WO2021111133A2

WO2021111133A2 - Microfluidic device for preparing and analysing a test liquid

Info

Publication number: WO2021111133A2
Application number: PCT/GB2020/053096
Authority: WO
Inventors: Clive Gavin Brown; Pedro Miguel Ortiz Bahamon; Rebecca Victoria BOWEN
Original assignee: Oxford Nanopore Technologies Limited
Priority date: 2019-12-05
Filing date: 2020-12-03
Publication date: 2021-06-10
Also published as: WO2021111133A3; GB201917832D0

Abstract

The invention resides in a microfluidic device for analysing a test liquid. The device has a preparation portion between an inlet and an upstream portion of the device, wherein said upstream portion is configured with a sensing chamber for housing a well array and for receiving via the inlet a test liquid to be sensed, and said preparation portion is configured having: a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion; and/or an actuator, operably configured to induce a test liquid from the inlet towards the upstream portion. Between the inlet and respective outlet lies a sole channel. The channel can extend parallel to the substrate, preferably along its entire length. The preparation portion can be configured in a first part, and the upstream portion can be configured in a second part, wherein said first part and second part are connectable such that a sample received and processed in the first part can be passed to the second part for analysis.

Description

MICROFLUIDIC DEVICE FOR PREPARING AND ANALYSING A TEST LIQUID

FIELD OF THE DISCLOSURE

The present invention relates to a microfluidic device, in particular a device comprising a sensor for sensing in wet conditions.

BACKGROUND

A variety of microfluidic devices and sensors are known. Sensors such as disclosed by W01999/013101 and WO 1988/008534 are provided in the dry state and a liquid test liquid applied to the device is transported to the sensor region within the device by capillary flow. Other types of sensors are known, such as ion selective sensors comprising an ion selective membrane.

Wet sensors are known from W02018/007819 and WO2019/106345, and an analysis apparatus incorporating means to provide amphiphilic membranes and nanopores to a sensor is disclosed by WO2012/042226, while a typical nanopore device provided in a ‘ready to use’ state comprises an array of amphiphilic membranes, each membrane comprising a nanopore and being provided across a well containing a liquid, as disclosed by WO2014/064443: each of these disclosures are hereby incorporated in their entirety by reference. Sample preparation technologies are known from papers including: “A Review on Macroscale and Microscale Cell Lysis Methods” by Mohammed Shehadul Islam et al - Micromachines 2017, 8, 83; doi:10.3390/mi8030083 - published 8 March 2017; “Stable and Simple Immobilization of Proteinase K Inside Glass Tubes and Microfluidic Channels”, by Andreas Kiichler - ACS Publication. ACS Appl. Matter. Interfaces 2015, 7, 25970-25980 - published 4 November 2015; “Electrical Lysis: Dynamics Revisited and Advances in On-chip Operation” by Bashir I. Morshed et al - Biomedical Engineering, Volume 41, Number 1, 2013:37-50; “DEP-on-a-Chip: Dielectrophoresis Applied to Microfluidic Platforms” by Haoqing Zhang et al - Micromachines 2019, 10, 423; doi:10.3390/mil0060423 - published 24 June 2019.Examples of products incorporating such means include the MinlON (RTM) and Flongle (RTM) available from Oxford Nanopore Technologies Ltd. Whilst useful, products associated with such disclosures are complex devices, relatively expensive and require a degree of end user skill and experience to configure and operate. Not only is it helpful to provide a device to the user in a ‘ready to use’ state, a lower cost solution and simpler analysis process involving such products is desired. In view of the forgoing, there remains a challenge to provide an easy to use microfluidic device that can be disposable or reusable, whilst supplied in a manner that is ready to use and simpler to operate. The present invention aims to at least partly reduce or overcome the problems discussed above.

SUMMARY

The invention generally resides in a sensing device having an integral preparation portion. The preparation portion can take an unprocessed test liquid, such as a bodily fluid, and prepare it for measurement. The preferred measurement method uses an array of nanopores that receive polynucleotides, proteins and the like, as described herein, that are derived from the test liquid and passes these components through nanopores supported on an array of nanopores. Reagent ports having, or configured to receive reagents, are provided in the preparation portion. Actuators, such as an electrowetting on dielectric (EWOD) interface can be provided to manipulate and process the test liquid. The actuator can be an electrowetting valve which can act as a fluid gate to permit or stop the flow of liquid. An example of a micromachined electrowetting microfluidic valve suitable for use in the invention is disclosed by US patent 8,037,903, hereby incorporated by reference in its entirety. According to an aspect of the invention, there is provided a microfluidic device for analysing a test liquid, said device comprising a body having a substrate and a cover, said cover connected to at least one side of the substrate. The body can be substantially planar. The substrate can be a printed circuit, such as a PCB. The cover can cover one or both sides of the body.

The substrate is configured having: a well array for supporting nanopores for the analysis of a portion of a test liquid; a microprocessor for processing signals derived from passing a portion of test liquid through a nanopore and sending said signals to an external controller for analysis. The functionality and, therefore, the cost, can be reduced by limiting the on-board processing of the device to managing the configuration of the array of nanopores for testing and deriving signals therefrom for transmission to a separate controller or analyser. The well array functions as a nanopore sensor.

The well array can be added on to the substrate, said well array being formable using moulded polymers, photolithography techniques are laser-machined plastics. Although the device herein is configured to a low cost and disposable device it is possible that the cover can be removed, and the well array replaced such that the device can be recycled by the manufacturer.

The cover and/or the substrate are configured with walls defining a channel, the channel having a fluid path between an inlet and an outlet of the device. The cover can be configured as a single piece. At least a portion of the fluid path can be entirely defined within the cover. At least a portion of the fluid path can be entirely defined in the cover with the substrate defining a closing surface. Alternatively, at least a portion of the fluid path can be defined by the substrate with the cover enclosing the path.

The fluid path has a preparation portion configured between the inlet and an upstream portion, said preparation portion being configured to receive and/or treat the test liquid; the upstream portion having a sensing chamber for housing the well array and for receiving via the inlet at least a portion of the liquid to be sensed, said upstream portion comprising a connection to a downstream portion that is positioned between the upstream portion and the outlet, said downstream portion for receiving liquid from the outlet channel of the upstream portion. The liquid can be sensed by the device using the well array sensor in the sensing chamber and the device can communicate the information detected to an off-device controller for subsequent analysis.

The fluid path can be configured such that the well array remains in a wet condition. The upstream portion can be filled with a liquid. The fluid path from inlet to outlet can be filled with a liquid.

A removably attachable seal can be configured to enclose the upstream portion and, when a liquid is provided in the upstream portion, inhibit flow of the liquid before removal of the seal, and after removal of the seal, permit liquid to pass from the from the upstream portion to the downstream portion. The fluid in the upstream portion can be balanced with fluid in the downstream portion such that a portion of a test liquid that comes in to contact with the upstream portion, such as after being passed through the preparation portion, it is drawn in to the sensing chamber for measurement by the well array.

A bridgeable barrier can be configured between the upstream portion and the downstream portion. The bridgeable barrier can be manually activatable to permit fluid flow from the upstream portion to the downstream portion. The bridgeable barrier can be closed with a seal. The bridgeable barrier can be configured as a fluidic switch, or valve, that conditionally lets fluid pass.

A bridge can be provided adjacent the bridgeable barrier. After removal of a seal the bridge can facilitate liquid to flow from the upstream portion to the downstream portion via or over the barrier. The seal can be removably attachable. The seal can be configured to inhibit liquid to flow from the inlet portion to the outlet portion.

The preparation portion is configured having: a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion. Additionally, or alternatively, the device can be configured with an actuator, operably configured to induce at least a portion of the test liquid into contact with the upstream portion such that the test liquid can be sensed.

The preparation portion can be an elongate channel forming part of the fluid path between the inlet and the upstream portion. The preparation portion can be formed in a linear fashion extending directly from the inlet to the upstream portion. Components of the preparation portion, such as a reagent port and/or actuator can require more space and/or isolation and, therefore, the fluid path of the preparation portion can be non-linear. The fluid path of the preparation portion can have a portion having at least one of a curved shaped, sawtooth shape or castellated formation. The preparation portion can extend in a planar direction. The fluid path, however, can extend out of plane and/or in a more than one plane to increase the path length and/or accommodate additional components.

A gate can be configured between the preparation portion and the upstream portion, said gate configured to controllably connect said preparation portion and said upstream portion. The gate can be a bridgeable barrier, fluidic switch, or valve.

The reagent port can have a sealable aperture for receiving a reagent. The reagent port can have a sealed interface with the fluid path. The reagent can be at least one of: a liquid deposited in the reagent port; a dried (lyophilised) reagent deposited in the reagent port; or a peg having a dried reagent thereon, said peg inserted in to the reagent port. The reagent can be releasable in to the preparation portion or activatable. Activation can be by way of manual intervention, such as by applying a force or breaking or removing a seal. US20160167047A1 hereby incorporated by reference in its entirety, discloses a microfluidic flow cell with a dry substance arranged in a cavity inside the flow cell for interaction with a fluid in the cavity. The actuator can be a manually operable pump. The pump can be configured to include a resilient flexible portion of the cover that, when moved or otherwise pulsed, causes a liquid in the fluid path to be displaced.

The actuator can be electro-mechanical. The actuator can be an electrowetting interface configured to induce movement of the test liquid or droplets of the test liquid. The electrowetting interface can induce a test liquid to travel along the length of the fluid path within the preparation portion. The actuator can be an electrowetting interface in the form of an array, such that droplets can be formed and manoeuvred. The actuator can be configured to at least one of split a droplet, lyse a cell or cause the contents of a cell to mix using dielectrophoresis. The actuator can have an electrowetting interface extending from the inlet to the upstream portion.

The or each reagent port can be configured with a corresponding electrowetting interface for manipulating a test liquid and cause interaction with a reagent provided in a reagent port.

According to another aspect, there is provided a microfluidic device for analysing a test liquid, said device comprising: a preparation portion as herein described. The preparation portion can reside between an inlet and an upstream portion of the device, wherein said upstream portion is configured with a sensing chamber for housing a well array and for receiving via the inlet a test liquid to be sensed, and said preparation portion is configured having: a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion; and/or an actuator, operably configured to induce a test liquid from the inlet towards the upstream portion.

According to another aspect, there is provided a system for analysing a test liquid, said system comprising: a microfluidic device as described and/or claimed; a controller configured to connect with the microfluidic device for operating the device and receiving and analysing signals derived from analysis of a test liquid processed by the microfluidic device. The system can include a dongle having one or more slots, the or each slot configured for receiving a microfluidic device, said dongle connectable to the controller.

The invention extends to methods of configuring and operating the devices herein. By way of example, after depositing a test liquid in the inlet, reagents can act upon the liquid before it is passed to the sensing chamber for measurement by the well array sensor. The test liquid can be at least one of blood, urine, saliva. After depositing the sample, it can be processed by being agitated and/or draw into the preparation portion. A preparation portion can be controlled by an external controller to feed the test liquid towards the upstream portion. When the test liquid has been prepared, a gate can be opened to allow the prepared test liquid into the sensing chamber. This can be complemented by a bridgeable barrier or other such control mechanism located between the upstream portion and downstream portion, which influences the balance of fluid in these chambers to induce fluid flow into the sensing chamber. Control of the fluid can be managed remotely via a controller operating actuator and/or by manual operation using the removal of seals and/or rotation of switches or valves.

Overall, the flowcell can be implemented with minimal functionality such that an ultra-low power ASIC can be used e.g. no on-board control electronics or analysis functionality is provided.

In another example there is provided a microfluidic device for preparing a test liquid for sensing of an analyte present therein, said device comprising a body having a substrate and a cover, said cover connected to at least one side of the substrate, wherein the cover and/or the substrate are configured with walls to define a channel between an inlet and an outlet.

The channel can define a sole fluid path between the inlet and the outlet. The channel can be branchless. The channel can have a sensing chamber having a well array for receiving least a portion of the prepared test liquid to be sensed. The test liquid is sensed through measurement. The device can have a plurality of channels and/or sensing chambers. The or each channel and/or sensing chamber can lie in a different plane within the device.

The substrate can be substantially planar. The channel can extend parallel to the substrate. The channel can extend parallel to the substrate along its entire length. The substrate can be a printed circuit on board, such as a PCB. The substrate can be a paper-based, such as cardboard.

The device can further comprise a sample section for receiving and preparing the test liquid configured in an upstream portion configured between the inlet and the sensing chamber. A plurality of upstream portions can connect to a single sensing chamber. The sensing chamber can comprise an outlet to a downstream portion for receiving liquid from the outlet of the sensing chamber, said downstream portion configured between the sensing chamber and the outlet.

The device can further comprise a well array for supporting nanopores for sensing the analyte. The device can be configured with an actuator, operably configured to induce at least a portion of the test liquid in the upstream portion to move towards the sensing chamber, such that the test liquid can be sensed. A plurality of actuators can be provided to at least one of: induce movement of a liquid to be sensed or measured towards the sensing chamber; concentrate the contents of the liquid to be sensed; release a sample preparation fluid to process the liquid to be sensed; agitate the liquid to be sensed.

The device can further include a microprocessor for (i) operating the or each actuator to move the sample along the channel, and/or (ii) process signals derived from the sensing of the analyte by the nanopores and sending signals to an external controller for analysis.

The preparation portion can be configured having an in-line treatment stage, said stage configured to transform the analyte or a derivative thereof for further treatment or sensing. An in line treatment stage can be provided with a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion.

The or each in-line treatment stage can be provided with an actuator. An actuator can be provided between the or each in-line treatment stage. An actuator can be at least one of: a mechanical finger-operable blister-pump; a pair of electrodes configured to induce electro-wetting movement; a pair of electrodes configured to induce dielectrophoresis; a pair of electrodes configured to induce electroporation; configured to induce peristaltic waves in the channel to move an analyte to be sensed from the inlet towards the outlet.

The channel can include at a stage that is at least one of: sample acquisition; cell lysis; DNA purification; library preparation; and adapter depletion. The reagent can be at least one of: a liquid deposited in the reagent port; a dried reagent deposited in the reagent port; or a peg having a dried reagent thereon, said peg inserted into the reagent port.

The preparation portion can be a stand-alone component, which can be connected to a further component having a sensing chamber. The preparation portion and the further components can be connected after the sample has been processed for sensing.

In another example there is provided a microfluidic device for analysing a test liquid, said device comprising: a preparation portion between an inlet and an upstream portion of the device, wherein said upstream portion is configured with a sensing chamber for housing a well array and for receiving via the inlet a test liquid to be sensed, and said preparation portion is configured having: a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion; and/or an actuator, operably configured to induce a test liquid from the inlet towards the upstream portion.

The preparation portion can be configured in a first part, and the upstream portion can be configured in a second part, wherein said first part and second part are connectable such that a sample received and processed in the first part can be passed to the second part for at least one of sensing, measurement and analysis. The first and second parts, when connected, can extend in parallel planes. The first and second parts can be separate and distinct components. The first part can be disposable and configured for single-use. The second part can be used for a plurality of test liquids.

The first and second parts can be powered and/or configured to operate independently. By way of example the sample can be processed for testing in the first part before being connected to the second part for analysis. The first part can be configured to removably and securably mount upon the second part.

In another example, there is provided a preparation portion for engagement with a second part having a sensing chamber. The preparation portion can be configured as a first part. The first part can be configured with an input port at one end of a channel and a connector at the other end of the channel. The connector at the opposite end of the channel functions as an outlet. The preparation portion can be configured with a connector for receiving power and/or control signals for controlling actuators and/or sensors in the preparation portion. The preparation portion can, optionally, have a sensing chamber. In another example, there is provided a system for analysing a test liquid, said system comprising: a microfluidic device according to any preceding claim; a controller configured to connect with the microfluidic device for receiving and analysing signals derived from analysis of a test liquid processed by the microfluidic device. The system can further comprise a dongle, said dongle having one or more slots, the or each slot configured for receiving a microfluidic device, said dongle connectable to the controller.

The ASIC can have up to 400 channels, such that it can be connected to well arrays having integer multiples of the number of channels on the ASIC e.g. 800, 1200, 1600 etc with the use of multiplexing.

The fluidic balance between the upstream and downstream can allow for a test liquid to be added without the need for a pipette.

Control of the flowcell can be implemented directly via a laptop computer, or mobile phone device or similar handheld device, although the functionality of the flowcell can be reduced further by providing a dongle to operate as an interface with a laptop of mobile phone device. A dongle can provide multiple input ports for enabling a laptop - via a wired or wireless connection - to interface with multiple flowcells inserted therein.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described below with reference to exemplary Figures, in which:

Fig. la is a schematic side-perspective view of a flowcell having an inlet port, upstream portion having a sensing chamber and downstream portion having and outlet port an inlet port, sensing chamber and outlet port configured in a Secure Digital (SD) card format;

Fig. lb shows a schematic cross-section of the flowcell of Figure la;

Figure 2 is a schematic perspective view of the flowcell of Figures la and lb inserted within a dongle that is connected to a laptop computer;

Figure 3 is a schematic perspective view of a multiple port dongle having a plurality flowcells of Figure la inserted therein, said dongle having a wired connection for interface with a remote computer;

Figure 4 is a schematic side-perspective view of a flowcell having an inlet port, preparation section, upstream portion having a sensing chamber and downstream portion having and outlet port, said flowcell having a Secure Digital (SD) (RTM) card format interface; Figures 5a, 5b and 5c are schematic perspective views of the flowcell of Figure 4 inserted within a dongle having different formats, namely a single port dongle and a multiple port dongle having, respectively, a single flowcell docked therein and multiple flowcells docked therein;

Figure 6 is a schematic of a sample section, having four stages, indicated by numbered zones as viewed, though which a sample is received for processing;

Figure 7 presents experimental results from the second stage of Figure 6;

Figure 8 presents further experimental results from the second stage of Figure 6;

Figures 9a and 9b are further experimental results from the second stage of Figure 6;

Figures 10a and 10b illustrate sequencing adapter deletion;

Figure 11 presents experimental results from the fourth stage of Figure 6; and

Figures 12a to 12e show various views of an example embodiment, in which the upstream portion and downstream portion are configured in separable components.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Figures la and lb show a microfluidic device 8 having body 10 of a flowcell having a substrate 12 covered, in part, by a cover 14 on the upper surface as viewed. In all the Figures herein the cover is clear, such that it appears that components are floating however these views best illustrate the fluid paths. In practice at least portions of the cover can be opaque. The body 10 is substantially planar. The substrate 12 is, typically, a printed circuit 16 having electronic components, including a microprocessor 18 that can communicate with an external controller via a connector 20. The cover 14 is connected to the substrate to leave the connector 20 exposed for interfacing with a reciprocal connection.

The body 10 of the flowcell is configured to receive a test liquid for analysis by a well array 22 configured to support nanopores for the analysis of a portion of a test liquid that passes through a nanopore in the well array 22. The well array has electrodes that are located at the bottom of each well and connected to the microprocessor 18. When a polymer, such as a polynucleotide, or protein passes through a nanopore the microprocessor detects a variation in a signal across the nanopore and sends the signal to an analysis device via the connector 20. The microprocessor can also receive signals via the connector for configuring the nanopores in the well array e.g. inserting a nanopore in each membrane suspended across each of the wells.

Overall, the microprocessor can function as an interface between the well array and external devices that at least one of control and/or perform analysis of the test liquid. To be clear, by moving the functions that control and/or perform analysis of the test liquid off the flowcell allows for the reduced the reduced cost and complexity of the flowcell.

Walls formed in the cover 14 and/or in the surface of the substrate 12 can, together define walls that define a channel 24 within the body 10 of the flowcell. The channel is configured to provide a fluid path 26 between an inlet 28 and an outlet 30. The outlet can be a vent port located at the end of a reservoir. Liquids do not necessarily flow out from the outlet. From the inlet 28, the fluid path 26 extends towards the upstream portion 32 having a sensing chamber 34 for housing the well array 22 and for receiving via the inlet at least a portion of the liquid to be sensed. The upstream portion comprises a connection 36 to a downstream portion 38 that is positioned between the upstream portion 32 and the outlet 30, said downstream portion for receiving liquid from the outlet channel of the upstream portion via the connection 36. The connection can include a valve, fluidic switch or bridgeable barrier.

The upstream portion, and at least the sensing chamber, can be filled with a liquid. The flowcell can be provided to a user prefilled and in a “wet chip” condition. The flowcell can be filled with a liquid from the inlet to the outlet. Or the flowcell can be filled with a liquid from the inlet to the outlet of the upstream portion. The device can be supplied with a liquid between the inlet and the upstream portion.

The inlet 28, which provides an input port 28, can have a removably attachable seal configured to enclose the upstream portion and, when a liquid is provided in the upstream portion, inhibit flow of the liquid before removal of the seal, and after removal of the seal, permit liquid to pass from the from the upstream portion to the downstream portion.

The body of the flowcell can be provided with fluid switches or valves to selectively connect the upstream portion 32 and downstream portion 38. A bridgeable barrier can be configured between the upstream portion and the downstream portion. The bridgeable barrier can be manually activatable to permit fluid flow from the upstream portion to the downstream portion. A bridge can be provided adjacent the bridgeable barrier, and after removal of the seal the bridge can facilitate liquid to flow from the upstream portion to the downstream portion via or over the barrier. The removably attachable seal can be configured to inhibit liquid to flow from the inlet to the downstream portion.

A first seal can be provided to cover the inlet that receives the test liquid. A second seal can be configured to cover the end of the sensing chamber 34 connection 36 to the downstream portion 38, thereby preventing liquid from flowing from the sensing chamber, over the barrier, into the liquid collection channel. The flowcell can be filled with a liquid from the first seal at the sample input port to the second seal at the end of the sensing chamber, such that the sensor is covered by liquid and unexposed to a gas or gas/liquid interface. The first and second seals can be removable to cause the liquid between the reservoir and the end of the sensing chamber outlet channel to flow so that some liquid flows over the barrier.

The capillary pressures at the downstream portion 38 and upstream portion 32 and inlet 30, which functions as an input port, are balanced such that following activation of the device, gas is not drawn into the sample inlet port, and the sample input port presents a wet surface to a test liquid. Following activation of the device and prior to addition of a test liquid, the device may be considered to be at equilibrium, namely wherein the pressure at the input port is equal to the pressure at the downstream collection channel. In this equilibrium state, liquid remains in the sensing chamber and gas is not drawn into the input port such that the input port presents a wet surface to a test liquid to be introduced into the device. The device is configured to ensure that balance of fluid forces is such that the sensing chamber remains filled with liquid and that liquid remains (at least partially) in the inlet, in the outlet and the liquid collection channel. If the equilibrium is disturbed by shifting the position of the liquid (without adding or removing liquid to the system) there is an impetus to return to that equilibrium. When the liquid is moved, it will create new gas/liquid interfaces. Thus, this balance of force and restoring of the equilibrium will effectively be controlled by the capillary forces at those interfaces.

The inlet 28 can be provided with reagents such that a preparation portion adjacent the inlet can prepare the test liquid for analysis within the sensing chamber.

Figures 2 and 3 show a dongle 40, the former having a single slot for receiving a flowcell 10 and the latter having multiple ports. The cost and functionality of the flowcell can be reduced by the processor 18 sending raw data off the flowcell to perform analysis of the test liquid. To further reduce the cost and functionality the dongle provides an interface to enable the flowcell to be controlled and to communicate data to an external analysis device, which in Figure 2 is illustrated, by way of example, as a laptop computer 42. The dongle of Figure 3 can accommodate a plurality of flowcells and communicate via a wired connection, as shown, or wireless connection to an analysis device. The dongle can be omitted by incorporating its functions in to either the flowcell or the analysis device to which the flowcell sends data.

Figure 4 shows a microfluidic device 8 having a flowcell 10 having a preparation portion 44, while Figures 5a, 5b and 5c show said flowcell engaging with a dongle. Like numerals refer to like features. For analysis using known flowcells a sample must be prepared before analysis. The preparation portion 44 enables sample preparation to be performed on the body 10 of the flowcell. Is configured having:

The preparation portion 44 is configured between the inlet 28 and an upstream portion 32. The preparation portion can have a reagent port 46 configured to receive a reagent for interacting with a test liquid to be analysed within the upstream portion and/or an actuator 48, operably configured to induce at least a portion of the test liquid into contact with the upstream portion such that the test liquid can be sensed.

The structure of the body of the flowcell of Figure 4 is analogous to the flowcell shown in Figure 1 accept that the length has been increased to accommodate and illustrate the functions of the preparation portion. In the example shown in Figure 4, four reagent ports 46 are provided in the fluid path 26 of the preparation portion 44, which snakes along the length of the body 10 and passes over five actuator 48 areas, illustrated by rectilinear pads located at the end of tracks on the printed circuit of the body.

The number of reagent ports 46 implemented and/or provided with reagents can be configured according to the required application. Similarly, the number of actuators can vary. The fluid path of the preparation portion can be provided with an actuator at either side of each reagent port implemented. The number of actuators implemented can be sufficient for a test liquid to be manoeuvred to interact with the reagent before analysis of the processed test liquid. Additionally, or alternatively, the preparation portion can be provided with reagents coated over the surface of the fluid path.

The or each reagent port 46 can have a sealable aperture for selectively injecting a reagent into the preparation portion. By way of example, the reagent can be a liquid deposited in the reagent port using a pipette. Alternatively, a dried reagent can be deposited in the reagent port and sealed during the manufacturing process. Further alternatively, the dried reagent can be provided on a peg that is inserted into the reagent port. In all the exampled the reagents can be sealed until required, or otherwise activatable via, for example, rehydration. The seal could be broken prior to use by a user, for example, applying pressure upon the port to break the seal.

The reagent can, for example, lyse a liquid to be tested before subsequently controllably dispensing a lysed portion of said processed fluid into the upstream portion 32 for measurements to be taken. The sample can be lysed through agitation, said agitation being caused manually or using an actuator 48, as described below. The preparation portion can contain at least one of a mechanical bead, chemical bead a36nd reagent for mechanically mixing and dispersing and selectively binding with nucleic acid, such as DNA, contained in the sample. A gate 50 can be provided between the preparation portion 44 and upstream portion 32 to controllably release a processed sample for analysis.

The flowcell can be configured to lyse a cell within the test liquid and release nucleic acids from within a cell. Chemical beads located within the preparation portion 44 can be provided for binding released nucleic acid. A mechanical bead can be movably configured to mechanically lyse a cell within the container. It can be shaken or agitated. The mechanical bead is type of a breaker. The breaker can be spherical. The breaker can be asymmetrical, irregular or granular. A combination is breakers having different shapes can be used. The mechanical bead or breaker can have an uneven surface. The breaker can have inert properties. A plurality of mechanical beads or breakers can be provided, such that multiple contacts between breakers and other between other surfaces in the container during manual agitation. The number of breakers typically used can be between 1 and 30, or between 1 and 5. The number of beads used is influenced by the size of the breaker and the size of the container. In light of the teaching herein an appropriate number of breakers and appropriate size or mix of breakers can be selected according to the size of the container and the sample type to be mixed. The beads can have an uneven surface to improve the mixing effect. The mechanical bead can be shaped to optimise agitation and lysing of the sample. E.g. it can be the shape of a prism, e.g. a five-sided prism. It is to be noted that the size of a breaker can be selected to inhibit entry to the outlet, or funnel, of the container. The mechanical bead is preferably of a high density relative to the liquid such that it can efficiently move through and mix the sample when manually moved. The mechanical bead may be metallic such as stainless steel. The beads may typically have a width or diameter cross section of between 0.1 and 2mm.

The reagent can be configured adapted to lyse cells and release the nucleic acids from within the nucleic acid analyte of interest in the sample and attach to the bead. In other words, the reagent can selectively bind with the nucleic acid analyte of interest.

The chemical beads can be a bead or matrix with a ligand that will preferably be pH switchable for the charge to capture nucleic acid. The chemical bead may be silica. The chemical bead can comprise 2-(2-Pyridyl)ethyl Silica Gel.

The beads and reagent can be provided on the interior surface of the walls of the container. The beads can be provided in a liquid. The liquid can be prefilled in the container.

The beads and reagent can be provided or coated on the exterior surface of the mechanical bead and releasable therefrom. By coating the surface of the mechanical bead or breaker can increased the rate of lysing and/or bonding to the nucleic acid of the sample. This can also apply to other surfaces on the interior of the container.

Reference herein to a nucleic acid or a polynucleotide include both naturally occurring nucleic acids, such as DNA or RNA and synthetic polynucleotides. The polynucleotide may be oxidized or methylated. The polynucleotide may be damaged. The polynucleotide may be single or double stranded.

It is to be noted that a reagent and mechanical and/or chemical beads can be provided in the flowcell of Figure 1.

The gate 50 can be configured to controllably connect said preparation portion and said upstream portion. The gate 50 can be implemented like the mechanism of Figure 2 in WO20 19/106345 or the barrier and bridge interface of WO2018/007819.

The actuator can be a manually operable pump, implemented by a flexible surface on the body 10 that can be pressed, perhaps repeatedly, to initiate movement of a test liquid within the preparation portion 44.

Additionally, or alternatively the actuator has an electrowetting interface configured to induce movement of the test liquid or droplets of the test liquid. Rectilinear electrodes shown in Figure 4 indicate that electrowetting forces can be induced at intervals along the length of the fluid path within the preparation portion. Electrowetting functionality can, however, be implemented along the substantial length of the fluid path of the preparation portion such that movement of the fluid to be tested and prepared for measurement can be controlled with the preparation sample.

The preparation portion can be configured with heaters to increase the number of samples for analysis using via amplification, such as polymerase chain reaction (PCR). The methods can include a polymerase, a template nucleic acid and a pool of canonical, and optionally non- canonical nucleotides. The test liquid can be prepared according to standard PCR techniques. Oligonucleotide matching may be used.

Electrowetting functionality can be implemented in light of the teaching disclosed in WO2019/I26715, WO2019/227013 and PCT/GB2019/053366, all of which are incorporated herein by way of reference. Sample preparation can adopt, by way of example, the techniques and procedures disclosed in PCT/GB2019/052456 - in particular the methods illustrated and described in relation to Figures 18a to 18k - all of which is incorporated herein by way of reference.

More generally, the preparation portion 44 described above can be adapted and applied to a microfluidic device for analysing a test liquid. Such a device can have a preparation portion between an inlet and an upstream portion of the device that is configured with a sensing chamber for housing a well array and for receiving via the inlet a test liquid to be sensed. The preparation portion can be configured having: a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion; and/or an actuator, operably configured to induce a test liquid from the inlet towards the upstream portion. The preparation portion 44 can extend in same plane of the device. Alternatively, layers of preparation portions 44 can be provided. A separate function and/or reagent can be provided in said layers.

The microfluidic device 8 can be configured in a system 52 and can include a dongle 40.

The microfluidic device when provided as a “wet-sensor” (i.e. a sensor that functions in a wet environment) can be stored in a state in which the sensor is kept wet, until it is needed. This is effectively achieved by providing a device that has an “inactive” state in which the sensor is kept wet, but in which the device cannot be used, and an “active” state in which the device can be used. In other words, an “inactive” state can be a state in which a flow path between a sample input port and a liquid collection channel is not complete.

The sensing chamber 34 is provided with sensing electrodes, not shown in the Figures. The sensor may be a component or device for analysing a liquid sample. For example, a sensor may be a component or device for detecting single molecules (e.g., biological and/or chemical analytes such as ions, glucose) present in a liquid sample. Different types of sensors for detecting biological and/or chemical analytes such as proteins, peptides, nucleic acids (e.g., RNA and DNA), and/or chemical molecules are known in the art and can be used in the sensing chamber. In some embodiments, a sensor comprises a membrane that is configured to permit ion flow from one side of the membrane to another side of the membrane. For example, the membrane can comprise a nanopore, e.g., a protein nanopore or solid-state nanopore. In some embodiments, the sensor may be of the type discussed and described in WO 2009/077734, the content of which is incorporated herein by reference. The sensor is connected to an electrical circuit, in use. The sensor may be an ion selective membrane provide directly over an electrode surface or over an ionic solution provided in contact with an underlying electrode.

The sensor may comprise an electrode pair. One of more of the electrodes may be functionalised in order to detect an analyte. One or more of the electrodes may be coated with a selectively permeable membrane such as NafionTM.

The digital control system is most conveniently configured on a field-programmable-gate- array device (FPGA). In addition, the FPGA can incorporate processor-like functions and logic required to interface with standard communication protocols i.e. USB and Ethernet.

In one example, the upstream portion 32 includes four stages, as shown in Figure 6. In this example, the upstream portion 38 of Figure 4 is shown schematically without the cover and/or the substrate that define the walls and the channel between an inlet and an outlet. As described above, the upstream portion provides a sample receiving section for receiving and preparing the test liquid configured in an upstream portion configured between the inlet and the sensing chamber. The output from the upstream portion would enter the sensing chamber. The upstream portion 38 is configured to receive complex biological samples, such as bodily fluids, mucus swabs and/or faecal matter, and is able to produce ready-to-sequence DNA libraries as output, with the nucleic acids of any organism found in the sample. The example below and associated results are derived from commercial equipment and lab grown bacteria (E.Coli.) together with surface activated beads.

Stage ‘ G includes an input port 54 that functions as an inlet 28. The port 54 can have a resealable cap or similar mechanism for receiving and reattaining a sample to be analysed. A sample is held in an elution chamber 56 prior to elution. The chamber can receive a swab or tip on an object holding a sample to be analysed, or the input port can receive a liquid sample.

Once the sample is held in the elution chamber 56 elution can be implemented by the release of a solubilisation liquid. This liquid can be stored, held, and controllably released from a store 58, which can be a blister 58, which holds the appropriate solution to maximise the number of cells in the resulting sample liquid. The store can be configured to controllably release solution. In the case of a blister, it can be burst to fill the chamber by the user directly, or by leveraging the force exerted by the user to insert the swab in the device or close its lid. The blister can be single use. Upon release of solution from the store or blister, the solution functions to wet, further wet or generally resuspend the contents in the channel. The solution can, for example, wet the swab and resuspend the cells from the swab matrix - once resuspended and free in solution the cells/viruses will diffuse randomly. The contents can settle under gravity.

The chamber can have electrodes 60 that controllably move e.g. resuspend cells by dielectrophoresis. This can be achieved by inducing charge on the surface of the cells. Movement can be further controlled by applying and/or adjusting a force by establishing an electric field. The electrodes can be configured to controllably concentrate cells and/or induce movement of cells in the sample. The electrodes can, for example, be patterned on one or more of the walls of the chamber 56. Using the electrodes, and a signal applied thereto, cells eluted from the sample can be concentrated near the chamber outlet. In this way, the solution passed on to the next stage can have an increased cell density, which would result in a more concentrated DNA sample.

In stage ‘2’, the solution containing the sample to be analysed is subjected to cell lysis and/or DNA purification. The solution derived from the sample is passed into a lysis chamber 62 that has a conductive plate 64 e.g. opposing side walls, such as floor and ceiling. The conductive walls can be used to establish an electric field across the chamber. The conductive walls can be a metal solid sheet, or a metal coated plastic. The conductive walls can be patterned. The pattern can include micropillars 66. Overall, the conductive walls or surfaces, such as parallel metal plates can be used to establish an electric field across the chamber.

By way of example, the lysis chamber 62 is configured to lyse cells. Lysis can be induced using electroporation. Electroporation is routinely used on transfection experiments to temporarily disrupt the cell membrane with an electric field - this results in temporary pores formed in the membrane. By extending the exposure of the sample to the electric field that causes electroporation, the cell membrane can be rendered unstable beyond repair and collapses, liberating the cell contents into the solution (lysate). The lysis can be performed with a commercially available piece of equipment (MicroPulser from BioRad), in which the cuvette where the lysis takes place is effectively a parallel plate system, such as the one described above.

Figure 7 shows the increase in UV absorbance at 260 nm (peak DNA absorbance point), for samples having undergone 5 cycles of electroporation, with respect to those that have not undergone electroporation. More particularly, it shows the UV absorbance spectra of cell culture supernatant (SN), before and after electroporation lysis with 5 pulses in a MicroPulser instrument and the peak at 260 nm indicates larger concentration of DNA after electroporation lysis.

A surface of the conductive walls 64 can be functionalised with a coating 68. The coating includes chemical means that facilitate DNA extraction and preparation. The chemical means can include at least one of: phenol chloroform, Sodium Dodecyl Sulfate (SDS), UREA, guanidine hydrochloride, serine proteases, Rnase or kits available from QIAGEN I. A coating, such as this enzyme, can rapidly digests the proteins in the lysate solution as the cell membranes collapse. The coating can be implemented in the form of enzyme activated beads that are mixed into a lysate. The walls of the lysis chamber 62 can be formed to maximise the surface area - for example, include at least one of patterning, micropatterned features or roughening - to increase the surface- area to volume ratio. Overall, enzyme functionalisation of the micropatterning can accelerate the protein digestion process. By way of example, the chemical means herein uses a surface bound proteinase K prior to use.

Figure 8 shows the results of protein electrophoresis performed on NuPAGE gel. Sample lanes/columns shown are, from left to right (i) ladder, (ii) proteinase K solution for control purposes, (iii) undigested lysate supernatant, (iv) lysate exposed to surface-bound proteinase K, (v) a non-specific absorbance control, using PMMA (acrylic beads), and (vi) a liquid enzyme control. Comparing lanes (iii) and (iv) clearly shows successful protein digestion after a few seconds of the lysate being exposure to the surface-bound proteinase K. Lane (iv) shows that the purifying effect is not caused by non-specific adsorption of proteins onto the beads’ surface. Lane (v) is a liquid proteinase K digestion control. Overall, an increase in the purity of the DNA sample can be achieved clearly appreciable.

Figures 9a and 9b compare the sequencing performance obtained from a rapid kit library prep using untreated (raw) lysate and lysate treated with surface-bound proteinase K beads - shown on the left side and right side of the different graphs, respectively. Figure 9a and 9b show a comparison between sequencing an untreated sample of lysate (raw lysate) and a sample of treated lysate (surf bnd proK). In Figure 9a, the comparison indicates how the number of strands of DNA from a sample have increased significantly. This can be seen by the increase in the lowermost data set in the graphs, wherein the treated lysate resulted in an increase of all sequencing time involving strands to be sampled - after 1 hour it is 14.5% and after 2 hours it is 22.5%. Similarly, in Figure 9b, a comparison between the total number of samples counted increases from 7416 (raw lysate) with untreated lysate to 46750 (surf bnd proK) with treated lysate. It is also to be noted that the x-axis indicated the number of counts while the y-axis indicates the read-length of the sample - which also increases with the treated sample.

As indicated in Figures 9a and 9b, including proteinase K immobilised in the lysis chamber has at least two functions. Firstly, it ensures that the proteins liberated by the collapse of the cell membranes are digested as soon as they are free. This is because amongst these proteins are nucleases, which degrade DNA and the presence of proteinase K inhibits the degradation of the DNA in the sample to be analysed. Secondly, the proteinase K removes the need for solid phase extraction of the DNA as means to purify the sample and avoid proteinase K contamination in the library preparation process further downstream. In standard molecular biology workflows this step usually involves beads, buffer exchanges and several washing steps, before the DNA is resuspended for library preparation.

It is to be noted that the use of proteinase K for treating a sample can be an alternative to a heat treatment - which can be costly and inefficient if resources are limited - thus reducing at least the energy required to process the sample and avoiding the need for a heating control system. Current rapid and field kits rely on a heat treatment step (e.g. 80C for 1 min) to inactivate the MuA transposase in the FRA tube. FRA is known from a kit SQK-RAD004 (rapid sequencing kit) available from Oxford Nanopore Technologies is the transpososome, this is a protein-DNA complex comprised of a specific DNA adapter and a transposase (MuA). This complex inserts into the gDNA (genomic DNA). A heat step is used to remove the transposase (MuA). The presence of the transposase (MuA) can decrease throughput during sequencing, its removal with proteinase K would be beneficial to remove the need for a heat step. Treating the sample at a constant ‘room’ temperature can allow the cost to be reduced such that the analysis device, or at least the upstream portion, can be manufactured to be single-use and disposable.

After lysis of the sample in stage ‘2’, the solution incubates in the lysis chamber 62 for between Is and 10 minutes, while digestion takes place. Thereafter it is moved to stage ‘3’.

In stage ‘3’, the solution is further prepared for sequencing e.g. library preparation. The channel 24 in stage ‘3’ in this example has three chambers, or ports 46, in series. Each port 46 holds a pellet of a lyophilised library prep kit e.g. SQK-LRK001 (field sequencing kit) from Oxford Nanopore Technologies. Each chamber is followed by a meandering portion of the where mixing of the solution takes place. The channel 24 can include actuators 48, as shown in Figure 4, although these are not shown in the schematic of Figure 6. Mixing enables a homogenous reaction to occur before the sample reaches the next port 46 for the subsequent library prep step.

In light of the teaching herein it would be understood that an alternative number of ports 46 and/or actuators 48 can be provided. In the example of Figure 6, three ports 46a, 46b, 46c are provided in the channel 24, each containing freeze-dried pellets of a reformulated kit. By way of example the kit can be one of those available from Oxford Nanopore Technologies, such as the “Field Sequencing Kit - SQK-LRK001” or “Rapid Sequencing Kit - SQK-RAD004”.

In the example, the ports 46a, 46b and 46c are populated with freeze-dried reagents in the form of pellets. Alternatively, the reagents can be supplied in non-freeze-dried version, which can result in an excess of sequencing adapter molecules with motor protein, and when the library is added to the sequencing flowcell it can be captured by the pore. This excess can result in a pore reading a known sequence of no analytical interest.

At the first port 46a, as shown, the DNA in the protein digested lysate is exposed to a ‘fragmentation’ pellet. The pellet contains a transpososome which inserts into the DNA (or target polynucleotide). The transpososome contains a DNA sequencing adapter with a ssDNA overhang complementary to the sequencing adapter, which can be referred to as a fragmented library. The fragmented library is then exposed to ‘motor’ pellet to be tagged with the necessary motor protein that enables nanopore sequencing. The motor pellet contains the sequencing adapter. A ssDNA overhang on the sequencing adapter hybridises to the complementary sequence on the fragmented library, and they are covalently joined by means of a chemical ligation, which can be referred to as the adapted library. Finally, the processed sample is exposed to a ‘buffer’ pellet, which can be complimented by RTB from a blister 58.

In stage ‘4’, excess sequencing adapter can be depleted. This can be achieved by the product of the library prep being exposed to a surface which is activated with DNA complementary probes that have a complementary sequence to the overhang in the sequencing.

Excess sequencing adapter (RAP) can be captured and sequenced by the nanopore, indicated by the ‘adapter samples’ section in Figure 11, decreasing the time available for sequencing the adapted library (gDNA / target polynucleotide) i.e. the ‘strand sample’ section in Figure 11. It is desirable to deplete the excess sequencing adapter to increase time available to capture and sequence adapted library. An increase in capture of adapted library is beneficial as it provides greater sensitivity e.g. shorter time to result. Sequencing adapter depletion can be achieved as described below. Two different lengths of overhang complementary probe 72 have been used so far. This can be achieved using a depletion chamber 70 and hybridisation probes, such as adapter depletion probes 72.

A depletion strategy schematic is shown in Figure 10a and 10b. In Figure 10a the complementary probes are complimentary to the ssDNA overhang strand of the sequencing adapter. For any adapter that has not hybridised to the fragmented library, the sequencing adapter should hybridise to the sequencing adapter and immobilise it on a surface. In Figure 10b the same applies, and the longer complementary sequence should result in more stable binding. The sequencing adapter 72 are configured on a wall of the depletion chamber 70 and takes the form of beads that are mixed in with the library. After a few minutes of incubation in the depletion chamber, to allow sequencing adapter hybridisation, the library is then ready to be infused in the flowcell of the sequencing platform. The device will have output port compatible with the flowcell input to the well array 22.

This can be achieved because the DNA in the protein digested lysate is exposed to a ‘fragmentation’ pellet. The fragmentation pellet contains a transpososome which inserts into the target polynucleotide. The transpososome contains a DNA adapter with a ssDNA overhang complementary to the sequencing adapter. This will be referred to as fragmented library. The fragmented library is then exposed to ‘motor’ pellet. The motor pellet contains the sequencing adapter. The ssDNA overhang on the sequencing adapter hybridises to the complementary sequence on the fragmented library, they are covalently joined by means of a chemical ligation. This can be referred to as the adapted library.

Figure 11 shows the percentage of time spent in different states throughout the sequencing run for a control sample (without sequencing adapter depletion), and for results obtained with the complementary probe 72 activated beads (see ‘short overhang’ and ‘extended overhang’). The sequencing adapter depleted libraries show a decrease in ‘unavailable samples’ and increase in ‘pore samples’, with respect to the control library. An adapted library is available for capture and sequencing during the ‘pore sample’ state and is not captured or sequenced when in the ‘unavailable sample’ state. The sequencing adapter depletion has resulted in an increase in the adapted library being sequenced, as shown by the increase in time spent in the ‘strand samples’ state, with respect to the control library.

In Figure 11, the average duty times (n=3) for libraries treated with either type of complementary probe, as well an untreated control. A discernible improvement in strand to adapter ratio can be seen, as well as a reduction in pore blocking (unavailable).

The results of sequencing adapter depletion (stage ‘4’) are demonstrated in Figure 11. In the control sample, without adapter depletion, 37.0% of time is spent sequencing the sequencing adapter, this is reduced to 23.9% and 23.7% of time with the short and long overhang complementary probes, respectively.

Figures 12a to 12e illustrate an example of a device 8 embodied in two parts - a first part 74 having a preparation portion 44, which can be a single-use disposable section, and a second part 76 having a base that contains a well array 22 in an upstream portion 32, sensing chamber 34 and a downstream portion. The base 76 in this example is provided by a flowcell from a MinlON device available from Oxford Nanopore Technologies. A connection 36 can be provided between the preparation portion 44 and the base.

Like features are provided with like numerals. The preparation portion 44 is additionally provided with a connector 78 that, when connected to a controller (not shown) can provide power and control instructions to features that can be provided, but not shown in Figure 12, such as actuators 48, electrodes 60, conductive plates 64 etc.

In the example of Figure 12, the device 8 is configured to receive a swab 80 having a sample to be analysed at an inlet 28 or input port 54 of the preparation portion 44 having an elution chamber 56. A blister 58 can be activated to facilitate elution of a sample. The sample can then be lysed before being passed along the channel 24 for exposure to the reagent ports 46a, 46b, 46c. A further blister 58 can be activated to facilitate preparation of the sample for output into a sensing chamber 34 connected to the output of the upstream portion.

In such a device, polymers such as polynucleotides or nucleic acids, polypeptides such as a protein, polysaccharides or any other polymers (natural or synthetic) may be passed through a suitably sized nanopore. In the case of a polynucleotide or nucleic acid, the polymer unit may be nucleotides. As such, molecules pass through a nanopore, whilst the electrical properties across the nanopore are monitored and a signal, characteristic of the particular polymer units passing through the nanopore, is obtained. The signal can thus be used to identify the sequence of polymer units in the polymer molecule or determine a sequence characteristic. A variety of different types of measurements may be made. This includes without limitation: electrical measurements and optical measurements.

The polymer may be a polynucleotide (or nucleic acid), a polypeptide such as a protein, a polysaccharide, or any other polymer. The polymer may be natural or synthetic. The polymer units may be nucleotides. The nucleotides may be of different types that include different nucleobases.

The nanopore may be a transmembrane protein pore, selected for example from MspA, lysenin, alpha-hemolysin, CsgG or variants or mutations thereof.

The polynucleotide may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), cDNA or a synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or other synthetic polymers with nucleotide side chains. The polynucleotide may be single-stranded, be double-stranded or comprise both single-stranded and double-stranded regions. Typically, cDNA, RNA, GNA, TNA or LNA are single stranded.

In some embodiments, the devices and/or methods described herein may be used to identify any nucleotide. The nucleotide can be naturally occurring or artificial. A nucleotide typically contains a nucleobase (which may be shortened herein to “base”), a sugar and at least one phosphate group. The nucleobase is typically heterocyclic. Suitable nucleobases include purines and pyrimidines and more specifically adenine, guanine, thymine, uracil and cytosine. The sugar is typically a pentose sugar. Suitable sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate.

The nucleotide can include a damaged or epigenetic base. The nucleotide can be labelled or modified to act as a marker with a distinct signal. This technique can be used to identify the absence of a base, for example, an abasic unit or spacer in the polynucleotide.

Of particular use when considering measurements of modified or damaged DNA (or similar systems) are the methods where complementary data are considered. The additional information provided allows distinction between a larger number of underlying states.

The polymer may also be a type of polymer other than a polynucleotide, some non- limitative examples of which are as follows.

The polymer may be a polypeptide, in which case the polymer units may be amino acids that are naturally occurring or synthetic.

The polymer may be a polysaccharide, in which case the polymer units may be monosaccharides.

A conditioning liquid provided in the device to maintain the sensor in a wet state may be any liquid that is compatible with the device (e.g., a liquid that does not adversely affect the performance of the sensor) By way of example only, when the sensor comprise a protein nanopore, it would be apparent to one of ordinary skill in the art that the conditioning liquid should be free of an agent that denatures or inactivates proteins. The conditioning liquid may for example comprise a buffer liquid, e.g., an ionic liquid or ionic solution. The conditioning liquid may contain a buffering agent to maintain the pH of the solution.

The sensor is one that needs to be maintained in a ‘wet condition’, namely one which is covered by a liquid. The sensor may comprise a membrane, such as for example an ion selective membrane or amphiphilic membrane. The membrane, which may be amphiphilic, may comprise an ion channel such as a nanopore. The membrane, which may be amphiphilic, may be a lipid bilayer or a synthetic layer. The synthetic layer may be a diblock or triblock copolymer.

The membrane may comprise an ion channel, such an ion selective channel, for the detection of anions and cations. The ion channel may be selected from known ionophores such as valinomycin, gramicidin and 14 crown 4 derivatives.

Some illustrative examples of the present disclosure are provided in the following enumerated clauses.

1. A microfluidic device for preparing a test liquid for sensing of an analyte present therein, said device comprising a body having a substrate and a cover, said cover connected to at least one side of the substrate, wherein: the substrate is configured having: a well array for supporting nanopores for sensing the analyte, a microprocessor for processing signals derived from the sensing of the analyte by the nanopores and sending signals to an external controller for analysis; and the cover and/or the substrate configured with walls defining a channel, the channel having a fluid path between an inlet and an outlet, the fluid path having: a sample preparation portion for preparing the test liquid configured between the inlet and a sensing chamber, the sensing chamber for housing the well array and for receiving least a portion of the prepared test liquid to be sensed, said sensing chamber comprising an outlet to a downstream portion for receiving liquid from the outlet of the sensing chamber.

2. A microfluidic device according to claim 1, wherein a removably attachable seal is configured to enclose an upstream portion, said the upstream portion including the sensing chamber for housing the well array and for receiving via the inlet at least a portion of the liquid to be sensed, said upstream portion comprising a connection to a downstream portion that is positioned between the upstream portion and the outlet and, when a liquid is provided in the upstream portion, inhibit flow of the liquid before removal of the seal, and after removal of the seal, permit liquid to pass from the from the upstream portion to the downstream portion.

3. A microfluidic device according to claim 1 or 2, wherein a bridgeable barrier is configured between the upstream portion and the downstream portion, and wherein said bridgeable barrier is manually activatable to permit fluid flow from the upstream portion to the downstream portion.

4. A microfluidic device according to claim 3, wherein a bridge is provided adjacent the bridgeable barrier, and wherein after removal of the seal the bridge facilitates liquid to flow from the upstream portion to the downstream portion via or over the barrier.

5. A microfluidic device according to any of claims 2, 3 or 4, wherein the removably attachable seal is additionally configured to inhibit liquid to flow from the inlet portion to the outlet portion.

6. A microfluidic device according to any preceding claim, wherein the preparation portion is configured having: a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion; and/or an actuator, operably configured to induce at least a portion of the test liquid into contact with the upstream portion such that the test liquid can be sensed.

7. A microfluidic device according to claim 6, further comprising a gate between the preparation portion and the upstream portion, said gate configured to controllably connect said preparation portion and said upstream portion.

8. A microfluidic device according to claim 6 or 7, wherein the reagent port has a sealable aperture for receiving a reagent.

9. A microfluidic device according to claim 6, wherein the reagent is at least one of: a liquid deposited in the reagent port; a dried reagent deposited in the reagent port; or a peg having a dried reagent thereon, said peg inserted in to the reagent port.

10. A microfluidic device according to claim 8 or 9, wherein said reagent is releasable into the preparation portion or activatable.

11. A microfluidic device according to claim 6 or 7, wherein the actuator is a manually operable pump.

12. A microfluidic device according to claim 6 or 7, wherein the actuator has an electrowetting interface configured to induce movement of the test liquid or droplets of the test liquid.

13. A microfluidic device according to claim 12, wherein the actuator has an electrowetting interface extending from the inlet to the upstream portion.

14. A microfluidic device according to claim 12 or 13, wherein or each reagent port is configured with a corresponding electrowetting interface for manipulating a test liquid and cause interaction with a reagent provided in a reagent port.

15. A microfluidic device according to any preceding claim, wherein the device is provided with a well array populated with nanopores and/or supplied with at least one reagent.

16. A microfluidic device for analysing a test liquid, said device comprising: a preparation portion between an inlet and an upstream portion of the device, wherein said upstream portion is configured with a sensing chamber for housing a well array and for receiving via the inlet a test liquid to be sensed, and said preparation portion is configured having: a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion; and/or an actuator, operably configured to induce a test liquid from the inlet towards the upstream portion. 17. A system for analysing a test liquid, said system comprising: a microfluidic device according to any preceding claim; a controller configured to connect with the microfluidic device for receiving and analysing signals derived from analysis of a test liquid processed by the microfluidic device.

18. A system according to claim 16, further comprising a dongle, said dongle having one or more slots, the or each slot configured for receiving a microfluidic device, said dongle connectable to the controller.

19. A microfluidic device for preparing a test liquid for sensing of an analyte present therein, said device comprising a body having a substrate and a cover, said cover connected to at least one side of the substrate, wherein the cover and/or the substrate are configured with walls to defining channel between an inlet and an outlet, said channel defining a sole fluid path between the inlet and the outlet, the channel having a sensing chamber having a well array for receiving least a portion of the prepared test liquid to be sensed, wherein the substrate is substantially planar, and the channel extends parallel to the substrate.

20. A microfluidic device according to claim 19, wherein the substrate is a printed circuit on board, such as a PCB.

21. A microfluidic device according to claim 19, further comprising: a sample section for receiving and preparing the test liquid configured in an upstream portion configured between the inlet and the sensing chamber, and said sensing chamber comprising an outlet to a downstream portion for receiving liquid from the outlet of the sensing chamber, said downstream portion configured between the sensing chamber and the outlet.

22. A microfluidic device according to claim 19, further comprising a well array for supporting nanopores for sensing the analyte.

23. A microfluidic device according to claim 19, further comprising actuators, operably configured to induce at least a portion of the test liquid into contact with the upstream portion such that the test liquid can be sensed.

24. A microfluidic device according to claim 19, further comprising a microprocessor for (i) operating the actuators to move the sample along the channel, and/or (ii) processing signals derived from the sensing of the analyte by the nanopores and sending signals to an external controller for analysis.

25. A microfluidic device according to claim 21, wherein the preparation portion is configured having an in-line treatment stage, said stage configured to transform the analyte or a derivative thereof for further treatment or sensing. 26. A microfluidic device according to claim 21, wherein an in-line treatment stage is provided with a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion.

27. A microfluidic device according to claim 21, wherein an in-line treatment stage is provided with an actuator.

28. A microfluidic device according to claim 21, wherein each in-line treatment stage is provided with at least one actuator.

29. A microfluidic device according to any of claims 23 to 28, wherein an actuator is at least one of: a mechanical finger-operable blister-pump; a pair of electrodes configured to induce electro-wetting movement; a pair of electrodes configured to induce dielectrophoresis; a pair of electrodes configured to electroporation.

30. A microfluidic device according to any of claims 21 to 29, wherein the channel includes at a stage that is at least one of: sample acquisition; cell lysis; DNA purification; library preparation; and adapter depletion.

31. A microfluidic device according to any of claims 26 to 30, wherein the reagent is at least one of: a liquid deposited in the reagent port; a dried reagent deposited in the reagent port; or a peg having a dried reagent thereon, said peg inserted into the reagent port.

32. A microfluidic device for analysing a test liquid, said device comprising: a preparation portion between an inlet and an upstream portion of the device, wherein said upstream portion is configured with a sensing chamber for housing a well array and for receiving via the inlet a test liquid to be sensed, and said preparation portion is configured having: a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion; and/or an actuator, operably configured to induce a test liquid from the inlet towards the upstream portion.

33. A microfluidic device according to claim 32, wherein the preparation portion is configured in a first part, and the upstream portion is configured in a second part, wherein said first part and second part are connectable such that a sample received and processed in the first part can be passed to the second part for analysis.

34. A microfluidic device according to claim 33, wherein the first and second parts extend in parallel planes when connected.

35. A system for analysing a test liquid, said system comprising: a microfluidic device according to any preceding claim; a controller configured to connect with the microfluidic device for receiving and analysing signals derived from analysis of a test liquid processed by the microfluidic device.

36. A system according to claim 35, further comprising a dongle, said dongle having one or more slots, the or each slot configured for receiving a microfluidic device, said dongle connectable to the controller.

Overall, the invention resides in a microfluidic device for analysing a test liquid. The device has a preparation portion between an inlet and an upstream portion of the device, wherein said upstream portion is configured with a sensing chamber for housing a well array and for receiving via the inlet a test liquid to be sensed, and said preparation portion is configured having: a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion; and/or an actuator, operably configured to induce a test liquid from the inlet towards the upstream portion. Between the inlet and respective outlet lies a sole channel. The channel can extend parallel to the substrate, preferably along its entire length. The preparation portion can be configured in a first part, and the upstream portion can be configured in a second part, wherein said first part and second part are connectable such that a sample received and processed in the first part can be passed to the second part for analysis. It will be understood that the invention is not limited to the embodiments above-described and various modifications and improvements can be made without departing from the concepts described herein. Except where mutually exclusive, any of the features may be employed separately or in combination with any other features and the disclosure extends to and includes all combinations and sub-combinations of one or more features described herein.

Claims

1. A microfluidic device for preparing a test liquid for sensing of an analyte present therein, said device comprising a body having a substrate and a cover, said cover connected to at least one side of the substrate, wherein the cover and/or the substrate are configured with walls to define a channel between an inlet and an outlet, said channel defining a sole fluid path between the inlet and the outlet, the channel having a sensing chamber having a well array for receiving least a portion of the prepared test liquid to be sensed, wherein the substrate is substantially planar, and the channel extends parallel to the substrate.

2. A microfluidic device according to claim 1, wherein the substrate is a printed circuit on board, such as a PCB.

3. A microfluidic device according to claim 1, further comprising: a sample section for receiving and preparing the test liquid configured in an upstream portion configured between the inlet and the sensing chamber, and said sensing chamber comprising an outlet to a downstream portion for receiving liquid from the outlet of the sensing chamber, said downstream portion configured between the sensing chamber and the outlet.

4. A microfluidic device according to claim 1, further comprising actuators, operably configured to induce at least a portion of the test liquid in the upstream portion to move towards the sensing chamber, such that the test liquid can be sensed.

5. A microfluidic device according to claim 1, further comprising a microprocessor for (i) operating the actuators to move the sample along the channel, and/or (ii) further comprising a well array for supporting nanopores for sensing the analyte and processing signals derived from the sensing of the analyte by the nanopores and sending signals to an external controller for analysis.

6. A microfluidic device according to claim 3, wherein the preparation portion is configured having an in-line treatment stage, said stage configured to transform the analyte or a derivative thereof for further treatment or sensing.

7. A microfluidic device according to claim 3, wherein an in-line treatment stage is provided with a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion.

8. A microfluidic device according to claim 3, wherein an in-line treatment stage is provided with an actuator.

9. A microfluidic device according to any of claims 4 to 9, wherein an actuator is at least one of: a mechanical finger-operable blister-pump; a pair of electrodes configured to induce electro wetting movement; a pair of electrodes configured to induce dielectrophoresis; a pair of electrodes configured to electroporation.

10. A microfluidic device according to any of claims 6 to 9, wherein the channel includes at a stage that is at least one of: sample acquisition; cell lysis; DNA purification; library preparation; and adapter depletion.

11. A microfluidic device according to any of claims 7 to 11, wherein the reagent is at least one of: a liquid deposited in the reagent port; a dried reagent deposited in the reagent port; or a peg having a dried reagent thereon, said peg inserted into the reagent port.

12. A microfluidic device for analysing a test liquid, said device comprising: a preparation portion between an inlet and an upstream portion of the device, wherein said upstream portion is configured with a sensing chamber for housing a well array and for receiving via the inlet a test liquid to be sensed, and said preparation portion is configured having: a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion; and/or an actuator, operably configured to induce a test liquid from the inlet towards the upstream portion.

13. A microfluidic device according to claim 12, wherein the preparation portion is configured in a first part, and the upstream portion is configured in a second part, wherein said first part and second part are connectable such that a sample received and processed in the first part can be passed to the second part for analysis.

14. A system for analysing a test liquid, said system comprising: a microfluidic device according to any preceding claim; a controller configured to connect with the microfluidic device for receiving and analysing signals derived from analysis of a test liquid processed by the microfluidic device.

15. A system according to claim 14, further comprising a dongle, said dongle having one or more slots, the or each slot configured for receiving a microfluidic device, said dongle connectable to the controller.