阅读说明：本技术 微流体装置 (Microfluidic device ) 是由 大卫·沃特曼 于 2018-11-23 设计创作，主要内容包括：一种用于分析测试液体的微流体装置包括：传感器(235),例如设置在感测室(237)中的具有纳米孔的膜；分别连接至感测室以使液体分别流入和流出感测室的感测室入口通道(261)和感测室出口通道(262),以及形成微流体装置的样品输入端口的容器(233),该容器与感测室入口通道(261)流体连通；液体收集通道(232)；在感测室出口通道262的末端与液体收集通道(232)之间的屏障(231)；第一密封件(251),其覆盖样品输入端口；第二密封件(252),其覆盖感测室出口通道(262)的末端,从而防止液体从感测室(237)越过屏障(231)流入液体收集通道(232)；其中微流体装置从在样品输入端口处的第一密封件(251)到第二密封件(252)填充有液体,使得传感器(235)被液体覆盖并且未暴露于气体或气体/液体界面；并且其中第一和第二密封件(251、252)是可移除的,以使液体在容器与感测室出口的末端之间并且越过屏障流动。(A microfluidic device for analyzing a test liquid comprising: a sensor (235), such as a membrane with nanopores disposed in a sensing chamber (237); a sensing chamber inlet channel (261) and a sensing chamber outlet channel (262) connected to the sensing chamber, respectively, for flowing liquid into and out of the sensing chamber, respectively, and a receptacle (233) forming a sample input port of the microfluidic device, the receptacle being in fluid communication with the sensing chamber inlet channel (261); a liquid collection channel (232); a barrier (231) between an end of the sensing chamber outlet channel 262 and the liquid collection channel (232); a first seal (251) covering the sample input port; a second seal (252) covering an end of the sensing chamber outlet channel (262) preventing liquid from flowing from the sensing chamber (237) across the barrier (231) into the liquid collection channel (232); wherein the microfluidic device is filled with a liquid from a first seal (251) at the sample input port to a second seal (252) such that the sensor (235) is covered by the liquid and not exposed to the gas or gas/liquid interface; and wherein the first and second seals (251, 252) are removable to allow liquid to flow between the container and the end of the sensing chamber outlet and across the barrier.)

1. A microfluidic device for analyzing a test liquid, comprising:

bridgeable barrier

An upstream portion located upstream of the bridgeable barrier for receiving a sensor arranged in a sensing chamber and for receiving a test liquid to be analysed, the upstream portion comprising an inlet channel and an outlet channel and being fillable with liquid between the inlet channel and the outlet channel;

a downstream portion downstream of the bridgeable barrier for receiving liquid from the outlet passage of the upstream portion;

a removably attached seal configured to enclose the upstream portion and when liquid is provided in the upstream portion,

-inhibiting the flow of said liquid before removing said seal, and

-allowing the liquid to pass through the barrier from the upstream portion to the downstream portion after removing the seal.

2. The microfluidic device of claim 1, wherein a bridge is disposed adjacent to the barrier, and wherein the bridge facilitates liquid flow from the upstream portion to the downstream portion via or over the barrier after removal of the seal.

3. The microfluidic device of claim 2, wherein the seal is further configured to inhibit liquid flow from the inlet portion to the outlet portion.

4. The microfluidic device of claim 2 or claim 3, wherein the surface of the bridge facing the barrier has a wetting contact angle with water of 90 ° or less, optionally 75 ° or less.

5. The microfluidic device of claim 4, wherein a surface of the bridge facing the barrier has a wetting contact angle with water of 20 ° or greater.

6. A microfluidic device according to claim 4 or claim 5 wherein the surface of the bridge facing the barrier is provided with a chemically hydrophilic layer or treatment, optionally a layer more hydrophilic than the untreated surface of the bridge or plasma treatment.

7. The microfluidic device of claim 4 or claim 5, wherein a surface of the bridge facing the barrier comprises a physical texture for increasing a surface area of the surface, optionally pillars, fins and/or grooves provided on the surface.

8. The microfluidic device according to any one of the preceding claims, wherein the upstream portion is filled with a liquid between the inlet channel and the outlet channel.

9. A microfluidic device for analyzing a test liquid, comprising:

a sensor disposed in the sensing chamber;

a sensing chamber inlet channel and a sensing chamber outlet channel respectively connected to the sensing chamber for respectively flowing liquid into and out of the sensing chamber, an

A receptacle forming a sample input port to the microfluidic device, the receptacle in fluid communication with the sensing chamber inlet channel;

a liquid collection channel;

a barrier between an end of the sensing chamber outlet channel and the liquid collection channel;

a first seal covering the sample input port;

a second seal covering the end of the sensing chamber outlet channel, thereby preventing liquid from flowing from the sensing chamber across the barrier into the liquid collection channel;

wherein the microfluidic device is filled with a liquid from a first seal at the sample input port to a second seal at the end of the sensing chamber outlet channel, such that the sensor is covered by liquid and not exposed to a gas or gas/liquid interface; and

wherein the first and second seals are removable to flow liquid between the container and the end of the sensing chamber outlet channel such that some liquid flows through the barrier.

10. The microfluidic device according to any one of the preceding claims, further comprising a barrier cover forming a bridging channel above the barrier for connecting the sensing chamber outlet channel to the liquid collection channel.

11. The microfluidic device of claim 10, wherein a surface of the barrier cover facing the barrier has a wetting contact angle with water of 90 ° or less, optionally 75 ° or less.

12. The microfluidic device of claim 11, wherein a surface of the barrier cover facing the barrier has a wetting contact angle with water of 20 ° or greater.

13. The microfluidic device of any one of claims 10 to 12, wherein the barrier cover is biased toward a position connecting the sensing chamber outlet channel to the liquid collection channel.

14. The microfluidic device of any one of claims 10 to 13, wherein the second seal is located below the barrier cover between the end of the sensing chamber outlet channel and the bridging channel.

15. The microfluidic device of claim 14, further comprising a release liner coupled to the second seal to aid in removal of the seal.

16. A microfluidic device according to claim 15 when dependent on claim 4 wherein the handle forms part of the release liner.

17. The microfluidic device of claim 16, wherein at least a portion of the release liner is positioned between the second seal and the barrier cover.

18. The microfluidic device of any one of claims 10-17, wherein the barrier cover further comprises a scoop extending from the bridging channel toward the sensing chamber outlet channel to facilitate flow to the bridging channel.

19. The microfluidic device of any one of claims 10 to 18, wherein the bridging channel comprises a bend connected to a downcomer alongside the barrier, and wherein the bend comprises a curved profile on at least one side.

20. The microfluidic device according to any one of the preceding claims, wherein the device is configured such that removal of the first and second seals does not result in exposure of the sensor to a gas or gas/liquid interface.

21. A microfluidic device as claimed in any one of the preceding claims wherein the first and second seals are connected such that they can be removed together.

22. The microfluidic device of claim 21, further comprising a seal handle attached to the first and second seals, the seal handle being pulled to remove the first and second seals.

23. The microfluidic device of any one of the preceding claims, wherein the liquid collection channel comprises a bend between a downcomer alongside the barrier and a main portion of the liquid collection channel, and wherein the bend comprises a curved profile on at least one side.

24. The microfluidic device of claim 15 or any claim dependent thereon, wherein the second seal is attached to a surface of the microfluidic device by a glue having a greater or lesser hydrophilicity than the surface.

25. The microfluidic device of claim 10 or any claim dependent thereon, wherein the barrier cover is biased to urge contact between the end of the sensing chamber outlet channel and the bridging channel.

26. The microfluidic device of claim 10 or any claim dependent thereon, wherein the barrier cover has a gasket to seal between the end of the sensing chamber outlet channel and the bridging channel.

27. A method of making a microfluidic device according to any one of the preceding claims, the method comprising removing the first and second seals, thereby causing liquid to flow between the container and the end of the sensing chamber outlet such that some liquid flows through the barrier to activate the device.

Technical Field

The present invention relates to microfluidic devices, in particular devices comprising sensors for sensing under wet conditions.

Background

Various microfluidic devices and sensors are known. Sensors such as those disclosed by WO99/13101 and WO88/08534 are provided in a dry state and a liquid test sample applied to the device is transported by capillary flow to the sensor area within the device. Other types of sensors are known, such as ion-selective sensors comprising ion-selective membranes.

Another example is provided by WO2009/077734, which discloses an apparatus for producing layers of amphiphilic molecules, now briefly discussed with reference to fig. 1.

Fig. 1 shows an apparatus 1 that can be used for forming layers of amphiphilic molecules. The device 1 comprises a body 2 having a layered structure, comprising a substrate 3 of non-conductive material supporting a further layer 4 of non-conductive material. The recess 5 is formed in the further layer 4, in particular as a hole extending through the further layer 4 to the substrate 3. The device 1 further comprises a cover 6 extending over the body 2. The lid 6 is hollow and defines a chamber 7, the chamber 7 being closed except for an inlet 8 and an outlet 9, both the inlet 8 and the outlet 9 being formed by openings through the lid 6. The lowermost wall of the chamber 7 is formed by the further layer 4.

In use, aqueous solution 10 is introduced into the chamber 7 and a layer 11 of amphiphilic molecules is formed across the recesses 5 to separate the aqueous solution 10 in the recesses 5 from the remaining volume of aqueous solution in the chamber 7. The use of a closed chamber 7 allows the aqueous solution 10 to flow very easily into and out of the chamber 7. This can be done simply by flowing the aqueous solution 10 through the inlet 8 until the chamber 7 is filled, as shown in figure 1. In the process, the gas (usually air) in the chamber 7 is displaced by the aqueous solution 10 and is discharged through the outlet 9.

The device comprises an electrode arrangement to allow measurement of an electrical signal across the layer 11 of amphiphilic molecules, which allows the device to be used as a sensor. The substrate 3 has a first conductive layer 20 deposited on the upper surface of the substrate 3 and extending below the further layer 4 to the recess 5. The portion of the first conductive layer 20 located below the recess 5 constitutes an electrode 21, and the electrode 21 also forms the lowermost surface of the recess 5. The first conductive layer 20 extends outside the further layer 4 such that a portion of the first conductive layer 20 is exposed and constitutes a contact 22.

The further layer 4 has a second conductive layer 23 deposited thereon and extending into the chamber 7 below the cover 6, the part of the second conductive layer 23 located inside the chamber 7 constituting an electrode 24. The second conductive layer 23 extends to the outside of the cover 6 so that a portion of the second conductive layer 23 is exposed and constitutes a contact 25. Electrodes 21 and 24 are in electrical contact with the recess 5 and the aqueous solution in the chamber 7. This allows the electrical signal across the layer of amphiphilic molecules 11 to be measured by connecting an electrical circuit to the contacts 22 and 25.

In practice, the device of fig. 1 may have an array of many such recesses 5. Each recess is provided with a layer 11 of amphiphilic molecules. Furthermore, each layer may be provided with nanopores to allow other molecules to pass through the layer (which affects the measured electrical signal). For example, one nanopore is provided per membrane. The extent to which this occurs depends in part on the concentration of nanopores in the medium applied to the membrane.

WO2012/042226 discloses an analytical device comprising means for providing an amphiphilic membrane and a nanopore to a sensor. The step of providing the amphiphilic membrane and the nanopore is typically performed prior to use of the device by an end user. However, it has the disadvantage that an extra step is required on the part of the consumer, and it is also necessary to provide the device with a complex fluid arrangement comprising a valve and a supply reservoir. Furthermore, providing such sensors for use by a user may be prone to error. The risk lies in: even if the system is set correctly, it will dry out, possibly damaging the sensor. The risk is also: excessive flow rates in the sample chamber may cause sensor damage. For more compact devices, this risk increases, bringing the sample input port closer to the sensor (and thus reducing the chance of system losses, thereby reducing the flow rate through the device).

It is therefore desirable to provide the user with a device in a "ready-to-use" state, wherein the amphiphilic membrane and the nanopore are pre-inserted and maintained in a humid condition. More generally, it is also desirable to provide a device in which the sensor is provided in a wet condition, for example to or by a user in a wet condition prior to detection of the analyte.

A typical nanopore device provided in a "ready-to-use" state comprises an array of amphiphilic membranes, each membrane comprising a nanopore and spanning a liquid-containing pore. WO2014/064443 discloses such a device and a manufacturing method. The test liquid to be analyzed is applied to the upper surface of the amphiphilic membrane. However, a device provided in a "ready-to-use" state also needs to take into account the additional factor that the sensor does not dry out, i.e. that liquid is not lost from the pores through the amphiphilic membrane, which may result in loss of performance or damage to the sensor. One solution to the problem of sensor dry-out is to provide the device with a buffer on the surface of the amphiphilic membrane so that any evaporation through the surface of the membrane is minimised and the liquids provided on either side of the membrane can have the same ionic strength to reduce any osmotic effects. In use, the buffer may be removed from the surface of the amphiphilic membrane and the test liquid to be analysed introduced to contact the surface. When the device contains a buffer, how to remove it and how to introduce the test liquid becomes a problem. Due to the presence of the buffer, i.e. the sensor is set in a "wet state", the capillary force provided by the dry capillary channel cannot be used to draw the test liquid into the sensor. Pumps may be used to displace the buffer and introduce the test liquid, but this results in increased complexity and cost of the device.

Ion-selective electrode devices comprising one or more ion-selective membranes are typically calibrated prior to use by solutions having known ion concentrations. The ion selective membrane may be provided in a capillary flow path connected to a fluid inlet through which a calibration solution may be introduced and caused to flow by capillary action through the ion selective electrode. Thereafter, the calibration solution may be displaced and the analyte solution flowed through the electrodes to perform the measurement. In large bench-top devices for measuring ions, for example, peristaltic pumps may be used to displace the liquid. However, for simple disposable devices, less complex solutions are more desirable.

In other devices, a pair of electrodes may be provided in a capillary passage, and a first test liquid may be drawn into the capillary passage by capillary action for electrochemical analysis. After measuring the first test liquid, it may be desirable to measure a second test liquid. However, since capillary forces are no longer available, additional force intervention is required in order to remove the first test liquid before introducing the second test liquid.

PCT/GB2017/052910, incorporated herein by reference, discloses a device 100 that can be used to form a layer of amphiphilic molecules similar to those in fig. 1 and 2, shown in fig. 10. However, in contrast to fig. 1 and 2, the apparatus 100 of fig. 10 is made of detachable components. Thus, the constituent components of the apparatus 100 may be provided as a kit.

The first component 110 forms the base of the device 100, while the second component 120 can be inserted into the base component 110 and removed from the base component 110. The base assembly 110 itself may be made up of multiple components 111, 112. When inserted, the first and second assemblies 110, 120 form connections between the first and second arrays of electrical connectors (discussed further below). This allows multiple second assemblies to be used with a single base assembly 110. The body of the second component is typically made of a plastics material having a degree of resilience. The plastic material may for example be polycarbonate.

In the device of fig. 10, a disposable flow cell is provided as a second component 120. The flow cell may be identical to the flow cell discussed in WO2014/064443, which is hereby incorporated by reference in its entirety. In the arrangement of fig. 4, the ability to provide a disposable flow cell 120 means that more expensive components of the assay device 100 can be incorporated into the first component 110, so that multiple experiments can be performed with different flow cells 120 relatively cheaply. As such, the flow cell 120 may include features corresponding to the notches and apertures 5 described with respect to fig. 1 and 2. Meanwhile, for example, the circuit element 61 and the rail 62 shown in fig. 2 may be provided in the base portion 110.

In view of the above, it remains a challenge to provide an easy to use microfluidic device that can be disposable or reusable while being provided in a ready-to-use manner.

Disclosure of Invention

The present invention aims to at least partially reduce or overcome the above problems.

According to one aspect of the present invention, there is provided a microfluidic device for analysing a test liquid, comprising one or more of: a bridgeable barrier; an upstream portion located upstream of the bridgeable barrier for receiving a sensor arranged in the sensing chamber and for receiving a test liquid to be analysed, the upstream portion comprising an inlet channel and an outlet channel and being fillable therebetween with the liquid; a downstream portion downstream of the bridgeable barrier for receiving liquid from the outlet passage of the upstream portion; a removably mounted seal configured to close the upstream portion and, when liquid is provided in the upstream portion, inhibit liquid flow prior to removal of the seal and allow liquid to pass through the barrier from the upstream portion to the downstream portion after removal of the seal. In this way, the device can retain liquid in the upstream portion before it is activated by removal of the seal. The liquid is retained in the upstream portion by the seal, preventing the liquid from flowing past the barrier or back from the inlet passage. Upon activation, liquid may flow through the barrier into the downstream portion.

Optionally, a bridge is provided adjacent the barrier, wherein the bridge facilitates the flow of liquid from the upstream portion to the downstream portion via or over the barrier after the seal is removed.

Optionally, the seal is further configured to inhibit liquid flow from the inlet portion to the outlet portion.

Optionally, the barrier-facing surface of the bridge has a wetting contact angle with water of 90 ° or less, optionally 75 ° or less. Optionally, the surface of the bridge facing the barrier has a wetting contact angle with water of 20 ° or more, although the contact angle may be as low as 0 °. In this way, the surface may suitably be hydrophilic to promote flow without causing undesired drainage of the sensing chamber and air ingress at the inlet.

Optionally, the barrier-facing surface of the bridge is provided with a chemically hydrophilic layer or treatment, optionally a layer more hydrophilic than the untreated surface of the bridge or the plasma treatment. The surface may be provided with one or more such layers, for example a layer of additional material and other chemical treatments, such as chemicals evaporated from a solvent. The surface may also or independently include a physical texture for increasing the surface area of the surface, optionally including posts, fins, and/or grooves disposed on the surface.

Optionally, the upstream portion may be filled with liquid between the inlet channel and the outlet channel.

According to another aspect, there is provided a microfluidic device comprising one or more of: a sensor disposed in the sensing chamber; a sensing chamber inlet channel and a sensing chamber outlet channel connected to the sensing chamber, respectively, for flowing liquid into and out of the sensing chamber, respectively, and a receptacle forming a sample input port of the microfluidic device, the receptacle being in fluid communication with the sensing chamber inlet channel; a liquid collection channel; a barrier between one end of the sensing chamber outlet channel and the liquid collection channel; a first seal covering the sample input port; a second seal covering the end of the sensing chamber outlet channel, thereby preventing liquid from flowing from the sensing chamber across the barrier into the liquid collection channel; wherein the microfluidic device is filled with a liquid from a first seal at the sample input port to a second seal at an end of the sensing chamber outlet channel such that the sensor is covered by the liquid and not exposed to the gas or gas/liquid interface; and wherein the first seal and the second seal are removable to allow liquid to flow between the reservoir and the end of the sensing chamber outlet channel, thereby allowing some liquid to flow through the barrier. Such a device reliably maintains the sensor in a state protecting the sensor before the seal is removed ("inactive" state), but it is simple for the user to activate it to the "active" state by removing the seal so that the device can be used for its sensing purposes.

The outlet channel may have a first end connected to the sensing chamber and a second end that may be covered by a second seal. The barrier may be between the second end of the sensing chamber outlet channel and the liquid collection channel.

Optionally, the surface of the barrier cover facing the barrier has a wetting contact angle with water of 90 ° or less, optionally 75 ° or less. Optionally, the surface of the barrier cover facing the barrier may have a wetting contact angle with water of 20 ° or more, but the contact angle may be as low as 0 °. In this way, the surface may suitably be hydrophilic to promote flow without causing undesired drainage of the sensing chamber and air ingress at the inlet.

The first seal may cover the container.

Optionally, the device is configured such that removal of the first and second seals does not cause exposure of the sensor to a gas or gas/liquid interface. This can be achieved by balancing the capillary forces on the device.

Optionally, the first and second seals are connected such that they can be removed together. Optionally, the device further comprises a seal handle attached to the first and second seals that can be pulled to remove the first and second seals. This allows the device to be activated by a simple single action.

Optionally, the device further comprises a barrier cover forming a bridging channel above the barrier for connecting the sensing chamber outlet to the liquid collection channel. The barrier cover may be biased towards a position connecting the sensing chamber outlet to the liquid collection channel. A second seal may be positioned below the barrier cover between an end of the sensing chamber outlet channel and the bridging channel. A release liner may be attached to the second seal to assist in removing the seal. The handle may form part of the release liner. A release liner may be positioned between the second seal and the barrier cover. Thus, in the active state, the barrier cover helps complete the fluid path through the device. The provision of a seal and/or release liner between the barrier and the barrier cover provides a convenient and easy to use way to deactivate the device, which can be easily reversed by the user to activate the device.

Optionally, the barrier cover further comprises a scoop extending from the bridge channel towards the sensing chamber outlet channel to facilitate flow into the bridge channel. The bridging channel may comprise a bend that connects to the downcomer alongside the barrier (in the direction in which the bridging channel is arranged above the barrier), and wherein the bend comprises a curved profile on at least one side. The liquid collection channel may comprise a bend between the downcomer alongside the barrier and a major portion of the liquid collection channel, and wherein the bend comprises a curved profile on at least one side. These features help ensure that the meniscus pinning (meniscuses pinning) does not impede flow through the device during activation and/or first use of the device.

Optionally, the second seal is attached to the surface of the microfluidic device by a glue that is more or less hydrophilic than the surface.

Optionally, the barrier cover is biased to promote contact between the end of the sensing chamber outlet channel and the bridging channel. The barrier cover may have a gasket to seal between the end of the sensing chamber outlet channel and the bridge channel. These features ensure that a good seal is provided in the active state.

According to another aspect, there is provided a method of making a microfluidic device according to any one of the preceding claims, the method comprising removing the first and second seals, thereby causing liquid to flow between the container and the end of the sensing chamber outlet such that some liquid flows through the barrier to activate the device.

Drawings

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

fig. 1 shows a prior art apparatus that may be used to form layers of amphiphilic molecules;

FIG. 2 shows an example of a microfluidic device;

FIG. 3 shows an exemplary design of a circuit;

fig. 4a shows a schematic view of a device corresponding to the microfluidic device of fig. 2;

FIG. 4b shows a schematic cross section along the flow path of the device of FIG. 4 a;

FIG. 5a is a schematic cross section of a sensing chamber and surrounding connections of a device such as that of FIG. 2 or FIG. 4;

figure 5b shows a scenario in which the activated device is tilted to cause fluid in the device to drain into the waste collection channel;

FIG. 5c shows the height difference between the inlet and the outlet;

5d to 5f show a scene of the sensing chamber;

FIG. 6 is a schematic plan view of a microfluidic device of an alternative construction;

FIGS. 7 and 8 illustrate an exemplary embodiment of the present invention;

fig. 9 shows an exemplary design of a guide channel for guiding a pipette to a sample input port;

fig. 10 shows a multi-part microfluidic device;

fig. 11 shows an alternative multi-part microfluidic device;

fig. 12 is a perspective view of the flow cell assembly of the multi-part microfluidic device of fig. 11 from above;

fig. 13 is a perspective view of the flow cell assembly of the multi-part microfluidic device of fig. 11 from below;

fig. 14 shows a schematic cross-sectional view of a flow cell assembly of the multi-part microfluidic device of fig. 11;

fig. 15 shows a schematic cross-sectional view of a barrier cover member of a flow cell assembly of the multi-part microfluidic device of fig. 11;

fig. 16 shows a schematic cross-sectional view of an alternative barrier cover element of a flow cell assembly;

FIG. 17 shows a perspective view of the flow cell assembly from above with the seal removed in FIG. 17a and replaced in FIG. 17 b; and

fig. 18 is a schematic cross-sectional view of adding a sample to a sample port.

Detailed Description

The present disclosure allows microfluidic devices that use "wet sensors" (i.e., sensors that function in a humid environment) to be produced and stored with the sensors remaining wet until it is needed. This is effectively achieved by providing a device having an "inactive" state in which the sensor remains wet but the device is not in use, and an "active" state in which the device can be used. In other words, the "inactive" state may be a state in which the flow path between the sample input port and the liquid collection channel is not complete, as described below. Conversely, an "activated" state may be a state in which the flow path between the sample input port and the liquid collection channel is complete. A particular benefit of keeping the sensor wet when considering a nanopore sensor (see more details below) is to ensure that the pore fluid does not escape through the membrane. The film is very thin and the sensor is very sensitive to moisture loss. The loss of moisture can create, for example, a resistive air gap between the pore fluid and the membrane, thereby breaking the electrical circuit between the electrodes disposed in the pores and in the sample. Moisture loss can also be used to increase the ionic strength of the pore liquid, which can affect the potential difference across the nanopore. The potential difference has an effect on the measurement signal and therefore any change will have an effect on the measurement value.

In any case, the device of the invention can remain in the "inactive" state for a long time until it is needed. During this time, for example, the device may be shipped (e.g., from a supplier to an end user) because the "inactive" state is robust and capable of maintaining the sensor in a wet state even when the device is in a non-standard orientation (i.e., an orientation in which the device is not used to perform its normal function). This is possible because the inactive state seals off the device interior volume containing the sensor from the surrounding environment. The internal volume (hereinafter referred to as the "saturation volume") is filled with liquid. The absence of any air gaps and/or bubbles means that the sensor does not have the potential to intersect the gas/air interface (which could impair the functionality of the sensor) even if the device is moved. Furthermore, even in the activated state, the device can keep the sensor in the wet state for a long time even if the device is activated and then not used.

Fig. 2 shows a top cross-sectional view of an example of a microfluidic device 30, with an inset showing a side cross-sectional view of a portion of the microfluidic device including a sample input port 33. The microfluidic device 30 comprises a sensing chamber 37 for housing the sensor.

The sensing chamber 37 is provided with a sensor, which is not shown in fig. 2. The sensor may be a component or a device for analyzing a liquid sample. For example, a sensor may be a component or device for detecting a single molecule (e.g., biological and/or chemical analyte, such as ions, glucose) present in a liquid sample. Different types of sensors for detecting biological and/or chemical analytes (e.g., proteins, peptides, nucleic acids (e.g., RNA and DNA)) and/or chemical molecules are known in the art and can be used in sensing chambers. In some embodiments, the sensor comprises a membrane configured to allow ions to flow from one side of the membrane to the other side of the membrane. For example, the membrane may comprise a nanopore, such as a protein nanopore or a solid state nanopore. In some embodiments, the sensor may be of the type discussed above with reference to fig. 1, described in WO2009/077734, the contents of which are incorporated herein by reference. In use, the sensor is connected to the electrical circuit. The sensor may be an ion selective membrane provided directly on the surface of the electrode or on an ionic solution in contact with the underlying electrode.

The sensor may comprise an electrode pair. One of the plurality of electrodes may be functionalized to detect the analyte. One or more of the electrodes may be coated with a permselective membrane, such as nafion (tm).

An exemplary design of such a circuit 26 is shown in fig. 3. The primary function of the circuit 26 is to measure the electrical signal (e.g. current signal) generated between the common electrode first body and the electrodes of the electrode array. This may simply be an output of the measurement signal, but may in principle also involve further analysis of the signal. The circuit 26 needs to be sensitive enough to detect and analyze the typically very low currents. For example, an open membrane protein nanopore can typically pass a current of 100pA to 200pA in the presence of a 1M salt solution. The selected ion concentration may vary and may be, for example, between 10mM and 2M. Generally, the higher the ion concentration, the higher the current under a potential or chemical gradient. The magnitude of the potential difference applied across the membrane will also affect the current through the membrane and may typically be chosen to be a value between 50mV and 2V, more typically a value between 100mV and 1V.

In this embodiment, the electrode 24 serves as an array electrode, and the electrode 21 serves as a common electrode. Thus, circuit 26 provides a bias voltage potential to electrode 24 relative to electrode 21, which is itself at virtual ground potential, and a current signal to circuit 26.

The circuit 26 has a bias circuit 40 connected to the electrode 24 and arranged to apply a bias voltage which is effectively present between the two electrodes 21 and 24.

The circuit 26 also has an amplifier circuit 41 connected to the electrode 21 for amplifying the current signal present between the two electrodes 21 and 24. In general, the amplifier circuit 41 is composed of two amplifier stages 42 and 43.

An input amplifier stage 42 connected to the electrode 21 converts the current signal into a voltage signal.

The input amplifier stage 42 may include a transimpedance amplifier, such as an electrometer operational amplifier configured as an inverting amplifier with a high impedance feedback resistor, e.g., 500M Ω, to provide the gain required to amplify current signals, typically having magnitudes on the order of tens to hundreds of pA.

Alternatively, the input amplifier stage 42 may comprise a switched integrating amplifier. This is preferable for very small signals because the feedback element is a capacitor and is almost noise free. In addition, the switched integrator amplifier has a wider bandwidth capability. However, the integrators do have a time lag, since they have to be reset before output saturation occurs. The skew can be reduced to about microseconds, so if the required sampling rate is much higher, it has no major impact. If the required bandwidth is smaller, the transimpedance amplifier is simpler. Typically, the switched integrating amplifier output is sampled at the end of each sampling period, followed by a reset pulse. Other techniques may be used to sample the start of integration to eliminate small errors in the system.

The second amplifier stage 43 amplifies and filters the voltage signal output by the first amplifier stage 42. The second amplifier stage 43 provides sufficient gain to raise the signal to a level sufficient for processing in the data acquisition unit 44. For example, given a typical current signal on the order of 100pA, with a 500M Ω feedback resistance in the first amplifier stage 42, the input voltage to the second amplifier stage 43 would be on the order of 50mV, and in this case the second amplifier stage 43 would have to provide a gain of 50 to increase the 50mV signal range to 2.5V.

The circuit 26 includes a data acquisition unit 44, which may be a microprocessor running a suitable program, or may include dedicated hardware. In this case, the bias circuit 40 is simply formed by an inverting amplifier which is supplied with a signal from a digital-to-analog converter 46, which may be a dedicated device or part of the data acquisition unit 44 and provides a voltage output in accordance with a code loaded into the data acquisition unit 44 from software. Similarly, signals from the amplifier circuit 41 are supplied to the data acquisition card 40 via an analog-to-digital converter 47.

The various components of circuit 26 may be formed from separate components or any of the components may be integrated into a common semiconductor chip. The components of circuit 26 may be formed from components disposed on a printed circuit board. To process the multiple signals from the electrode array, the circuit 26 is modified essentially by duplicating the amplifier circuit 41 and the a/D converter 47 of each electrode 21 to allow signals to be taken from each well 5 in parallel. Where the input amplifier stages 42 comprise switched integrators, they will require a digital control system to process the sample-and-hold signal and reset the integrator signal. Digital control systems are most conveniently configured on field programmable gate array devices (FPGAs). In addition, FPGAs may contain similar processor functionality and logic required to interface with standard communication protocols (i.e., USB and ethernet). Since the electrode 21 is kept grounded, it is practically provided to be shared by the electrode array.

In such systems, a polymer such as a polynucleotide or nucleic acid, a polypeptide such as a protein, a polysaccharide or any other polymer (natural or synthetic) may be passed through a nanopore of appropriate size in the case of a polynucleotide or nucleic acid, the polymer units may be nucleotides, so the molecule passes through the nanopore while monitoring the electrical properties across the nanopore and obtaining a signal that is characteristic of the particular polymer unit passing through the nanopore, so this signal may be used to identify the sequence or determine the sequence characteristics of the polymer unit in the polymer molecule various different types of measurements may be made including, but not limited to, electrical and optical measurements.american chemical association (j.am.chem.soc.) 2009, 1312. abo discloses one suitable optical method involving fluorescence measurement.possible electrical measurements include current measurement, impedance measurement, tunneling measurement (ivanovovap et al. ("Nano Kuaisi (na L ett.)) transmembrane current measurement (12; day 11) and 279. RTM.: 279, WO 279, 92. RTM.) (WO 32. RTM.) (see [ RTM.) (for example: electrical current measurement in conjunction with the application No. (WO 2010. 29.) (r.) (WO) (electrical current measurement).

The polymer may be a polynucleotide (or nucleic acid), a polypeptide (such as a protein), a polysaccharide, or any other polymer. The polymers may be natural or synthetic. The polymer unit may be a nucleotide. The nucleotides may be of different types including different nucleobases.

The nanopore may be a transmembrane protein pore selected from, for example, MspA, lysenin, α -hemolysin, CsgG, or a variant or mutation thereof.

Polynucleotides can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), cDNA, or synthetic nucleic acids known in the art (e.g., Peptide Nucleic Acid (PNA), Glyceric Nucleic Acid (GNA), Threose Nucleic Acid (TNA), locked nucleic acid (L NA), or other synthetic polymers with nucleotide side chains).

In some embodiments, the devices and/or methods described herein can be used to identify any nucleotide. Nucleotides may be naturally occurring or artificial. Nucleotides typically contain a nucleobase (which may be abbreviated herein as a "base"), a sugar, and at least one phosphate group. Nucleobases are usually heterocyclic. Suitable nucleobases include purines and pyrimidines, more particularly 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 nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides typically contain a monophosphate, diphosphate or triphosphate.

Nucleotides may include damaged or epigenetic bases. Nucleotides may be labeled or modified to serve as labels with different signals. This technique can be used to identify deletions of bases, such as abasic units or spacers in a polynucleotide.

Particularly useful when considering measurements of modified or damaged DNA (or similar systems) is a method that considers supplementary data. The additional information provided allows to distinguish a large number of underlying states.

The polymer may also be a class of polymers other than polynucleotides, some non-limiting examples of which are as follows.

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

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

The conditioning fluid provided in the device for maintaining the sensor in a wet state may be any fluid compatible with the device (e.g., a fluid that does not adversely affect the performance of the sensor). By way of example only, when the sensor comprises protein nanopores, it will be apparent to one of ordinary skill in the art that the conditioning fluid should be free of agents that denature or inactivate the protein. The conditioning liquid may, for example, comprise a buffer, such as an ionic liquid or an ionic solution. The conditioning solution may contain a buffer to maintain the pH of the solution.

The sensor is one that needs to be maintained in a "wet state" (i.e., a sensor covered by a liquid). The sensor may comprise a membrane, such as an ion selective membrane or an amphiphilic membrane. The membrane, which may be amphiphilic, may include ion channels (e.g., nanopores).

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 as an ion selective channel, for detecting anions and cations. Ion channels can be selected from known ionophores (e.g., valinomycin, gramicidin, and 14 crown 4 derivatives).

Returning to fig. 2, the sensing chamber has a liquid inlet 38 and a liquid outlet 39 for respectively passing liquid into and out of the sensing chamber 37. In the inset of fig. 2, the inlet 38 is shown in cross-section of the device 30 in fluid communication with the sample input port 33. The sample input port 33 is configured for introducing (e.g., transporting) a sample to the microfluidic device 30, e.g., for testing or sensing. A seal 33A (e.g., a plug) may be provided to seal or close the sample input port 33 when the device 30 is in its inactive state, thereby preventing any fluid from entering or exiting through the sample input port 33. In this way, the seal 33A may be disposed within the sample input port 33 in an inactivated state. Preferably, the seal 33A is removable and replaceable. The sample input port may be desirably located near the sensing chamber, for example, as shown in fig. 2, where the port is disposed directly at the sensing chamber. This reduces the volume of sample liquid that needs to be applied to the device by reducing the volume of the flow path.

Downstream of the outlet 39 of the sensing chamber 37 is a liquid collection channel 32. The liquid collection channel may be a waste collection container and is used to receive fluid that has been drained from the sensing chamber 37. At the most downstream end (e.g., end) of collection channel 32 is a vent port 58 to allow venting of gas as collection channel 32 receives liquid from the sensing chamber and is filled with liquid.

In the example shown in fig. 2, upstream of the sensing chamber 37 is an optional liquid supply port 34. The liquid supply port provides an opportunity to supply liquid (e.g., buffer) into the device once the device 30 is in its activated state. It may also be used to deliver larger volumes of sample, if desired, and to flush/prime the previous sample from the sensing chamber 37 in large quantities before delivering a new sample.

As described in more detail below, the device is configured to accept a sample at the sample input port, which is then actively drawn into the sensing chamber without the aid of external forces or pressure (e.g., by capillary pressure as described below). This eliminates the need for the user to introduce the test liquid into the device under a positive applied pressure.

In fig. 2, the device 30 is in an inactive state. This is achieved by providing a valve 31 and a seal 33A on the sample input port 33, the valve 31 being configured to be in a closed state, which is a state in which fluid is not allowed to flow between the liquid collection channel 32 and the sensing chamber 37, the seal 33A sealing or closing the sample input port 33. In the inactive state, as shown in fig. 2, flow through sensing chamber 37 is not possible. The valve 31 in the closed state is a structure serving as a flow path interruption between the liquid outlet 39 of the sensing chamber 37 and the liquid collection channel 32 to prevent upstream liquid (e.g., liquid from the sensing chamber 37) from flowing into the liquid collection channel 32. Similarly, the valve 31 in the closed state is a structure serving as a flow path interruption between the supply port 34 and the sensing chamber 37 to prevent upstream liquid (e.g., liquid introduced through the supply port) from flowing into the sensing chamber 37. In this way, the sensing chamber 37 is isolated from the supply port 34 and the waste collection container (in the form of the liquid collection channel 32, which may be open to the atmosphere). Furthermore, the provision of a plug 33A sealing the sample input port 33 ensures that the sensing chamber 37 is completely isolated. The plug 33A may also serve additional purposes: when removed, it may create a "suction" in the inlet 38, ensuring that the port 33 becomes wetted (and thus ready to receive sample fluid) when the plug 33A is removed. Thus, the stopper 33A provides a priming action. The priming action may draw fluid from the liquid collection channel or a separate priming reservoir (see examples below) (e.g., indirectly, displacing fluid into sensing chamber 37 and thus into inlet 38 and port 33).

In some embodiments, the valve 31 serves a dual function. For example, as shown in fig. 2, the valve 31 may be configured such that it acts on the state of the activation system. The activation system may complete the flow path between liquid outlet 39 and liquid collection channel 32 (and between supply port 34 and sensing chamber 37). Furthermore, as discussed in more detail below, such activation occurs without draining sensing chamber 37 of liquid. That is, the sensor 37 remains unexposed to the gas or gas/liquid interface after activation. In the example of fig. 2, this is achieved by rotating the valve 31 by 90 ° within the valve seat 31A (from the orientation shown). This causes the valve channel 31B to complete the flow path interruption 36 between the liquid outlet 39 and the liquid collection channel 32 and between the buffer liquid input port 34 and the sensing chamber 37. In this activated state, liquid may flow from the buffer supply port 34 (also referred to herein as a "purge port") through the sensing chamber 37 and into the liquid collection channel 32. However, as discussed in more detail below in connection with fig. 5a to 5f, such flow does not occur freely.

As a result, the sensing chamber 37 can be pre-filled with a conditioning fluid (e.g., buffer) prior to rotating the valve 31 to the position shown in fig. 2. It should be noted that the type of conditioning fluid according to the present invention is not particularly limited, but should be suitable according to the nature of the sensor 35. Assuming that plug 33A has been inserted and sensing chamber 37 is properly filled so that there are no air bubbles, the sensor has no chance of coming into contact with the air/liquid interface that could damage the sensor. In this way, the device 30 can be operated robustly without fear of damaging the sensor itself.

Fig. 4a shows a schematic view of a device 30 corresponding to the device of fig. 2. In fig. 4, the fluid channels are simply shown as lines. Furthermore, the valves 31 are shown as two separate valves 31 upstream and downstream of the sensing chamber 37. This is for clarity, but in some embodiments it may be desirable to have two separate valves 31 as shown.

Figure 4b shows a schematic cross section of the device of figure 4a along a flow path. This may not be a "true" cross-section in the sense that the flow path may not be linear in the manner shown in fig. 4 b. Nevertheless, this schematic is useful for understanding the flow paths available for liquid in the device 30. In particular, it can be seen that the upstream buffer supply/wash port 34 is separated from the sensing chamber by the upstream valve 31. Another downstream vent port 58 can be seen separated from the sensing chamber 37 by the downstream valve 31. Thus, it is apparent that sensing chamber 37 may be filled with fluid and isolated from upstream port 34 and downstream port 58. Furthermore, by providing a seal on the sample input port 33, the sensing chamber can be completely isolated.

It is also beneficial to consider the scale of the features shown in fig. 4a and 4 b.

The wash port 34 and the sample input port 33 may be of similar design, as both are configured to receive a fluid to be delivered to the device 30. In some embodiments, ports 33 and/or 34 may be designed to accommodate the use of a liquid delivery device (e.g., pipette tip) to introduce liquid into the ports. In a preferred embodiment, both ports have a diameter of about 0.4 to 0.7mm, which allows for wicking of fluid into the ports while also limiting the possibility of free drainage of liquid by the device 30 (discussed in more detail below). In contrast, the size of the downstream vent port 58 is less critical because in conventional use, it is not intended to accept a liquid delivery device (e.g., a pipette) or to deliver liquid.

The size of the sensor may vary and depends on the type and number of sensing elements (e.g., nanopores or ion-selective electrodes) disposed in the sensor the size of the sensor 35 may be about 8 × 15 mm.

The "saturation volume" of the device 30 is the volume of the flow path connected between the valves 31 that can be filled with liquid and sealed from the surrounding environment, for example, when a plug 33a is present to seal the sample input port 33 and the valves 31 are configured in a closed state, one valve controlling the flow between the liquid outlet 39 and the liquid collection channel 32 and the other valve controlling the flow between the buffer input port 34 and the sensing chamber 37 for the valves 31. In one embodiment, the saturation volume may be about 200 μ Ι, which may vary depending on the design of the flow path in the devices described herein. However, smaller volumes are more preferred (e.g., to reduce the size of the sample required), and preferably the saturation volume is 20 μ l or less. In other configurations, it may not be necessary to provide a purge port 34 (and a fluid path connected to the sensing chamber 37), in which case the saturation volume would extend from the sealed sample input port 33 to the sensing chamber 37 and through the liquid outlet 39 to the flow path disruption 36.

Conversely, it is desirable that the liquid collection channel 32 have a volume greater than the saturation volume, e.g., at least 3 times greater, e.g., at least 4 times greater, at least 5 times greater, at least 10 times greater, or at least 15 times greater, so that it can collect liquid drained from the saturation volume over several test and flush cycles. In one embodiment, the liquid collection channel 32 may have a volume of 2000 μ l, with the hydraulic radius of the liquid collection channel typically being 4mm or less.

The size of the valve 31 is not particularly critical (and, as described below, alternative flow path interruptions may be provided). They serve to isolate the saturated volume connected to the plug 33 a.

Furthermore, even in the activated state, the device is resistant to the sensing chamber 37 becoming dry. This is discussed below with reference to fig. 5a, which is a schematic cross section of a sensing chamber 37 according to an embodiment and a surrounding connection of the device 30, e.g. of fig. 2 or 4.

In fig. 5a, the sensor 35 is arranged in a sensing chamber 37. Sensing chamber liquid inlet 38 is connected upstream of sensing chamber 37 for ease of presentation (i.e., although liquid inlet 38 is shown entering sensing chamber 37 from above in fig. 2 and 4, the change in position in fig. 5a does not affect the results of the underlying analysis). Fig. 5a shows another constriction 38a of the diameter before the liquid inlet reaches the sensing chamber 37. This may be due to, for example, an enlargement of the input port 33 to facilitate sample collection/presentation. Downstream of the sensing chamber 37 is a liquid outlet 39 leading to the liquid collection channel 32.

In the figure, several parameters and dimensions are shown. The height (in meters) is indicated by the symbol h. The radius of curvature (in meters) is indicated by the symbol R. The radius of the tubular member (in meters) is indicated by the symbol r. The surface tension (in N/m) is indicated by the symbol γ. Liquid Density (in kg/m)³In units) is denoted by the symbol ρ. Flow velocity (in m)³In units of/s) is represented by the symbol Q. The contact angle (in degrees) of the liquid/gas meniscus with the wall of the device 30 is indicated by the symbol theta. Subscript "i" is used to indicate conditions at the inlet, subscript "c" is used to indicate conditions at the constriction, and subscript "o" is used to indicate conditions at the constrictionIndicating the conditions at the outlet.

The fluid behavior in the depicted system is controlled by capillary and/or laplace bubble pressure and Poiseuille pressure drop to limit flow rate. As is well known, the capillary pressure at the meniscus can be calculated using the following equation:

equation 1

Wherein R is₁And R₂Is the radius of curvature in the vertical direction. In the case of tubes, such as capillaries, the radius of curvature R₁And radius of curvature R₂The same and the radius of curvature is related to the radius of the tube by the following equation:

equation 2

Further, in the rectangular channel, R₁And R₂Instead, the radius of curvature is given by the following equation:

equation 3

Where a is, for example, the width of the rectangular cross section and b is the height of the rectangular cross section.

For incompressible Newtonian fluids, the pressure loss can be calculated according to the Hagen-Poiseuille formula, assuming that the non-accelerated laminar flow is much longer than its diameter in a constant circular cross section pipe:

equation 4

Where μ is the viscosity of the liquid (in N.s/m)²In meters), l is the length of the pipe (in meters) over which the flow occurs, and r is the radius of the pipe (in meters).

Finally, the static pressure is calculated according to the following formula:

equation 5

P_h＝ρgh

Wherein g is the acceleration of gravity (9.8l m/s)²) And h is the height of the fluid column.

Fig. 5b shows a scenario in which the activated device 30 is tilted to cause fluid in the device 30 to drain into the liquid collection channel 32. When considering whether a fluid will remain at the opening of the inlet 38 (i.e., the sample input port 33), the capillary pressure (P) at the inlet can be understood_ci) Must be equal to or greater than the sum of any differences in the capillary pressure at the outlet and the hydrostatic pressures resulting from the inlet and outlet not being at the same level, (the difference in height being denoted as h in figure 5b and the equations below) to avoid free drainage. This is set forth in the following equation:

P_ci≥P_co+ρg.h

from this equation, in combination with equations 1 and 2, the maximum height difference h before free discharge can be derived (assuming the contact angles θ at the inlet and outlet are the same):

replacing typical values of the relevant variables (e.g. r)_i＝0.4mm，r₀＝3.0 mm，θ＝82°，ρ＝1000kg/m³And γ ═ 0.072N/m), indicating that a height differential of about 4mm can be achieved before inlet dehumidification.

Taking this into account further, and as shown in figure 5c, if the height difference exceeds the critical value, the meniscus at the input port 33 will recede to the inlet of the sensing chamber. In the limit before the meniscus separates from the inlet (i.e. gas is allowed to enter the sensing chamber 37), the meniscus will have a maximum radius of curvature equal to the radius of the inlet (ignoring any constrictions 38 a). In this case, the contact angle θ will be zero, so the non-emission scenario is described as:

P_ci≥P_h+P_co

and in the limit:

again, using the above typical values, this indicates that the allowable height difference between the inlet of the sensing chamber and the downstream meniscus and waste outlet can be of the order of 36 mm. As a result, the sensing chamber 37 is less likely to de-wet in normal use, even if the inlet port 33 itself is not kept wet, as this is a significant height difference, which indicates an unusual amount of tilt.

Furthermore, the sensing chamber is less likely to dewet by dripping out of the inlet. As shown in fig. 5d, the other extreme of the previously considered scenario is the limit before liquid starts to drip from the inlet. Also in this case, the radius of curvature of the meniscus (in the other direction at this time) is equal to the radius of curvature of the inlet capillary itself. In this case, assuming h is the height difference between the inlet meniscus and the outlet meniscus, and the outlet is raised to encourage flow out of the inlet, the non-dripping scenario is described as:

P_ci≥P_h-P_co

and in the limit:

again, an alternative typical value indicates that the maximum allowed h is of the order of 37 mm. Again, this is within the tolerance of normal processing in use.

Thus, from the above analysis, it can be seen that once the device 30 switches from the inactive state to the active state, the liquid sensor 35 will remain wet under normal conditions. Furthermore, even if the input port 33 becomes dehumidified, this does not necessarily result in the sensor being exposed to a gas/liquid interface, as this interface is likely to be fixed at the entrance of the sensing chamber 37.

It is also contemplated how this stability affects the ability to deliver the sample to the sensing chamber 37. In fig. 5e, the first extreme of wicking fluid from the "puddle" into the input port 33 is considered. In this case, the capillary pressure for drawing in the fluid is balanced by laminar flow losses in the inlet (having a length l):

using typical values (including μ ═ 8.9x10^-4N.s/m²And l ═ 3mm), a flow rate of 25. mu.l/s can be obtained. This is sufficient when the sample volume is low, for example in a microfluidic device with a total volume of about 200 μ l.

In the other extreme, as shown in fig. 5f, the sample may be supplied to the input port 33 as a droplet (e.g. a drop of blood from a finger or a drop from a pipette). In this case, the driving force is the laplace bubble pressure of the droplets:

for a 1mm droplet, the pressure is about 144 Pa (typical values are used). Compared to a puddle wicking scenario, 2D approximation shows that this is about 20 times greater, so a flow rate of about 500. mu.l/s can be expected for the same viscous drag.

As a result, it can be seen that the device 30 (e.g., the inlet 38 and outlet 39 and the size of the liquid collection channel 32) can not only be configured to robustly maintain a wetted state in the sensing chamber 37, but can also be easily operated to draw fluid into the sensing chamber 37. When the sample has been supplied, the device 30 returns to a new equilibrium where the device will not dehumidify/drain. That is, the device 30 is configured to avoid free draining of the sensing chamber 37. In particular, the sample input port 33, sensing chamber inlet 38 and liquid collection channel 32 are configured to avoid such venting, such that when the activation system is operated to complete the flow path downstream of the sensing chamber 37, the sensor 35 remains unexposed to the gas or gas/liquid interface, even when the device 30 is tilted. In other words, sensing chamber inlet 33 and liquid collection channel 32 are thus configured to balance capillary pressure and flow resistance to avoid free draining of sensing chamber 37 when the flow path is completed.

In considering how to configure the sensing chamber inlet and liquid collection channel to balance capillary pressure and flow resistance, it is helpful to consider how the device actually works. Priming of the device to its "activated state" is achieved by completing the flow path between the liquid outlet and the liquid collection channel 32. The capillary pressures at the downstream collection channel and the sample input port are balanced so that upon activation of the device, gas is not drawn into the sample inlet port and the sample input port presents a wetted surface to the test liquid. If the capillary pressure at the fluid collection channel is greater than the capillary pressure at the sample input port, the device will vent upon activation and buffer fluid is drawn into the collection channel.

After activation of the device and before addition of the test liquid, the device can be considered to be in an equilibrium state, i.e. where 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, so that the input port presents a wetted surface to the test liquid to be introduced into the device. The device is configured to ensure a balance of forces such that the sensing chamber remains filled with liquid and the liquid remains (at least partially) in the inlet, outlet and liquid collection channels. If the equilibrium is disturbed by changing the position of the liquid (without adding or removing liquid from the system), the return to the equilibrium is urged. As the liquid moves, it will create a new gas/liquid interface. Thus, the balance of forces and the restoration of equilibrium will be effectively controlled by capillary forces at these interfaces.

Ideally, the balance of forces is such that upon activation or addition of a volume of liquid, the liquid fills the sample input port and presents a wetted surface. However, some adjustment may be required after activation/priming in order to provide a wetted surface at the sample input port. In any case, the inlet port is configured such that after the test liquid is added to the port, the capillary pressure at the input port is less than the capillary pressure at the downstream collection channel. This provides a driving force to draw test liquid into the device, thereby moving liquid from the sensing chamber into the liquid collection channel. This continues until the pressures at the sample input port and the liquid collection channel again reach equilibrium. This driving force may be provided by changing the shape of a volume of liquid applied to the input port, as shown in equation 1, where a volume of fluid applied to the port (e.g., with a particular radius of curvature as shown in fig. 5 f) "collapses" into the port, thereby reducing the effective rate of bending and providing a laplace pressure (e.g., due to the head of the volume of test liquid, there may also be other components of the total driving pressure that may decrease over time as the volume is introduced into the device). The liquid inlet diameter is advantageously smaller than the diameter of the liquid collection channel so that the fluid is at the input port and on the sensor and the liquid is present in the device as a continuous phase rather than a discrete phase separated by a gas.

An additional volume of sample may then be applied to the device to further displace the buffer from the sensing chamber. This may be repeated multiple times such that the buffer is removed from the sensor in the sensing chamber and replaced with the test liquid. The number of times required to completely displace the buffer from the sensor will be determined by the internal volume of the device, the volume of test sample applied, and the degree of driving force achievable.

Thus, in this particular embodiment, the test liquid can be drawn into the device and the buffer displaced, for example by using a pipette, without the need for the user to apply an additional positive pressure. This simplifies the application of the test liquid to the device. Surprisingly and advantageously, the present invention provides a device that can be provided in a "wet state" in which a liquid can be removed from the device by applying only another liquid to the device.

Furthermore, the above analysis only considers linear configurations. Fig. 6 is a schematic plan view of an exemplary microfluidic device 30 in an alternative configuration. In this configuration, the waste collection channel 32 downstream of the outlet 39 of the chamber 37 is disposed in a tortuous or tortuous path to maintain the channel 32 within a defined maximum radius from the sample input port 38. Such a configuration allows for a large length (and therefore volume) of the waste collection channel 32 while maintaining the maximum distance of the downstream meniscus within a maximum radius. The maximum allowed radius is determined by the allowed height difference between the input port 38 and the downstream meniscus, which does not result in the sensing chamber 37 being vented. In other words, a purely linear arrangement will result in the meniscus reaching the maximum allowable height difference after a certain amount of use, but in a curved arrangement the meniscus is shifted back closer to the input port 33, and thus the critical condition is not reached. This is because the curved arrangement keeps the downstream meniscus closer to the input port, requiring a larger angle of inclination to achieve the same height difference (for any given amount of liquid in the downstream channel, only the path of the channel changes, provided the dimensions of the channel do not change).

Furthermore, even if the sample input port 33 does dehumidify, the device 30 may be operable to re-activate the system. In the examples of fig. 2 and 4, additional liquid may be supplied directly to the inlet 38 through the sample input port 33. Alternatively, rewetting may be facilitated by drawing liquid from outlet 39 and sensing chamber 37 back into inlet 38 and sample input port 33. Another alternative is to provide additional fluid through the buffer supply port 34.

However, in other embodiments, at least the downstream portion of the valve 31 of the embodiment of fig. 2 may be omitted and replaced by another form of flow path disruption. For example, the downstream waste channel 32 may be isolated from the saturation volume by a surface treatment (e.g., a hydrophobic substance), which will effectively form a barrier to upstream liquids until the disruption is removed by forced flow induced by a priming or flushing action. This surface treatment is in fact a trap. In fact, the disruption 36 may be any flow obstruction that may be removed or overcome by the activation system.

Fig. 7 and 8 are exemplary embodiments of the devices described herein.

Fig. 7 shows a device 30 in which a pipette 90 is used to supply a sample to the input port 33. In this example, the port 33 is centrally disposed above the sensor in the sensing chamber 37. In this example and the example of FIG. 8, a valve 31 of the type shown in FIG. 2 is provided (i.e., a single valve that opens and closes both the upstream and downstream passages of sample chamber 37).

In fig. 8, the main image of the device 30 shows the presence of a plug or seal 33A on the sample input port. The expanded image shows the plug 33A removed, revealing the underlying sample input port 33. In this example, the sample input port 33 is disposed at the most upstream end of a sensing chamber 37 containing a sensor 35. This is advantageous because in the activated state with the upstream wash port 58 closed, the sample chamber 37 can be quickly filled by forcing sample through port 33 in order to displace buffer already downstream of the sample chamber (i.e. upstream displacement is not possible due to the closed wash port 58).

Some operational scenarios of the microfluidic device 30 of the present invention (i.e., as shown in fig. 8) will now be discussed.

In the first configuration, valve 31 is open and sample port 33 is also open (i.e., plug 33A is not present). The wash port/buffer supply port 34 is closed. In this configuration, a pipette may be used at the vent port 38 to withdraw all liquid, including withdrawing liquid from the sample cell. Alternatively, if liquid is supplied to the port, it displaces fluid through the waste reservoir 32 into the sensing chamber 37 and out the sample port 33.

In another configuration, the valve 31 and the sample input port 33 are open and the vent port 58 is sealed. In this scenario, the pipette may provide fluid into the purge port 34, forcing the fluid through the sample cell into the sample chamber 37 (i.e., to a saturated volume) and downstream into the reservoir 32. This also results in the sample input port 33 becoming wet if it has been de-wetted. Alternatively, if a pipette is used to expel liquid, the sensing chamber and the upstream portion of the device may expel liquid.

In another configuration, the valve 31, purge port 34, and vent port 58 are all open. In this configuration, a pipette may be supplied to the sample input port 33 to provide a sample into the sensing chamber. Alternatively, the sensing chamber 37 may drain liquid if a pipette is used to drain liquid from the sample input port 33. If this is done slowly, the liquid may also be withdrawn from the waste reservoir 32.

In another scenario, the valve 31 and purge port 34 are open, while the vent port 58 is closed. In this scenario, fluid may be applied through sample input port 33 to force fluid out of wash port 34, if desired. Alternatively, extracting liquid from the sample input port 33 will draw air into the sample cell via the purge port.

In another configuration, the valve 31 and vent port 58 are open, while the purge port 34 is closed. In this scenario, fluid supplied to the sample input port 33 can be pushed into the sample cell more quickly without fluid escaping from the wash port. Alternatively, if done quickly, extracting fluid from the sample input port 33 in this scenario will cause the sample cell and downstream waste reservoir to drain.

In the other two configurations, the valve 31 is closed. In some configurations, closing the valve 31 may connect the upstream purge port 34 to the downstream waste reservoir 32 while isolating the sensing chamber (i.e., in the arrangement of fig. 2, the upstream purge port 34 is not so connected to the downstream waste reservoir 32, but increasing the length of the valve channel 31B may result in such a connection). When such a connection is made, waste may be filled from the vent port 58 (i.e., causing any liquid to overflow from the purge port 34) or from the purge port 34 (i.e., causing any liquid to overflow from the vent port 58). In addition, waste can be emptied by withdrawing liquid from either the purge port 34 or the vent port 58 (assuming the other is open).

Fig. 9 shows an exemplary design of a guide channel 91 extending from a sample input port 92 of a portion of the device 90. The guide channel tapers outwardly from the port and is used to guide a pipette tip 100 applied to the channel to the sample input port. The guide channel also slopes downward toward the sample input port, which facilitates movement of the pipette tip to the port. Once the pipette tip is directed to the sample input port, the user can apply a liquid sample from the pipette tip to the port. The collar 93 serves to define the area of the channel and serves as a support for the pipette tip applied directly to the sample input port. Due to the size of the port (e.g., the diameter may be 1mm or less), it can be challenging for a user to position the pipette tip directly at the sample input port itself. The outwardly tapering channel region provides a larger target area for a user to position and direct the pipette tip to the sample input port, if desired.

Fig. 11 shows a device similar to fig. 10. The device 200 has a first component 210 that forms the base of the apparatus 200, while a second component 220 can be inserted into the base component 210 and removed from the base component 210. The base component 210 itself may be made up of multiple components 211, 212. The first and second components 210, 220 each have a respective array of electrical connectors that form a connection with each other when the first and second components 210, 220 are connected. This allows multiple second assemblies to be used with a single base assembly 210. The body of the second component 220 is typically made of a plastic material having a degree of elasticity. The plastic material may for example be polycarbonate.

The second component 220 in fig. 11 is a microfluidic device, i.e. a flow cell. The flow cell 220 is shown in perspective view in fig. 12 and 13. Fig. 12 shows a view from above, while fig. 13 shows a view from below. In fig. 13, a connector array (not shown) forms the bottom of the sensor 235. The base 210 of fig. 11 may have a corresponding array of electrical connectors connected to the array on the flow cell 220.

Fig. 14 shows a schematic cross section through a flow cell 220. Sensor 235 is disposed in sensing chamber 237. A liquid (e.g., a buffer or sample to be tested) may be supplied to the sensing chamber via the inlet channel 261. Similarly, liquid may exit the sensing chamber through outlet channel 262. The inlet channel 261 and the outlet channel 262 are separate channels to allow fluid to flow continuously through the sensing chamber from the inlet channel 261 to the outlet channel 262.

The flow cell 220 may be configured such that the flow path through the device is made of a material with good liquid retention properties. That is, the material is substantially liquid impermeable and may also be non-porous. This applies in particular to the upstream part comprising the wetting volume before activation, i.e. the part comprising the inlet channel 261, the chamber 237 and the outlet channel 262. Downstream portions, such as the bridging channels discussed below, do not require such high liquid retention characteristics because they are not exposed to the fluid until activated. In any case, examples of suitable barrier materials include Cyclic Olefin Copolymers (COC) or Cyclic Olefin Polymers (COP), which are highly rigid and transparent. Other suitable materials, although softer and translucent rather than transparent, include Polyethylene (PE) based and polypropylene (PP) based materials. However, the flow cell 220 may also include additional coatings, co-extrusions, laminates or portions made of the lower barrier material (optionally in combination with an auxiliary barrier as part of the device packaging). That is, the surface of the flow path may be made of a material having good liquid retaining properties, and the surrounding material may be different.

The inlet channel 261 communicates with a receptacle 233 that serves as a sample input port for the flow cell 220. In other words, the receptacle 233 (when the first seal 251 is removed, see below) is open to the surroundings of the flow cell 220, as can be seen in fig. 12. This allows a user to place a sample to be tested into the receptacle 233 in the activated state of the flow cell 220. By providing a large (e.g. 5mm diameter) port 233, it is easy for a user to provide a sample to the input port 233 without introducing any gas into the flow cell 220.

That is, the geometry of the port 233 is such that it provides a container during the inactive state (prior to removal of the seals 251 and 252, see below). It is also possible to provide the container instantaneously, if or when the sample is added during the active state, at a speed faster than the speed at which it is drawn into the flow cell.

Once activated, the liquid/air interface at the sample inlet end of the flow path is biased to rest at the corner between the inlet channel 261 and the port/container 233. The liquid/air interface at the other end of the flow path rests freely along the waste channel 232, the position of which is determined by the volume of fluid. Due to capillary action, even if the pool fluid evaporates, this is true no matter at which liquid/air interface the evaporation occurs-the interface at the sample inlet end remains static, while the waste end retracts as the amount of fluid decreases.

To add the sample to the flow cell 220, the user need only contact the sample at the liquid/air interface of the sample inlet end (i.e., at the transition between the inlet channel 261 and the port/container 233). This may be direct or by adding the sample to the receptacle forming region of port 233 and allowing the sample to move toward and contact the interface (e.g., under gravity flow). The inlet diameter of the sample input port 233 is larger than the droplet diameter and may advantageously be disc-shaped. Thus, a droplet may be added to the device, able to move by gravity to the bottom of the disk, and contact the fluid at the top of the inlet channel 261 at the interface with the sample input port. The tapered sides of the sample input port 233 allow for focusing of the droplets on the inlet channel and minimize the introduction of gas into the flow cell by preventing the formation of voids. The sample input port 233 may also have a shape other than a disk, such as a shallow cone.

The addition of sample is further illustrated in fig. 18, which shows sample fluid 291, flow cell molding 292, sensor 293, and cell fluid 294. The sealing surface 295 has a sample port opening/receptacle 296 with a radius greater than the sample droplet radius 297. This allows sample fluid 291 to contact cell fluid air interface 298 rather than bridging across the opening and trapping an air gap between the fluid interfaces. During manufacture, due to the fixation on sharp rounded edge 299 formed by the closing surface in the mold tool, pool fluid air interface 298 is biased to rest at transition point 298 due to capillary action. If the pool fluid air interface 298 is forced away from the edge 299, the taper of surfaces 284 and 285 toward the edge 299 increases the capillary force that acts to return the pool fluid air interface 298 to the edge 299. In the extreme case where the pool fluid air interface 298 is forced away from the rim 299, the attachment to the rim 286 increases the laplace bubble pressure to prevent further air flow to the sensor 293.

Since container 233 is located on the top surface of flow cell 220, it is located above sensing chamber 235. However, this is not necessary in a direct sense (i.e. the reservoir does not have to be directly above the sensing chamber) or in an absolute sense (i.e. the reservoir does not have to be at a higher level than the sensing chamber) as liquid will be drawn through the device by capillary flow (as described below). Container 233 can be located at the same height as the sensing chamber or below sensing chamber 237.

The flow cell 220 is also provided with a waste liquid collection channel 232. In use, collection channel 232 receives liquid that exits sensing chamber 237 through outlet channel 262.

However, immediately between the outlet channel 262 and the collection channel 232 is the flow barrier 231. The flow barrier 231 is a wall separating the outlet channel 262 from the collection channel 232. In other words, without the barrier 231, the flow path upstream of the barrier 231 ending with the outlet channel 262 and the flow path downstream of the barrier 231 starting with the collection channel 232 would be directly connected to each other. In the illustrated construction, the barrier 231 (and thus the end of the outlet channel 262) is raised above the level of the sensing chamber 237. However, this is not necessary as the liquid will be drawn through the device by capillary flow, as described below.

In the active or activated state, liquid may pass through the barrier 231 and into the waste collection channel 232. However, as shown in FIG. 14, the flow cell is in an inactive state. In this state, the first seal 251 covers the sample input port 233, and the second seal 252 covers the end of the sensing chamber outlet channel 262. In the illustrated embodiment, both the first seal 251 and the second seal 252 are provided as part of the same integral seal element 250. As shown, the integral sealing member 250 may also cover the inlet of the waste collection channel 232 in the inactive state. The sealing member 250 may be attached to the surface of the flow cell 220 by a glue having a higher or lower hydrophilicity than the surface. In particular, when the first and second seals 251, 252 are removed, such glue may be left behind, thereby imparting advantageous wetting characteristics to the surface (e.g., preventing liquid from flowing out of the reservoir 233 or encouraging liquid to flow into the bridging channel 241 discussed below).

The end of the outlet channel 262 and the inlet of the waste collection channel 232 may be connected to the barrier 231 via the barrier cover 240 in the active state. The barrier cover 240 may include a bridging channel 241 for connecting the outlet channel 262 and the collection channel 232, and is discussed in further detail below.

Sealing member 250 may further include a release liner portion 253. A release liner 253 is attached to the second seal 252. The release liner 253 can either extend beyond the second seal 252 (extending further below the barrier cover 240 as shown) or can be flipped back over the seal to include the handle portion 254.

In this arrangement, pulling the handle 254 provides a simple way to remove both seals 251 and 252. That is, by pulling the handle 254, the release liner 253 is pulled back from under the barrier cover 240, whereupon the seal 252 is also peeled back in the same direction. In this way, any adhesive remaining on the underside of the second seal 252 does not contact the barrier 240 when peeled and exposed, but is covered by the release liner 253 when it is pulled back from under the barrier cover 240 simultaneously with the second seal 252. When the handle 254 is pulled further, the first seal 251 is also removed from the sample input port 233.

The barrier cover 240 is preferably sprung up so as to be pushed towards the body of the flow cell 220. As shown in fig. 12, the barrier cover 240 may be biased into position by a securing means such as a bolt or screw 245. In other arrangements, the barrier cover 240 may be formed as one piece, with the body forming the fluid passage. In either arrangement, the cover 240 may be flexible to allow removal of the second seal 252 and then for the cover 240 to adjust and support on the exposed surface below the seal 252.

As a result, when the sealing element 250 is removed, the bridge channel 241 of the barrier cover 240 is pushed into place to form a connecting channel between the outlet channel 262 and the waste collection channel 232. the bridge channel 241 may be surrounded by a gasket 244, as shown in FIG. 15, to ensure a good seal between the outlet channel 262 and the waste collection channel 232. however, a seal may also be formed without a gasket by securing a fluid around the bridge channel 241. alternatively, the barrier cover 240 may have a body made of a spring material (e.g., metal or a suitable plastic material), but the bridge channel 241 may be made of another material that helps form a seal, such as an elastomeric material.

Thus, once the sealing element 250 is removed, a continuous flow path is created through the flow cell 220 from the port 233, through the inlet channel 261 to the sensor chamber 237, then to the outlet channel 262 and through the bridging channel 241 into the waste collection channel 232. Completion of this flow path between the upstream and downstream portions on either side of the barrier 231 places the flow cell 220 in an "active state". That is, the active state is a state in which liquid can pass from the input port 233 through the sensor chamber into the waste collection channel 232. The bridge channel 241 has a capillary dimension to facilitate the passage of liquid from the collection channel 232 to the outlet channel 262.

Prior to removing the sealing element 250 (and thus the first and second seals 251, 252 are still in place), the flow cell 220 is in an "inactive state". In this state, there is a sealed fluid volume, or "saturation volume," that is formed from the first seal 251, through the closed input port 233, the inlet passage 261, the sensing chamber 232, and the outlet passage 262 to the surface of the second seal 252. In other words, the flow path upstream of the barrier 231 is closed. In the inactive state, the flow cell is filled with liquid from the first seal 251 at the sample input port 233 to the second seal 252 at the end of the sensing chamber outlet channel 262. By filling the volume with a liquid, such as a buffer, the sensor 235 is prevented from being exposed to a gas or gas/liquid interface. This in turn protects the delicate components of the sensor 235, such as any membrane provided with nanopores.

The benefit of providing an inactive state in which the flow cell 220 is filled with liquid from the first seal 251 to the second seal 252 is that the flow cell can be prepared for use and then conveniently transported without destroying the sensor array. In particular, by excluding any gas, and thus any gas/liquid interface, from the interior volume, bubbles do not have a chance to damage the surface of the sensor 235 when the flow cell 220 moves and may change direction during transport.

Conversely, by configuring the flow cell to an "active" state by removing sealing element 250, sample can be added to port 233 and liquid can flow through sensing chamber 237 and into waste collector 232. However, the arrangement of input port 233 and barrier 231 relative to sensing chamber 237 means that liquid is not freely drained from sensing chamber 237 even in the active state. This is because the size of the inlet channel 261 and the outlet channel 262 means that capillary forces determine the movement of the fluid.

That is, the initial removal of the sealing element 250 may result in some liquid flowing out of the original saturated volume, i.e., out of the outlet channel 262, and into the bridging channel, and possibly into the waste channel 232. That is, removal of the sealing element 250 may have a "priming" effect, drawing some liquid through the device. However, due to the balance of capillary forces, such priming will not result in free flow of fluid resulting in drainage of the sensing chamber 23.

In use, liquid is drawn from the reservoir 233 into the inlet channel 261 by capillary action. To assist in drawing fluid through the flow cell 220, particularly from the outlet channel 262 and into the bridging channel 241, the barrier cover 240 may be provided with scoops 242 and 243, which are, for example, protrusions of circular profile, although other shapes are possible. The first scoop 242 extends from the barrier cover 240, through the bridge channel 241 and into the outlet channel 262. The second dipbucket 243 extends from the barrier cover 240, through the bridging channel 241, and into the waste collection channel 232. In some embodiments, only the scoop 242 may be provided into the outlet passage 262. In other embodiments, only the dip-bucket 243 into the waste collection channel 232 may be provided. In other embodiments, as shown, both of the leach buckets 242 and 243 may be provided.

The dip legs 242 and 243 help overcome any meniscus "pinning" that may counteract capillary action during liquid flow through the cell 220. In other words, as the liquid approaches the end of the outlet channel 262, the dip-bucket penetrates into the liquid before the meniscus reaches the end of the outlet channel 262. This helps capillary action to continue drawing liquid into the bridging channel 241. Similarly, the provision of the dip-bucket 243 helps to introduce liquid into the collection channel 232 without the liquid experiencing a meniscus that is fixed at the entrance of the liquid collection channel 232.

The flow from the bridging channel 241 into the waste collection channel 232 may also be assisted by providing rounded corners at the ends of the bridging channel 241, thereby reducing the number of sharp edges and hence the possibility of fixation. This rounded corner 263 is shown in fig. 14, and a rounded edge at the entrance of the downcomer 264 (which also aids in the travel of liquid to the channel) can also be seen. Similarly, rounded corners 265 may be provided between the downcomers 264 of the waste collection channels 232 (i.e., the inlet portions of the channels 232 proximate the barrier 231) into the main channels 266 of the waste collection channels 232. This is shown in fig. 15. The rounded corner 265 is located opposite the sharp edge/corner on the other side of the channel. Although the corners 265 are rounded, the cross-section of the channel in a direction perpendicular to the flow direction may be rectangular. This combination allows the fluid to be secured on the sharp edge while the fluid can travel around the bend where it impedes flow. This is because, with one point of contact fixed, as the fluid progresses along the channel, it can form its natural contact angle with the curved surface without "stretching" the exposed fluid surface (i.e., requiring work to be done on the surface).

Fig. 16 shows an alternative arrangement to fig. 15, with only one leaching bucket 242. Additional details regarding how channels are formed by the upper and lower mold pieces-flow cell assembly molding upper 271 and flow cell assembly molding lower 272-are also shown. The figure shows the configuration after removal of the sealing element (not shown) from the sealing surface 274 (n.b. the sealing surface 274 extends continuously from left to right in the figure, although it is clear that it is interrupted in certain sections through the port). A seal 275 is made between the barrier cover 240 and the flow cell upper molding 271, closing the bridging channel 241 between the cell outlet channel 262 and the waste inlet channel 232. The surfaces 279 of the bridging channels may be hydrophilic to aid capillary action. The protruding portion of the barrier cover 240 forms a dip bucket 242 that passes over the sealing surface 274 and contacts the pool fluid air interface secured to the rim 281. The protrusion 282 of the flow cell assembly molded lower portion 272 extends up to a port in the flow cell assembly molded upper portion 271, but does not intersect the sealing surface 274, so that the sealing element lies flat on the sealing surface 274. However, radius 283 prevents immobilization so that flow cell fluid may travel along surface 274 and contact protrusion 282. Once the fluid contacts the flow cell assembly molded lower portion at protrusion 282, capillary action draws it down a continuous surface of radius 265 so that the fixation to flow cell assembly molded upper rim 285 does prevent the fluid front from advancing along the channel.

To further facilitate flow around the barrier 231, the bridging channel 241 and/or the surface of the barrier facing the bridging channel 241 may be provided with suitable surface wetting properties. This may also apply to the waste channel to avoid that the flow of liquid through the device is immobilized in the waste channel. To promote capillary action, the contact angle within the flow path is preferably less than 90 °. Thus, the wetting contact angle of the surface in question with water may be 90 ° or less. Optionally, the surface may be more hydrophilic than pure water to account for changes in wetting properties of the sample, e.g., a wetting contact angle with water of 75 ° or less.

However, in certain arrangements, it may be desirable to ensure that these surfaces are not too hydrophilic to avoid the resulting capillary effect overcoming the entrapment of fluid at the input port and drawing liquid through the device and allowing air to enter, potentially exposing the sensor. Considering the arrangement of fig. 5c and the pressure balance discussed above, it can be considered that the contact angle at the inlet is zero, resulting in the smallest bubble radius, from which it can be seen that air will enter only if: the effective radius of the waste channel is smaller than the radius of the input port (assuming the same height of the fluid surface). In practice, the effective radius of the waste channel may be at least twice the size of the input port. Nevertheless, the device is not always horizontal, and therefore the effect of the hydrophilic or low contact angle waste surface is to reduce the pressure head that can be sustained due to the tilting of the device. As a result, the contact angle with water is optionally 10 ° or more, further optionally 20 ℃ or more.

The surface properties can be controlled by physical or chemical treatments. As mentioned above, this is particularly applicable for the bridging channel 241, since it is easily accessible during production, but may also be applicable for other components, such as the surface of the barrier facing the bridging channel 241 and the waste channel.

In terms of physical processing, the bridging channel 241 may be designed to have increased capillary action by increasing the area of the hydrophilic surface to overcome the localized area of hydrophobicity. That is, the surface area may be increased compared to a flat/non-textured surface. This may be achieved by, for example, texturing on the surface facing the barrier 231 to provide micro-roughness and/or macro-features. Such macro features may be provided as pillars, fins or channels/grooves, for example. Additionally or alternatively, a non-periodic and non-deterministic pattern may be generated on the surface. Such micro-features may be provided by forming the surface of the bridging channel with a molding tool having a spark coating and/or by etching the surface. Such features may be, for example, about 0.2mm deep. Such features may be created as part of the mold that bridges the channels 241.

Another form of physical treatment may include providing a physically porous element in the bridging channel 241. Such elements may assist in wicking liquid into the bridging channel 241 and subsequently through the bridging channel. Such elements may fill the bridging channel 241. Such an element may be a sponge, for example made of cellulose, or made of fabric or fiber. In some embodiments, the porous element may be dissolved in the liquid flowing through the device (after removal of the seal) as the porous element will serve its purpose once the liquid has been assisted through the bridging channel.

In terms of chemical treatment, the bridging channel 241 may be coated with a suitable chemical to increase the hydrophilicity of the channel. Such chemicals may be commercial hydrophilic coatings, typically applied in a carrier solvent that evaporates leaving behind a layer of hydrophilic ingredients, e.g. Jonnin (r) ((r))Denmark) P100 and S100. Other solutions that evaporate to leave a layer of the hydrophilic component, such as a saline solution, may also be used.

Another form of chemical treatment may be achieved by providing a layer of a different material, such as a solid or gel layer, between the seal and the upper surface of the barrier 231, the further layer having a material that is more hydrophilic than the underlying material of the barrier 231. The additional layer may be bonded or fused to the underlying material substrate, or may be overmolded. The advantage of this approach is that different materials may provide different benefits-for example the primary substrate may be a material with good water vapour barrier properties to ensure containment of the necessary fluid within the device, while the additional layer may be made of a material that is more hydrophilic than the substrate (since materials with good vapour barrier properties are typically relatively hydrophobic rather than hydrophilic) to promote flow over the barrier 231. Examples of such methods include using shaped nylon 6 (polycaprolactam) as an additional layer, which forms a contact angle with water of about 63 °, or a thin layer of PET (polyethylene terephthalate), which has a contact angle with water of about 73 °. Other materials exhibiting suitable hydrophilicity include polyvinyl alcohol (PVOH), contact angle of about 51 °; polyvinyl acetate (PVA), contact angle of about 61 °; polyethylene oxide (PEO)/polyethylene glycol (PEG), contact angle of about 63 °; nylon 6,6, contact angle about 68 °; nylon 7,7, contact angle about 70 °; polysulfone (PSU) with a contact angle of about 71 °; polymethyl methacrylate (PMMA), contact angle about 71 °; or nylon 12, contact angle of about 72 deg..

The balance of capillary forces across the flow cell 220 means that fluid is not free to flow from the sensing chamber into the bridging channel 241 and the waste collection element 232 without some additional driving force. The driving force may be to provide additional fluid to the input port 233. It is also possible that fluid is present in the input port container 233 when the seal 251 is removed. In either case, such flow only occurs when the upstream liquid/air interface stagnates at the transition between inlet channel 261 and port/reservoir 233 due to the balance of capillary forces as described above. In this way, activating flow cell 220 does not expose sensor 235 to a gas or gas/liquid interface. In other words, activating flow cell 220 does not cause liquid to drain through flow cell 220 such that sensor chamber 237 empties and sensor 235 is exposed to air. In addition, by immobilizing the fluid at the edge between the chamber 237 and the inlet channel 261, for example during excessive tilting or acceleration of the flow cell 220, further protection against air entering the cell 220 is provided. Once this transient event is over, the interface will move by capillary action from the edge back to the transition between the inlet channel 261 and the port/reservoir 233.

After the sample is added, the seals can be replaced on the sample and waste ports to reduce evaporation. This is shown in fig. 17. Fig. 17a shows the cell 220 with the sealing element 250 removed to expose the sample port 233. It also shows a fluid waste port 267 and an air waste port 268. These ports allow fluid to be completely withdrawn and removed from the flow cell 220. The port 267 serves as an entry point for removing fluid from the waste channel 232. Although the fluid is in communication with sensor 235, when the fluid is removed, air preferentially replaces the fluid extracted from downstream through port 268 rather than the fluid from upstream sensor chamber 237 and sample port 233. Fig. 17b shows how sealing element 250 is replaced after sample is supplied to port 233 to reduce evaporation and protect port 233 from contamination. Sealing member 250 may also have a waste port cap 269 that similarly helps reduce evaporation from ports 267, 268 and also helps prevent contamination. The seal may have a transmission window in the region of the sample port and/or waste port to assist in port inspection.

It will be understood that the present invention is not limited to the embodiments described above, and that various modifications and improvements may be made without departing from the concepts described herein. Any feature may be used alone or in combination with any other feature except where mutually exclusive, and the invention extends to and includes all combinations and sub-combinations of one or more of the features described herein.