can be omitted.
  • the light shield 19 and the valve 17 are separate components.
  • the light shield 19 may be integrally formed with the valve 17 such that the light shield 19 and the valve 17 are not separate components.
  • the light shield 19 has a light shield projection 207
  • the valve handle 163 has a slot 191 which receives the light shield projection 207 to couple the valve 17 to the light shield.
  • the slot 191 may be provided in the light shield 19, and the projection may be provided on the valve 17.
  • the light shield 19 moves linearly in a direction 251 which is a parallel to the width axis 6, and the valve 17 rotates. In other examples, the light shield 19rotates and/or the valve 17 moves linearly.
  • the light shield 19 when in the uncovered position, does not overlie any part of the array of nanopore sensing elements 133, so the light shield 19 does not block any of the light from outside the flow cell 3 from reaching the array of nanopore sensing elements 133. In other examples, when in the uncovered position, the light shield 19 overlies some of the total surface area 153 of the array of nanopore sensing elements 133, such as no greater than 20% of the total surface area 153 of the array of nanopore sensing elements 133 when viewed in a plane which is parallel to the horizontal plane 8.
  • the light shield 19 when in the covered position, completely overlies the total surface area 153 of the array of nanopore sensing elements 133 when viewed in a plane which is parallel to the horizontal plane 8 such that substantially no light from outside the flow cell 3 can reach the array of nanopore sensing elements 133. In other examples, when in the covered position, the light shield 19 overlies less than 100% of the total surface area 153 of the array of nanopore sensing elements 133, such as no less than 80% of the total surface area 153 of the array of nanopore sensing elements 133 when viewed in a plane which is parallel to the horizontal plane 8.
  • the sequencing instrument 1 has a single flow cell 3. In other examples, the sequencing instrument 1 has multiple flow cells 3.
  • the valve 17 has first and second projections 165,167, and the main body 7 of the flow cell 3 has first and second overhangs 43,45.
  • the valve 17 has a different number of projections 165,167, such as one projection 165, and the main body 7 of the flow cell 3 has an equal number of overhangs 43,45, such as one.
  • Figure 25 shows a flow chart which details a method 501 of manufacturing the main body 7 of the flow cell 3.
  • the main body 7 of the flow cell 3 is manufactured from two parts: an upper part 601 and a lower part 603 (shown in Figure 26 and Figure 27).
  • the upper part 601 defines an upper portion of the sensing chamber 27, the connection channel 29, the waste collection channel 31, and the waste channel 33.
  • the upper part 601 has a joining surface 605, which is the surface of the upper part 601 intended to abut and join with a corresponding joining surface of the lower part 603.
  • the joining surface 605 of the upper part 601 extends around a periphery of the upper part and the peripheries of the upper portions of the sensing chamber 27, the connection channel 29, the waste collection channel 31, and the waste channel 33.
  • the lower part 603 of the main body 7 defines a lower portion of the sensing chamber 27, the connection channel 29, the waste collection channel 31, and the waste channel 33.
  • the lower part 603 of the main body 7 has a joining surface 607, which is the surface of the lower part 603 of the main body 7 intended to abut and join with the joining surface 605 of the upper part 601 of the main body 7.
  • the joining surface 607 of the lower part 603 extends around a periphery of the lower part 603 and the peripheries of the lower portions of the sensing chamber 27, the connection channel 29, the waste collection channel 31, and the waste channel 33.
  • the joining surface 607 of the lower part 603 comprises a bead 611 which projects upwards from the joining surface 607 of the lower part 603.
  • the entire lower part 603 is injection moulded in a single operation such that the bead 611 is integrally formed with the joining surface 607 of the lower part 603.
  • the method commences with providing 503 the upper part 601 and the lower part 603.
  • the upper part 601 is placed on the lower part 603 such that the joining surface 607 of the lower part 603 abuts 505 the joining surface 605 of the upper part 601.
  • the bead 611 is melted 507 using a laser to weld the joining surface 607 of the lower part 603 to the joining surface 605 of the upper part 601.
  • the laser is aimed through the upper part 601, which is formed of a transparent material.
  • Figure 28 shows a flow chart which details a first method 701. Firstly, a flow cell for use as part of a nanopore array device is provided 713.
  • the flow cell is the same as the flow cell 3 described above.
  • the valve is moved 705 between: a first position in which the valve allows the liquid to flow through the sensing chamber inlet and the liquid to flow through the sample chamber outlet; and a second position in which the valve inhibits the liquid from flowing through the sensing chamber inlet and the liquid from flowing through the sample chamber outlet.
  • Figure 29 shows a flow chart which details a second method 711. Firstly, a flow cell for use as part of a nanopore array device is provided 713. The flow cell is the same as the flow cell 3 described above.
  • the flow cell has a main body defining a sensing chamber, the main body comprising a surface through which light can pass into the sensing chamber, an array of nanopore sensing elements located within the sensing chamber; and a light shield for inhibiting passage of light through the surface.
  • the light shield is moved 715 relative to the sensing chamber to vary an amount of light that can reach the array of nanopore sensing elements.
  • Figure 30 shows a flow chart which details a third method 801.
  • a flow cell for use as part of a nanopore array device is provided 713.
  • the flow cell is the same as the flow cell 3 described above.
  • the valve is connected 805 to the main body by aligning 807 the projection with the gap; with the projection aligned with the gap, moving 809 the projection through the gap, and moving 811 the valve such that the projection is aligned with the overhang and received within the recess.
  • the valve When the valve is connected to the main body, the valve is moved 813 between an open position and a closed position. When the valve is in the open position, the valve allows at least one of the sample liquid to enter the sensing chamber inlet and the waste liquid to exit the sensing chamber outlet. When the valve is in the closed position, the valve inhibits at least one of the sample liquid from entering the sensing chamber inlet and the waste liquid from exiting the sensing chamber outlet.

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Abstract

Cuve à circulation destinée à être utilisée en tant que partie d'un dispositif à réseau de nanopores, la cuve à circulation comprenant une chambre de détection avec un réseau d'éléments de détection à nanopores pour détecter un liquide, une entrée de chambre de détection pour permettre au liquide d'entrer dans la chambre de détection, une sortie de chambre de détection pour permettre au liquide de sortir de la chambre de détection, et une soupape. La soupape est mobile entre une première position dans laquelle la soupape permet au liquide de s'écouler à travers l'entrée de chambre de détection et le liquide à s'écouler à travers la sortie de chambre d'échantillon et une seconde position dans laquelle la soupape empêche le liquide de s'écouler à travers l'entrée de chambre de détection et le liquide de s'écouler à travers la sortie de chambre d'échantillon.
PCT/GB2025/051068 2024-05-17 2025-05-16 Cuve à circulation à nanopores et procédé Pending WO2025238376A1 (fr)

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GBGB2407099.7A GB202407099D0 (en) 2024-05-17 2024-05-17 A flow cell and a method
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