Apparatus and method for controlling the insertion of membrane channels into a membrane - Patents.com

Abstract

An apparatus for controlling the insertion of a membrane channel into a membrane is provided, the apparatus comprising a first bath for holding a first liquid in contact with a first surface of the membrane, a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids, a first electrode configured to contact the first liquid, a second electrode configured to contact the second liquid, and a drive circuit configured to apply a potential difference across the membrane via the first and second electrodes to promote the insertion of the membrane channel from the first or second liquid into the membrane. Various configurations of the drive circuit are described that allow for effective promotion of the membrane channel insertion while reducing the risk of damage to the membrane. Corresponding methods are also described.

Description

[Technical field]

[0001] The present invention relates to a device and a method for controlling the insertion of a membrane channel into a membrane. The channel may be a nanopore. The nanopore may be a protein channel. [Background technology]

[0002] The channel formed across the membrane can be used to detect molecular entities. Interaction between the molecular entity and the channel can cause a characteristic modulation of the signal. By monitoring this signal, it is possible to detect the characteristic modulation and thereby detect the molecular entity. Various techniques have been proposed based on this principle, as disclosed in WO2008 / 102120, WO2009 / 035647, WO2000 / 79257, WO2001 / 42782, and WO2007 / 057668. One example is the measurement of a current signal due to the flow of ions through a membrane channel. The membrane separates two solutions, and the membrane channel provides a transport path through the membrane between the solutions. The membrane is highly resistive, such that the only transport path between the solutions is through one or more membrane channels. A molecular entity of interest can be made to interact with the channel, for example, to translocate through the channel.

[0003] Detection of molecular entities using this technique provides a method for the direct identification of single molecules and molecular entities without the need for fluorescent labeling and detection. There is a wide range of possible applications, such as sequencing of DNA or other nucleic acids, detection of chemical or biological molecules for safety and defense, detection of biological markers for diagnostics, ion channel screening for drug development, and label-free analysis of interactions between biological molecules.

[0004] To provide sufficient throughput, an array of individual membranes can be provided, each membrane containing a membrane channel.

[0005] The membrane is typically amphiphilic and can be bilayer.Techniques for forming amphiphilic layers are well known, as disclosed by Montal and Mueller Proc Natl Acad Sci USA.1972 Dec,69(12):3561-3566, WO-2008 / 102120, WO2008 / 012552, and WO2014 / 064444.The thickness of the resulting membrane can vary depending on factors such as the nature of the membrane material, the inclusion of solvent, the geometry of the membrane, and the preparation method.

[0006] In some systems, membrane channels do not spontaneously insert into the membrane in the absence of additional stimuli, and often do so very slowly. Choosing such a system can be advantageous, as it allows control over the process of membrane insertion. Techniques to aid in the insertion of membrane channels are known, the most common being the application of a potential difference across the membrane. It is believed that this potential difference stretches and thins the membrane, facilitating insertion. Voltage-assisted insertion can also be used to control the number of membrane channels inserted into the membrane, where the applied potential can be actively lowered in response to membrane channel insertion and corresponding detection of current through the membrane, lowering the applied potential difference after insertion of a membrane channel reduces the likelihood of insertion of the next channel. The lowering of the applied potential can be done by the user, which is common practice for many laboratories forming membranes and performing single channel recordings. A drawback of this approach is that it requires the user to observe the current (or a similar observable parameter, such as resistance) of the channel and to manually react to changes in that parameter. This is practical for single channel recordings, but not practical when dealing with large arrays. It is therefore desirable to automate this process.

[0007] Various methods have been disclosed to automate the control of the applied potential over an array of membranes. One uses computer control to react to the detection of an increase in current observed as a result of nanopore insertion, as disclosed in US2016 / 0289758. Another teaches the use of electrode-mediated air bubbles to assist membrane formation and agitation stimulation to achieve a similar effect, as disclosed in US2012 / 0052188. The number of channels required for each membrane may vary depending on the measurement technique used. For ion flow measurements, it is desirable to provide one channel per membrane.

[0008] Although the use of computer-controlled automation of stimuli such as applied potentials is beneficial over manual methods of controlled pore insertion, they have many undesirable characteristics. One such characteristic is the need for a computer or similar decision-making circuit to perform the operation of removing or reducing the pore insertion stimulus. Such circuitry is expensive and may not be practical when dealing with many arrays of membranes. Each membrane must undergo this computer-controlled process to ensure good single-channel yields, and must be connected to a dedicated computer controller or share the computer controller among other membranes. Providing a dedicated computer controller for each channel is expensive. Connecting multiple membranes to a control unit reduces this cost, resulting in pore insertion occurring sequentially as the control unit cycles around each membrane in turn. This can increase the overall amount of time required for pore insertion across the array (selective multiplexing) or reduce the response time of the device (time-division multiplexing). Furthermore, it was found that inserting channels in a sequential manner by adding protein nanopores to a solution in contact with the membrane resulted in adsorption of the nanopores to the walls of the container due to the increased time that the nanopores were in solution before being inserted, which could reduce the yield of inserted nanopores across the array.

[0009] One approach to address this problem is to use a voltage reduction unit. US2020 / 179920(A1) discloses "passive pore insertion" in which the voltage applied across the membrane to perform pore insertion is adjusted by a voltage reduction unit placed in series with a voltage source, and the adjustment is made according to the current sourced / sunk through the membrane / pore arrangement. However, this approach is not very flexible and many of the parameters depend on the characteristics of the current source used. Summary of the Invention

[0010] An approach that overcomes the problems detailed above and is described in aspects of the invention is to use circuitry that essentially reduces the potential applied across the membrane when a channel is inserted. In some embodiments, this does not require decision-making electronics and can be inexpensively deployed across large arrays. Other advantages may be obtained by combining the circuitry with other components and configurations.

[0011] According to an aspect of the invention, an apparatus for controlling insertion of a membrane channel into a membrane is provided, comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; and a drive circuit configured to apply a potential difference across the membrane via the first and second electrodes to facilitate insertion of the membrane channel from the first or second liquid into the membrane, the drive circuit being configured such that the applied potential difference before insertion of the membrane channel is defined by a reference voltage and the applied potential difference after insertion of the membrane channel is defined by a selected current provided by a current source and a resistance between the first and second electrodes, the voltage limit of the current source being independent of the reference voltage, and the applied potential difference after insertion of the membrane channel is lower than the applied potential difference before insertion of the membrane channel.

[0012] The independence of the reference voltage and the voltage limits of the current sources provides improved flexibility in component selection while allowing the reference voltage to be selected to reach a range sufficient to promote channel insertion without damaging the membrane.

[0013] In some embodiments, the drive circuit comprises at least one power rail defining a reference voltage and at least one diode in series between the at least one power rail and the first electrode or the second electrode, the at least one power rail and the at least one diode being configured to limit an applied potential difference to less than or equal to the reference voltage. In some embodiments, the at least one power rail and the at least one diode are configured to limit the magnitude of the voltage applied to the first electrode or the second electrode to less than or equal to the reference voltage. The diode configuration is easy and inexpensive to implement, especially when an array of membranes may be used. Limiting the applied potential difference ensures that the membrane is not exposed to voltages that risk damaging the membrane.

[0014] In some embodiments, the drive circuit comprises a comparator configured to compare a reference voltage to the voltage at the first electrode or the second electrode, and the output from the comparator determines whether the applied potential difference is defined by the reference voltage or the selected current. The comparator is a low-cost, robust means of providing a switching function for reducing the applied potential difference after membrane channel insertion.

[0015] In some embodiments, the selected current is in the range of 50-300 pA. Currents in this range provide a potential difference across the membrane that is not so large that there is a risk of damaging the membrane after membrane channel insertion.

[0016] In some embodiments, the drive circuitry is configured, preferably by logic control, to allow the selected current to be selected from a number of available current values, allowing greater flexibility in selecting the potential difference across the membrane after membrane channel insertion.

[0017] In some embodiments, the drive circuit is configured to apply a gradually increasing potential difference across the membrane, which helps ensure that only a single membrane channel is inserted into the membrane by starting at a low potential difference that is highly unlikely to cause multiple channel insertions and increasing until a potential difference sufficient to insert the membrane channel into the particular membrane is reached.

[0018] In some embodiments, the device is configured to periodically measure one or more electrical properties of the membrane, and in response to the one or more electrical properties indicating that a membrane channel has been inserted into the membrane, to block the subsequent application of a potential difference across the membrane by the driving circuit.By measuring the electrical properties of the membrane, a more accurate indication of when the membrane channel has been inserted can be obtained.Blocking the potential difference after the membrane channel is inserted reduces the possibility of the membrane being damaged, for example, by the occurrence of a high potential difference due to blocking of the membrane channel.

[0019] In some embodiments, the device comprises a measurement circuit configured to perform periodic measurements of one or more electrical properties of the membrane, the device being configured to switch from a drive state to a measurement state for each measurement of one or more electrical properties of the membrane, the drive state being a state in which the drive circuit is electrically connected to the first electrode or the second electrode in such a way that a potential difference can be applied across the membrane, and the measurement state being a state in which the drive circuit is not electrically connected to the first electrode or the second electrode in such a way that a potential difference can be applied across the membrane. This scheme ensures that the performance of the drive circuit is not affected by the measurement circuit, and vice versa.

[0020] In some embodiments, the device is configured to block subsequent application of the potential difference by not switching from the measurement state to the drive state, which provides a convenient way to block application of the potential difference to reduce the possibility of damage to the membrane.

[0021] In some embodiments, the drive circuitry comprises a latch arrangement configured to automatically latch the applied potential difference across the membrane after insertion of the membrane channel into the membrane at a lower value than the potential difference applied before insertion of the membrane channel into the membrane.

[0022] According to one aspect of the invention, an apparatus for controlling insertion of a membrane channel into a membrane is provided, comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; and a drive circuit configured to apply a potential difference across the membrane via the first and second electrodes to facilitate insertion of the membrane channel from the first or second liquid into the membrane, the drive circuit configured to automatically reduce the potential difference in response to insertion of the membrane channel into the membrane, the drive circuit comprising a latch arrangement configured to automatically latch the potential difference applied across the membrane after insertion of the membrane channel into the membrane at a value lower than the potential difference applied before insertion of the membrane channel into the membrane.

[0023] The latch configuration can be used to automatically disconnect the applied potential after membrane channel insertion without having to use any kind of logic control to evaluate whether to disconnect the drive circuitry or the applied potential. This can provide the advantage of reducing the possibility of damage to the membrane while simplifying the circuitry involved, thereby reducing cost and manufacturing complexity.

[0024] In some embodiments, the latch configuration is configured to be triggered by a drop in an applied potential difference caused by the insertion of a membrane channel into the membrane, which causes the resistance of the membrane (and thereby the potential difference across the membrane) to drop suddenly, and therefore this is a convenient trigger to use for a latch circuit.

[0025] In some embodiments, the drive circuit is further configured to regenerate the mediator after measuring the interaction between the molecular entity and the membrane channel inserted in the membrane, and regeneration of the mediator is performed by applying a potential difference across the membrane that is opposite in polarity to the potential difference applied during measurement of the interaction between the molecular entity and the membrane channel.

[0026] According to one aspect of the invention, an apparatus for controlling insertion of a membrane channel into a membrane is provided, comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; and a drive circuit configured to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel from the first or second liquid into the membrane, wherein the drive circuit is further configured to regenerate a mediator after measuring an interaction between a molecular entity and the membrane channel inserted into the membrane, the regeneration of the mediator being performed by applying a potential difference across the membrane that is opposite in polarity to the potential difference applied during the measurement of the interaction between the molecular entity and the membrane channel.

[0027] After insertion of the membrane channel, the membrane can be used to perform measurements of molecules passing through the membrane channel. This process can cause depletion of the buffer in the first bath and / or the second bath. The circuit used to facilitate the membrane channel insertion is configured to apply a potential difference across the membrane and can therefore be used to apply a potential difference to regenerate the buffer. Using the same circuit for both purposes has the advantage of reducing the cost and overall complexity of the device used to perform measurements of molecules passing through the membrane channel.

[0028] In some embodiments, the drive circuitry is configured to modulate the potential difference applied across the membrane with an AC waveform of smaller amplitude than the average amplitude of the potential difference.

[0029] According to an aspect of the invention, an apparatus for controlling insertion of a membrane channel into a membrane is provided, comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; and a drive circuit configured to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel from the first or second liquid into the membrane, the drive circuit configured to modulate the potential difference applied to the membrane with an AC waveform having an amplitude smaller than the average amplitude of the potential difference.

[0030] Applying small modulations in the potential difference across the membrane can help promote membrane channel insertion at lower average applied potential differences and / or reduced times of application of high potential differences, thereby reducing the likelihood of damage to the membrane. Without wishing to be bound by theory, it is believed that at least some contribution to the observed effects arises from modulations that cause disturbances or ripples leading to small local changes in membrane geometry and / or surface tension that can trigger insertion.

[0031] In some embodiments, the AC waveform has an amplitude in the range of 0-100 mV and / or a frequency in the range of 0-10 kHz. These amplitude and frequency ranges have been found to be particularly effective for promoting insertion of membrane channels.

[0032] In some embodiments, the device is configured to measure one or more electrical properties of the membrane and set or adjust a potential difference applied across the membrane prior to insertion of the membrane channel into the membrane based on the measured one or more electrical properties.

[0033] According to an aspect of the invention, an apparatus for controlling insertion of a membrane channel into a membrane is provided, comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; and a drive circuit configured to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel from the first or second liquid into the membrane, wherein the apparatus is configured to measure one or more electrical properties of the membrane based on the measured one or more electrical properties and to set or adjust the potential difference applied across the membrane prior to insertion of the membrane channel into the membrane.

[0034] By setting the applied potential difference prior to membrane channel insertion based on the electrical properties of the membrane, the potential difference can be selected to effectively promote membrane channel insertion, taking into account variations between membranes. This can improve the speed and effectiveness of membrane channel insertion, for example, by allowing the correct potential difference to be determined and applied more quickly, rather than having to increase the potential difference from a low initial value.

[0035] In some embodiments, the drive circuitry is configured to facilitate simultaneous insertion of the membrane channel into the membrane at multiple different pixels, each pixel associated with a first electrode and a second electrode driven by the drive circuitry. Providing multiple pixels is advantageous to increase throughput and allow more membranes to be processed at the same time.

[0036] In some embodiments, the drive circuitry is configured to apply a common potential difference to pixels whose membrane channels have not yet been inserted into the membrane, which simplifies the design and operation of devices having multiple pixels since each pixel does not need to be individually addressed.

[0037] In some embodiments, the drive circuitry is configured to incrementally increase the common potential difference and to control the incremental increase based on detection of membrane channel insertion in other pixels, allowing an effective potential difference to be reached more quickly by determining whether the applied potential difference is promoting membrane channel insertion across multiple pixels.

[0038] In some embodiments, the drive circuitry is configured to control the incremental increase based on the detected rate of membrane channel insertion, which is a useful measure of how effectively the currently applied potential difference is promoting membrane channel insertion across the plurality of pixels.

[0039] In some embodiments, the gradual increase comprises a series of steps, and the drive circuit is configured to initiate a step to a higher potential difference when the detected membrane channel insertion rate drops below a predefined threshold, the gradual increase being meant to maintain the potential difference at a level that drives an appropriate membrane channel insertion rate that does not increase the risk of membrane damage.

[0040] In some embodiments, the driving circuitry is configured to measure one or more electrical properties of the membrane at each pixel, and set or adjust a potential difference applied across the membrane at each pixel prior to insertion of the membrane channel into the membrane of the pixel based on the measured one or more electrical properties. Since the membrane properties may vary, the appropriate potential difference to effectively promote membrane channel insertion may also vary. This allows for a more rapid setting of the appropriate potential difference based on the membrane properties, without having to start increasing the potential difference from a low initial value.

[0041] In some embodiments, each pixel is associated with a first electrode and a second electrode that are driven by a drive circuit, which results in a well-defined correspondence between the pixel and the first and second electrodes.

[0042] In some embodiments, two or more of the plurality of pixels are associated with respective second electrodes and share a common first electrode. By sharing a common first electrode, device and circuit construction can be simplified by allowing one common connection for applying a potential difference to the pixels.

[0043] In some embodiments, two or more of the plurality of pixels are associated with a first bath and a plurality of respective second baths, the common first electrode being configured to contact a first liquid in the first bath and the respective second electrodes being configured to contact a second liquid in the respective second baths. Sharing a common first bath can further simplify the structure of the device and improve performance by allowing all pixels to share a single first bath having a larger capacity.

[0044] In some embodiments, the second electrodes are individually addressable by drive circuitry, allowing for the provision of appropriate potential differences to promote membrane channel insertion at each pixel based on the various properties of each membrane, while reducing the risk of damage to the membrane.

[0045] In some embodiments, the pixels are arranged in a two-dimensional grid, with the second electrode associated with each pixel being connected to the second electrodes associated with other pixels on the same row or column of the grid, such that each second electrode is individually addressable using a combination of rows and columns. This type of grid arrangement allows each pixel to be individually addressed using a simple structure of electrical connections.

[0046] In some embodiments, the second electrodes are each provided at the base of a respective second bath, and the drive circuitry is provided in an integrated circuit integral with the second baths, the second electrodes being electrically connected to the drive circuitry. Integrating the drive circuitry may simplify the construction of the device.

[0047] In some embodiments, the second electrode is removable from the drive circuit. Making the drive circuit removable can be advantageous in devices where some components, such as the second bath, are disposable, thus reducing waste by reusing the drive circuit.

[0048] In some embodiments, the drive circuitry is provided by application specific integrated circuits. These dedicated chips can simplify manufacturing and reduce production costs.

[0049] According to one aspect of the present invention, there is provided a sequencing flow cell comprising an apparatus in which a drive circuit is provided by an application specific integrated circuit, the sequencing flow cell further comprising a control circuit configured to control the drive circuit, a sensor array configured to define a first bath and a second bath and to support a membrane, a housing having one or more fluid loading ports, one or more fluid channels fluidly connecting the fluid loading ports to the first bath and / or the second bath, and an electrical connector for connecting the flow cell to a measurement instrument.

[0050] According to one aspect of the present invention, there is provided a sequencing apparatus comprising one or more of the devices described above, further comprising instrumentation electronics configured to interface with the one or more devices, a processor configured to control drive circuitry of the one or more devices via the instrumentation electronics and to calculate a sequence of the nucleotide molecule based on measurements from the devices, and optionally temperature control means configured to control the temperature of the first liquid and the second liquid in the one or more devices.

[0051] In some embodiments, the one or more devices are sequencing flow cells as described above, wherein the electrical connector of each of the one or more sequencing flow cells is configured to connect to instrumentation electronics, and the processor is configured to control drive circuitry of the one or more flow cells via the control circuitry of the respective flow cell.

[0052] According to one aspect of the invention, there is provided a method of controlling insertion of a membrane channel into a membrane using an apparatus comprising a first bath for holding a first liquid in contact with a first surface of a membrane, a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids, a first electrode configured to contact the first liquid, and a second electrode configured to contact the second liquid, the method comprising controlling a drive circuit to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel from the first or second liquid into the membrane, the applied potential difference before insertion of the membrane being defined by a reference voltage and the applied potential difference after insertion of the membrane being defined by a selected current provided by a current source and a resistance between the first and second electrodes, a voltage limit of the current source being independent of the reference voltage, and the applied potential difference after insertion of the membrane channel being lower than the applied potential difference before insertion of the membrane channel.

[0053] The independence of the reference voltage and the voltage limits of the current sources provides improved flexibility in component selection while allowing the reference voltage to be selected to reach a range sufficient to promote channel insertion without damaging the membrane.

[0054] According to one aspect of the invention, there is provided a method of controlling insertion of a membrane channel into a membrane using an apparatus including a first bath for holding a first liquid in contact with a first surface of the membrane, a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids, a first electrode configured to contact the first liquid, and a second electrode configured to contact the second liquid, the method comprising controlling a drive circuit to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel from the first or second liquid into the membrane, the drive circuit being configured to automatically lower the potential difference in response to insertion of the membrane channel into the membrane, the drive circuit comprising a latch arrangement configured to automatically latch the potential difference applied across the membrane at a value lower than the potential difference applied prior to insertion of the membrane channel into the membrane.

[0055] The latch configuration can be used to automatically disconnect the applied potential after membrane channel insertion without having to use any kind of logic control to evaluate whether to disconnect the drive circuitry or the applied potential. This can provide the advantage of reducing the possibility of damage to the membrane while simplifying the circuitry involved, thereby reducing cost and manufacturing complexity.

[0056] According to one aspect of the invention, there is provided a method of controlling insertion of a membrane channel into a membrane using an apparatus comprising a first bath for holding a first liquid in contact with a first surface of a membrane, a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids, a first electrode configured to contact the first liquid, and a second electrode configured to contact the second liquid, the method comprising controlling a drive circuit to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel from the first or second liquid into the membrane, and regenerating a mediator after measuring an interaction between a molecular entity and the membrane channel inserted into the membrane, the regeneration of the mediator being performed by applying a potential difference across the membrane that is opposite in polarity to the potential difference applied during the measurement of the interaction between the molecular entity and the membrane channel.

[0057] After insertion of the membrane channel, the membrane can be used to perform measurements of molecules passing through the membrane channel. This process can cause depletion of the buffer in the first bath and / or the second bath. The circuit used to facilitate the membrane channel insertion is configured to apply a potential difference across the membrane and can therefore be used to apply a potential difference to regenerate the buffer. Using the same circuit for both purposes has the advantage of reducing the cost and overall complexity of the device used to perform measurements of molecules passing through the membrane channel.

[0058] According to an aspect of the invention, there is provided a method of controlling insertion of a membrane channel into a membrane using an apparatus comprising a first bath for holding a first liquid in contact with a first surface of a membrane, a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids, a first electrode configured to contact the first liquid, and a second electrode configured to contact the second liquid, the method comprising controlling a drive circuit to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel from the first or second liquid into the membrane, and modulating the potential difference applied across the membrane with an AC waveform that is smaller than the average amplitude of the potential difference.

[0059] Applying small modulations in the potential difference across the membrane can help promote membrane channel insertion at lower average applied potential differences and / or reduced times of application of high potential differences, thereby reducing the likelihood of damage to the membrane. Without wishing to be bound by theory, it is believed that at least a contribution to the observed effect is due to modulations causing disturbances or ripples that lead to small local changes in membrane geometry and / or surface tension that can trigger insertion.

[0060] According to one aspect of the invention, there is provided a method of controlling insertion of a membrane channel into a membrane using an apparatus comprising a first bath for holding a first liquid in contact with a first surface of a membrane, a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids, a first electrode configured to contact the first liquid, and a second electrode configured to contact the second liquid, the method comprising controlling a drive circuit to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel from the first or second liquid into the membrane, and setting or adjusting the potential difference applied across the membrane prior to insertion of the membrane channel into the membrane based on one or more measured electrical properties.

[0061] By setting the applied potential difference prior to membrane channel insertion based on the electrical properties of the membrane, the potential difference can be selected to effectively promote membrane channel insertion, taking into account variations between membranes. This can improve the speed and effectiveness of membrane channel insertion, for example, by allowing the correct potential difference to be determined and applied more quickly, rather than having to increase the potential difference from a low initial value.

[0062] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the accompanying drawings in which corresponding reference symbols indicate corresponding parts and in which several embodiments of the present disclosure are shown. [Brief description of the drawings]

[0063] [Figure 1] 1 illustrates an apparatus according to one embodiment. [Diagram 2] 1 illustrates an apparatus according to one embodiment showing an exemplary layout of pixels in a grid arrangement. [Diagram 3] FIG. 1 is an electrical schematic of the device along with an electrical representation of the membrane. [Figure 4] FIG. 1 is a circuit diagram of one implementation of a pore insertion circuit. [Diagram 5] 5 shows the behavior of the voltage applied to the second electrode by the circuit of FIG. 4 as a function of the load resistance. [Figure 6] FIG. 13 is a circuit diagram of an alternative implementation of the pore insertion circuit using a comparator. [Figure 7] FIG. 13 is a circuit diagram of an alternative implementation of a pore insertion circuit that uses a comparator and can output multiple values ​​of selected current. [Figure 8] 6a and 6b show implementations for ramp-controlled ramping of the reference voltage and selected current. [Figure 9] 6a and 6b show implementations for increasing the reference voltage and the selected current with step control. [Figure 10] 13 shows step control of the reference voltage in response to a device detecting a drop in pore insertion rate. [Figure 11]1 illustrates switching between a pore insertion circuit and a measurement circuit according to one embodiment. [Figure 12] 12 illustrates a step control of the reference voltage as an alternative to the ramp control of the reference voltage shown in FIG. [Figure 13] 1 shows a flow diagram of an exemplary embodiment in which the potential difference applied to the membrane prior to membrane channel insertion is set according to measurements of the membrane's electrical properties. [Figure 14] FIG. 11 is a schematic diagram of a pore insertion circuit in which a latch arrangement latches the value of the potential difference after insertion of a membrane channel. [Figure 15] FIG. 13 is a schematic diagram of an embodiment in which the device is also configured to regenerate the mediator. [Figure 16] The current supplied by the pore insertion circuit is shown before and after membrane channel insertion, during sequencing measurements, and during mediator regeneration. [Figure 17] FIG. 13 is a circuit diagram of an implementation in which the pore insertion circuit is further configured to regenerate a mediator. [Figure 18] FIG. 1 is a schematic diagram of an embodiment in which a drive circuit applies a potential difference modulated by an AC waveform. [Figure 19] 19 shows an example of an AC waveform applied to the first electrode in the embodiment of FIG. 18. [Figure 20] FIG. 1 is a schematic diagram of an apparatus according to one embodiment used as part of a sequencing flow cell. [Figure 21] FIG. 1 is a schematic diagram of a sequencing instrument that uses a sequencing flow cell that includes a device according to one embodiment. [Figure 22] FIG. 1 is a diagram of an exemplary sequencing flow cell product in which the apparatus may be implemented. [Diagram 23] FIG. 1 is a diagram of an exemplary sequencing instrument in which the sequencing flow cell comprising the device may be used. [Figure 24] FIG. 1 is a diagram of an exemplary sequencing instrument in which the sequencing flow cell comprising the device may be used. [Diagram 25] FIG. 1 is a diagram of an exemplary sequencing instrument in which the sequencing flow cell comprising the device may be used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] Figure 1 shows an apparatus for controlling the insertion of a membrane channel into a membrane 4 according to one embodiment. The apparatus of Figure 1 can also be used for single molecule detection by nanopore sensing. As explained above, the membrane 4 is typically amphiphilic and can be a bilayer. The membrane channel inserted into the membrane 4 can be any suitable membrane channel and can also be referred to as a pore or nanopore.

[0065] The device comprises a housing 206 and a first bath 6 contained within the housing 206. The first bath 6 holds a first liquid. The first liquid contacts a first surface of each membrane 4.

[0066] The device further comprises four second baths 8 (also referred to as wells) contained in a housing 206. In other embodiments, a different number of second baths 8 may be provided. Each second bath 8 is sealed by a membrane 4.

[0067] Each second bath 8 holds a second liquid. The second liquid in each second bath 8 contacts a second surface of the membrane 4 to seal that second bath 8. The membrane thereby separates the first liquid and the second liquid. Typically, the membrane has a thickness in the range of 1 nm to 5 nm.

[0068] The first electrode 14 provided in the first bath 6 is configured to be in contact with the first liquid. The second electrode 16 is configured to be in contact with the second liquid in each second bath 8. Each second bath 8 has its own second electrode 16. Configured to be in contact with the liquid means that the first electrode 14 and the second electrode 16 are at least in electrical contact with the first liquid and the second liquid, respectively. This can be achieved by the first electrode 14 and the second electrode 16 being in direct contact with the first liquid and the second liquid. Alternatively, the first electrode 14 and the second electrode 16 can be in electrical contact with the first liquid and the second liquid through an intermediate layer, such as an oil or lipid layer.

[0069] As described below, the first electrode 14 and the second electrode 16 may be used to control the insertion of the membrane channel into the membrane 4. In certain embodiments, at a later time after the membrane channel is inserted, the first electrode 14 and the second electrode 16 may also be used to sense molecular entities via their interaction with the membrane channel.

[0070] The device further comprises a drive circuit configured to apply a potential difference (which may also be referred to as membrane voltage) across the membrane 4 via the first electrode 14 and the second electrode 16. The applied membrane voltage may be such as to promote insertion of a membrane channel from the first liquid 6 or the second liquid 8 into the membrane 4. Thus, a membrane channel (which may for example comprise a membrane protein) is provided in the first liquid or in the second liquid, or in both. The membrane voltage promotes insertion of the membrane channel. As mentioned in the introductory part of the specification, and without wishing to be bound by theory, it is believed that the voltage aids the insertion by stretching and thinning the membrane 4.

[0071] Although the housing 206 is shown with a single first bath 6 and four second baths 8 with associated membranes 4, this is not required and only a single first bath 6 and second bath 8 may be provided. In other embodiments, even more first baths 6 and second baths 8 and corresponding first electrodes 14 and second electrodes 16 may be provided. The number of second baths may be any integer between 1 and 100,000. It may be 100 or more, 1000 or more, or 10,000 or more.

[0072] Each second bath 8 and second electrode 16 may be considered to be part of a pixel 106. The drive circuitry is configured to facilitate simultaneous insertion of the membrane channel into the membrane 4 in a number of different pixels 106. Each pixel 106 is associated with a first electrode 14 and a second electrode 16 that are driven by the drive circuitry. The drive circuitry comprises a pixel circuit 20 that is unique to each pixel 106 and replicated between each pixel 106, and any common circuitry 15 that is shared between some or all of the pixels 106. The device may also comprise a control circuitry 50 that is used to control the drive circuitry.

[0073] In some embodiments, each pixel 106 is associated with a respective pair of first electrodes 14 and second electrodes 16 driven by a drive circuit. More preferably, however, two or more of the plurality of pixels 106 are associated with respective second electrodes 16 and share a common first electrode 14, as shown in FIG. 1. In FIG. 1, four pixels 106 are associated with a common first electrode 14. Two or more of the plurality of pixels 106 are associated with a first bath 6 and a plurality of respective second baths 8. The common first electrode 14 is configured to contact the first liquid in the first bath 6, and the respective second electrodes 16 are configured to contact the second liquid in the respective second baths 8. This allows a plurality of pixels to share a single first bath 6 and first electrode 14.

[0074] In some embodiments, such as in FIG. 1, the second electrodes 16 are each provided at the base of a respective second bath 8. The drive circuitry may be provided in an integrated circuit integral with the second bath 8. The second electrodes 16 may be electrically connected to the drive circuitry, i.e. the second electrodes 16 may form part of the integrated circuit in which the drive circuitry is provided. Alternatively, the second electrodes 16 may be detachable from the drive circuitry. This may be advantageous in some circumstances, for example when the second bath 8 and the second electrodes 16 are provided as part of a disposable component and the drive circuitry is reused between multiple disposable components.

[0075] Preferably, the second electrodes 16 are individually addressable by drive circuitry, for example via the corresponding pixel circuit 20. This means that the insertion of the membrane channel into each membrane 4 can be controlled for each pixel 106, thereby reducing the chances of inserting multiple membrane channels into the same membrane 4 or causing damage to any membrane 4.

[0076] The drive circuitry may be provided by an application specific integrated circuit (ASIC) 108. Other parts of the electronics and electrical connections within the device may also be provided by the ASIC 108, such as the second electrode 16.

[0077] Figure 2 shows a high-level layout of an exemplary design of such an ASIC 108. Figure 2 shows an embodiment in which the pixels 106 are arranged in an array comprising a two-dimensional grid, allowing them to be individually addressed.

[0078] As mentioned above, the size of the array on the ASIC 108 (i.e., the number of pixels 106) depends on the application. For example, the size may depend on the amount of sequencing data to be measured by the sequencing instrument in which the device is used. The array may comprise, for example, 100 pixels, 1000 pixels, 10,000 pixels, or 100,000 pixels or more. In the examples shown herein, each pixel 106 is associated with a single second bath 8 and second electrode 16. However, in some embodiments, multiple second baths 8 and second electrodes 16 may be connected to each pixel via an isolation switch, for example as described in WO2010 / 122293(A1).

[0079] The second electrodes 16 associated with each pixel 106 are connected to the second electrodes 16 associated with other pixels 106 on the same row or column of the grid. This connection may be achieved using circuitry 110, 112 for addressing and data readout of the pixels 106 on each row and column, such that each pixel 106 and second electrode 16 therein is individually addressable using a combination of row and column. Similarly, as shown in FIG. 1, the first electrodes 14 may be shared between all pixels 106 in the array, or at least some groups of pixels 106 in the array may share the first electrodes 14.

[0080] The size of each pixel 106 may range from 40 μm to 400 μm, or more typically range from 200 μm to 300 μm, depending on factors such as the desired form factor of the overall product, the number of pixels, the configuration of the sensor array 202, and the circuit design of the ASIC 108.

[0081] The ASIC 108 may further include additional circuitry for programming set voltages and currents, e.g., reference voltages and selected currents, within each pixel 106. Circuitry may also be included for digitizing and reading out measured signals from each pixel 106, and multiplexing outputs from multiple pixels 106 into a convenient format for reading from the ASIC. Further description of exemplary ASIC configurations may be found in WO2016 / 181118(A1).

[0082] The ASIC 108 may further include means for measuring the temperature of all or part of the device, e.g., the temperature of the first and second liquids in the first bath 6 and second bath 8. The ASIC 108 may further include means for heating and / or cooling all or part of the device, e.g., the temperature of the first and second liquids in the first bath 6 and second bath 8. The ASIC may further include a non-volatile memory and means for reading / writing from / to the non-volatile memory. Such memory may be used to store and recall information relevant to the use, e.g., calibration or test information, information about electrical properties associated with each pixel 106 (e.g., electrical properties of the membrane 4) and the associated sensor array structure above the pixel, information about the lot ID, measurement data, measurement conditions (time and date, measured temperature, user input data, etc.), error codes, etc.

[0083] Each pixel 106 comprises a measurement circuit 104 for measuring an electrical signal (typically a current) containing the sequencing data, and a pore insertion circuit 102 for providing an electrical signal to facilitate the insertion of a pore into the membrane. The pixels 106 may further include circuitry (not shown) for several additional functions, including calibration, self-test, unblocking, and a reverse bias mode for mediator regeneration.

[0084] 3 shows an electrical representation of the configuration for pore insertion at each pixel 106 of the nanopore array. Each pixel 106 has an associated second electrode 16 to which is connected a pixel circuit 20 comprising a measurement circuit 104 and a pore insertion circuit 102.

[0085] The measurement circuit 104 and the pore insertion circuit 102 can be configured to be disconnected from the second electrode 16 by switches S2 and S1, respectively. The electrical properties of the membrane 4 and its surroundings are measured by the capacitors C m and resistor R m It is represented by R p represents the resistance of the nanopore (membrane channel) and the insertion of the nanopore into the membrane 4 can be represented electrically by a closed switch S3. The first electrode 14 can be common to some or all of the pixels 106 in the array, as discussed above.

[0086] The potential difference across the membrane may be controlled by varying the voltage at one or both of the first electrode 14 and the second electrode 16. Preferably, however, the voltage at the first electrode 14 is held constant and the applied potential difference is controlled by controlling the voltage at the second electrode 16. For example, the first electrode 14 may be connected to a DC bias power supply. The bias power supply holds the voltage at the first electrode 14 at a fixed level, for example 0V.

[0087] Various circuit configurations of the pore insertion circuit 102 will now be described according to embodiments.

[0088] A first example of a pore insertion circuit 102 is shown in FIG. 4. The pore insertion circuit 102 includes a current source. This can be any suitable current source of standard design, and supplies a current I S Using this design of the pore insertion circuit 102, the drive circuit is configured such that the applied potential difference before the insertion of the membrane channel is defined by the reference voltage. The applied potential difference after the insertion of the membrane channel is defined by the selected current provided by the current source and the resistance between the first electrode 14 and the second electrode 16. The drive circuit is configured such that the voltage limit of the current source is independent of the reference voltage and the applied potential difference after the insertion of the membrane channel is lower than the applied potential difference before the insertion of the membrane channel.

[0089] This can be achieved by a drive circuit comprising at least one power rail defining a reference voltage and having at least one diode D1, D2 in series between the at least one power rail and the first electrode 14 or the second electrode 16. The at least one power rail and the at least one diode D1, D2 are configured to limit the applied potential difference to less than or equal to the reference voltage. The power rails can be implemented, for example, using a DC power source or a connection to a DC power source. The diodes D1, D2 can be any components that act as diodes when connected in an appropriate manner. For example, D1 and D2 in FIG. 4 can be implemented using normal two-terminal diodes, but also using transistors. In the case of a transistor, the gate / base and emitter of the transistor can be used as the two diode terminals.

[0090] At least one power rail and at least one diode D1, D2 may be configured to limit the magnitude of the voltage applied to the first electrode 14 or the second electrode 16 to below a reference voltage.

[0091] In the embodiment of FIG. 4, a pair of diodes D1, D2 are arranged in parallel at the output of the current source. The diodes are connected to the output voltage (V OUT ) to V CLAMP and -VCLAMP where V is the voltage at which the CLAMP represents the reference voltage. If the output voltage is not clamped at any extreme value, the output voltage is determined by the selected current from the current source and the DC load resistance, i.e., the resistance between the first electrode 14 and the second electrode 16.

[0092] Overall, the circuit functions as either a voltage source or a current source, depending on the DC load impedance presented at its output. In the embodiment of FIG. OUT is applied to the second electrode 16. However, as discussed above, in alternative embodiments, this voltage may be applied to the first electrode 14.

[0093] In Figure 4, the output voltage V OUT is given by the following equation: V OUT =I S *R load (I S *R load <V CLAMP in the case of) V OUT =V CLAMP (I S *R load >V CLAMP in the case of) Output current I OUT is given by the following equation: I OUT =I S (I S *R load <V CLAMP in the case of) I OUT =V CLAMP / R load (I S *R load >V CLAMP in the case of)

[0094] V as a function of DC load OUT The behavior of this is shown in Figure 5, which shows the two operating modes of the circuit, where the output is a reference voltage (V CLAMP ) or at a constant selected current (IS )

[0095] Reference voltage V CLAMP The value of V can be selected according to the properties and / or materials of the membrane channel and / or membrane to provide favorable conditions for pore insertion. For example, the reference voltage V CLAMP The value of may be between 0.2V and 0.6V, optionally between 0.3V and 0.5V, and optionally about 0.4V.

[0096] The selected current I S I S *R m >V CLAMP , whereby the circuit is in a voltage-limited mode (output V CLAMP However, the selected current must also be I S *R m R p / (R m +R p ) should be selected so as not to be so large as to significantly stress the membrane and thereby create a risk of bursting, and so high a voltage.

[0097] Therefore, I S The optimal value of is determined by the other system parameters (V CLAMP ,R m ,R p ) In a typical system, I S may range from 50 to 300 pA, optionally from 50 to 250 pA, optionally from 100 to 150 pA, optionally about 130 pA. The membrane channel resistance may typically range from 0.1 to 10 GOhm, more typically from 0.5 to 2 GOhm, even more typically greater than or about 1 GOhm.

[0098] In one particular embodiment, the design of the integrated circuit is CLAMP and I S This may facilitate V being set differently for different pixels in the array. CLAMP and IS can be set globally for the entire array. For example, some or all of the pixels 106 can share their pixel circuits 20 and corresponding pore insertion circuits 102.

[0099] The advantages of this embodiment are that it is simple to implement, requires few circuit components and consumes low power, making it suitable for miniaturization in small pixels.

[0100] An alternative circuit configuration for the pore insertion circuit 102 is shown in FIG. 6. In this embodiment, the drive circuit comprises a comparator U1 configured to compare a reference voltage with the voltage at the first electrode 14 or the second electrode 16. The output from the comparator U1 determines whether the applied potential difference is defined by the reference voltage or by the selected current. The reference voltage may be applied to one of the inputs of the comparator. Each transistor has one terminal connected to a supply rail VCCA.

[0101] In FIG. 6, U1 is a comparator that has a high output when the voltage at the positive input (+) exceeds the voltage at the negative input (-) and a low output when the voltage at (+) is less than the voltage at (-). Comparator U1 can be any conventional comparator component. In some embodiments, comparator U1 may comprise an op-amp, with a reference voltage applied to one of the inputs of the op-amp. As with the embodiment of FIG. 4, the circuit of FIG. 6 operates in a manner such that the output voltage is V CLAMP The input is configured to act as a current source that is clamped to

[0102] In current source mode (after insertion of the membrane channel), V OUT <V CLAMP This means that the output of comparator U1 is high. Therefore, T3 is off, so the gates of T1 and T2 are low and these transistors are on. The transistor pair T1 and T2 are configured to act as a current mirror in standard design, so that the current through T2 is equal to the reference current I REF T1 and T2 = I Smultiplied by the relative size (W / L ratio) of the

[0103] In voltage mode (before the insertion of the membrane channel), V OUT is V CLAMP V tends to be low, and the output of comparator U1 is low. Therefore, T3 is on, so the gates of T1 and T2 are high and these transistors are off. OUT The value of V is set by the feedback loop of comparator U1. CLAMP is maintained.

[0104] Figure 7 shows a variation of the embodiment of Figure 6 in which the drive circuit is configured to allow the selected current to be selected from a number of available current values. In Figure 7, the output current in the current mode (after insertion of the membrane channel) is selectable according to one of three values.

[0105] The selected current is preferably selected by a logic control. In Fig. 7, the logic control (not shown) is configured such that either or both of the switches S2a, S2b are closed. The current mirror is therefore configured such that the output device is either T2a, T2b, or both T2a and T2b in parallel. The output current provided through OUT is therefore selectable between three values ​​according to the configuration of the switches S2a and S2b.

[0106] Those skilled in the art will appreciate that the selection of different selected currents in the current mode (after membrane channel insertion) can be determined by adding additional load devices T2 in parallel and allowing different configurations of T2 to be connected between the rails VCCA and OUT by control of appropriate switches.

[0107] The advantage of the embodiment of FIG. 7 (e.g., compared to the embodiment of FIG. 6) is that V CLAMPThe advantage of this approach is that it allows accurate definition of V and is insensitive to transistor process variations. Changes in transistor characteristics due to process variations can mean that the exact voltage across different transistors can vary slightly for the same selected current. It is therefore possible to ensure that the voltage across the membrane is always V when desired. CLAMP To ensure that the selected current is greater than the ideal current, it may be necessary to use a selected current that is higher than the ideal current to account for possible variations in transistor characteristics. The embodiment of FIG. 7 allows for effective averaging of transistor characteristics between different transistor selections, or having different selections of S2a and S2b. This is because the selected current is proportional to the reference voltage V CLAMP This allows the amount of heat to be set to be applied more accurately and reliably.

[0108] The drive circuitry can be configured to apply a common potential difference to pixels 106 where the membrane channel has not yet been inserted into the membrane. The voltage at which membrane channel insertion occurs is different for each membrane 4. It has been found that different membranes 4 behave differently according to natural manufacturing variations in membrane thickness and capacitance. Thus, the probability that a pore will be inserted into the membrane 4 (within a given period of time) has a different voltage dependence for different membranes. This means that V CLAMP This can be utilized in embodiments of the driver circuit by allowing the value of V to vary over time. Thus, the driver circuit 15 can be configured to gradually increase the membrane voltage (common potential difference) until insertion occurs.

[0109] In any of the embodiments of Figures 4, 6 and 7, the drive circuitry may be configured to apply a gradually increasing potential difference across the membrane. To effect pore insertion, the output V OUT It may be advantageous to adjust the value of . This may involve gradually increasing the reference voltage that defines the applied potential difference prior to insertion of the membrane channel.

[0110] 8a and 9a show the reference voltage V CLAMP8a) or step (FIG. 9a) functions.

[0111] The advantage of this embodiment is that a high percentage of the membranes 4 can successfully have single pores inserted into them. Thinner membranes 4 have a low V CLAMP Starting from a lower V CLAMP At this time, it is more likely to have the pore inserted without bursting. After pore insertion, the applied potential difference is changed to a lower value (e.g., lowering the reference voltage V CLAMP By cutting off (I S This reduces the risk of the membrane 4 subsequently bursting (due to the membrane potential resulting from the application of .alpha. across the membrane) and also reduces the possibility of an unwanted second pore being inserted into the membrane 4.

[0112] Changing the applied potential difference to a lower value after membrane channel insertion also reduces the risk of a high voltage being suddenly and unexpectedly applied across the membrane 4 if the membrane channel is subsequently completely or partially blocked. Such blocking would abruptly increase the membrane channel resistance and increase the voltage generated across the membrane 4. This can be particularly problematic if the base voltage is increased in the period between pore insertion and pore blocking to insert pores in other membranes 4, whereby the suddenly increased voltage exceeds the voltage appropriate for the particular membrane 4 that was blocked. A further advantage of this embodiment is that it reduces the set-up time required to perform pore insertion in a targeted percentage of the membranes 4 in the array.

[0113] In a further refinement of this embodiment, as shown in Figs. 8b and 9b, the current I S can also be ramped or stepped over time.

[0114] Reference voltage V CLAMPThe timing associated with the steps in may be determined as a function of the measured number of pore insertions. In other words, the drive circuitry may be configured to control the gradual increase in the applied potential difference based on the detection of membrane channel insertion in other pixels 106. This means that the potential difference applied to each pixel 106 prior to membrane channel insertion may be based at least in part on the effectiveness of the current value of the potential difference in facilitating channel insertion.

[0115] This is shown in Figure 10. CLAMP A value of is set and the number of successfully inserted pores is monitored at discrete time steps. In particular, in FIG. 10, the driving circuit is configured to control the incremental increase in the number of channel insertions N based on the detected membrane channel insertion rate. P appears to be saturated (i.e., dN P / dt becomes smaller), and V CLAMP (The value of is stepped to the next value). If the gradual increase includes a series of steps, the drive circuitry may be configured to initiate a step to a higher potential difference when the detected membrane channel insertion rate drops below a predetermined threshold. Alternatively, other schemes may be employed. For example, the drive circuitry may be configured to ramp the applied potential difference more rapidly when the detected membrane channel insertion rate falls below a predetermined threshold.

[0116] This embodiment has the advantage of improving the inefficiency of pore insertion while simultaneously reducing the time required to insert pores into a high percentage of the membranes 4 in the array. A more consistent rate of channel insertion can be achieved by controlling the applied potential difference to pixels 106 that do not yet have membrane channels inserted based on membrane channel insertion into other pixels 106, and in particular based on the rate of channel insertion. This allows for successful channel insertion in a desired percentage of the membranes 4 in the array in a short period of time, with less risk of damage to any particular membrane 4.

[0117] In another embodiment in which the drive circuit is configured to apply a gradually increasing potential difference across the membrane 4, the membrane 4 may be periodically monitored to ascertain whether a pore has been inserted, for example, by measuring the DC impedance of the pore. In such an embodiment, the device is configured to periodically measure one or more electrical properties of the membrane 4, and in response to the one or more electrical properties indicating that a membrane channel has been inserted into the membrane 4, to interrupt subsequent application of the potential difference across the membrane 4 by the drive circuit.

[0118] 3, the device comprises a measurement circuit 104 configured to perform periodic measurements of one or more electrical properties of the membrane 4. The device is configured to switch from a drive state to a measurement state after each measurement of one or more electrical properties of the membrane 4. This can be achieved using switches S1 and S2.

[0119] The drive state is a state in which the drive circuit is electrically connected to the first electrode 14 or the second electrode 16 in such a way that a potential difference can be applied across the membrane 4. The measurement state is a state in which the drive circuit is not electrically connected to the first electrode 14 or the second electrode 16 in such a way that a potential difference can be applied across the membrane 4. For example, in Figure 3, the drive state is a state in which the switch S1 is closed and the measurement state is a state in which the switch S1 is open.

[0120] Preferably, in the actuated state, the measurement circuit 104 is not electrically connected to the first electrode 14 or the second electrode 16 so as not to affect the application of a potential difference to the membrane by the actuated circuit. In Fig. 3, this can be achieved by opening switch S2 in the actuated state. Switch S2 is then closed in the measurement state so that the measurement circuit 104 can perform periodic measurements of one or more electrical properties of the membrane 4.

[0121] Figures 11 and 12 show an implementation of this embodiment. Periodically, for example every 0.1, 0.5, 1 or 5 seconds, the device switches from the drive state to the measurement state. In the measurement state, the pore insertion circuit 102 is disconnected from the membrane 4 by opening switch S1, and the measurement circuit 104 is connected by closing switch S2. The DC current through the membrane is measured by the measurement circuit 104, thus determining whether the pore has been successfully inserted into the membrane 4.

[0122] Preferably, the device is configured to block further application of the potential difference by not switching from the measurement state to the drive state. In the examples of Figures 3, 11 and 12, once successful pore insertion is detected, switch S1 is configured to remain open for the remainder of the pore insertion procedure.

[0123] This embodiment, in combination with a gradual increase in the applied potential voltage, ensures that pore insertion occurs initially above the reference voltage V CLAMP If pore insertion is successful, the pore insertion circuit 102 is disconnected, thereby preventing damage to the membrane due to continued application of the potential difference. If pore insertion is not successful, the reference voltage V CLAMP The value of is gradually increased and periodically retries are performed (either ramp controlled as in FIG. 11 or step controlled as in FIG. 12).

[0124] Additionally or alternatively to the embodiments described above, measurements of one or more electrical properties of the film 4 may be used to determine an appropriate reference voltage. In one embodiment, the drive circuitry is configured to measure one or more electrical properties of the film. The drive circuitry may measure one or more electrical properties of the film 4 at each of the pixels 106 when multiple pixels are present. This may be accomplished using the measurement circuitry 104.

[0125] The drive circuit is then configured to set or adjust the potential difference applied across the membrane prior to insertion of the membrane channel into the membrane based on the one or more measured electrical properties. In the case of multiple pixels, the drive circuit may set or adjust the potential difference applied across the membrane 4 at each pixel 106 prior to insertion of the membrane channel into the membrane 4 of the pixel 106 based on the one or more measured electrical properties. The advantage of this approach is that the electrical conditions generated by the drive circuit to promote pore insertion can be adapted according to the membrane properties. These may include different material properties of the membrane or pore, or chip-to-chip or pixel-to-pixel variations associated with manufacturing. This may allow the reference voltage applied across the membrane for each pixel 106 to be more quickly set to a value that is likely to effectively promote pore insertion.

[0126] This approach can be implemented, for example, in the embodiments shown in Figures 4, 6, or 7. The reference voltage V used to perform pore insertion CLAMP and / or the selected current I S The value of is determined by a measured property of the membrane 4, e.g., capacitance C m and resistor R m The workflow is shown diagrammatically in FIG.

[0127] Referring to FIG. 3, the electrical properties of the membrane 4 can be determined by switching the device to the measurement state by closing switch S2 and opening switch S1. Then, C m and R m The electrical properties of the membrane, such as can be measured using measurement circuitry 104.

[0128] Appropriate electrical parameters, such as reference voltages and selected current values ​​associated with the pore insertion circuit 102, may then be defined according to the measured values ​​of Cm and Rm, either globally or on a pixel-by-pixel basis.

[0129] In one embodiment as shown in FIG. CLAMP and / or the selected current I Sis selectively programmed for each pixel. Alternatively, the reference voltage V CLAMP and / or the selected current I S is globally defined for all pixels 106. CLAMP and / or the selected current I S can be stepped between different values ​​with different configurations of switch S2 being opened in different steps.

[0130] The membrane resistance R represents the leak path through or around the membrane 4. m The variation of R m Since the value of may determine whether the pore insertion circuit 102 operates to limit the voltage or limit the current (i.e., in a voltage limiting mode or a current limiting mode), the current I S Typically, I S The value of I S *R m is the clamp (reference) voltage V CLAMP (so that the drive circuitry can generate a V across the membrane before pore insertion occurs). CLAMP (I) is applied, but not so large that a significant potential is created across the membrane 4 after the pore is inserted. This reduces the risk of damage to the membrane 4 after the membrane channel is inserted. Depending on the configuration and size of the pixel 106, I S Typical values ​​for may range from 0 to 300 pA, optionally range from 100 to 200 pA, and optionally about 150 pA.

[0131] Alternatively or in addition to the embodiments described above, the drive circuitry may comprise a latch arrangement configured to automatically latch the potential difference applied across the membrane after insertion of the membrane channel into the membrane at a lower value than the potential difference applied before insertion of the membrane channel into the membrane.

[0132] In such an embodiment, the drive circuit and pore insertion circuit 102 are configured to use a circuit arrangement configured such that upon a reduction in the voltage across the membrane (associated with pore insertion causing a drop in the effective resistance across the membrane), the pore insertion circuit 102 is automatically disconnected from the membrane. The latch arrangement is configured to be triggered by a drop in the applied potential difference caused by the insertion of the membrane channel into the membrane 4.

[0133] This embodiment achieves similar advantages to the embodiment of Figures 10 to 12. S *R m This reduces the risk of damage or rupture due to the insertion of the second pore into the membrane 4 and reduces the possibility of an unwanted second pore being inserted into the membrane 4. Furthermore, the pore may be completely or partially blocked after insertion. This is because p , further increasing the applied potential. However, the latching arrangement disconnects the pore insertion circuit 102 after pore insertion, reducing the risk of high voltages across the membrane 4 in the event of pore blocking.

[0134] An additional advantage is that the means for disconnecting the pore insertion circuit 102 can be integrated into the pore insertion circuit 102 itself, allowing for instantaneous disconnection after pore insertion. This allows for the use of external logic control to periodically disconnect the pore insertion circuit 102 and reduce the total membrane resistance R p R m / R m +R p It becomes unnecessary to measure

[0135] An exemplary pore insertion circuit 102 for achieving disconnection during pore insertion is shown in Figure 14. According to the operation of this exemplary circuit, upon successful pore insertion, V OUTdrops and the output of comparator U1 transitions from low to high. U2 is also a comparator, whose output also transitions. DFFR is a resettable D-type flip-flop. When the output of U2 (connected to input D of DFFR) transitions high, the output QB of DFFR becomes a digital low and is latched in this state. Thus, switch S3 (implemented using a transistor in the example) is turned off and the output OUT is isolated from the rest of the circuit. QB remains low and S3 remains switched off until DFFR is reset by applying a logic high level to input RESET. Thereby, the pore insertion circuit 102 is automatically disconnected after the membrane channel (pore) insertion into the membrane 4 and latched in the disconnected state.

[0136] Alternatively or additionally to the above embodiments, the drive circuit may be further configured to regenerate the mediator after measurement of the interaction between the molecular entity and the membrane channel inserted in the membrane 4. This is a further advantage of the nanopore-based measurement circuit, namely the ability to operate with a reverse bias applied to the membrane 4 to regenerate the mediator.

[0137] After pore insertion, the membrane 4 can be used to perform measurements of molecular entities such as DNA molecules or other biomolecules. The measurements can be, for example, sequencing measurements to sequence DNA molecules. The measurements are achieved by performing measurements of the interactions between the membrane channel and the molecular entities as they pass through the membrane channel. This process can cause depletion of ions in the mediator that forms the first and / or second liquid in the first bath 6 and / or second bath 8. To continue measurements of the molecular entities, the mediator needs to be periodically regenerated.

[0138] Regeneration of the mediator is performed by applying a potential difference across the membrane 4 which is opposite in polarity to the potential difference applied during the measurement of the interaction between the molecular entity and the membrane channel. This forces a current through the membrane channel which is opposite to the current used during the measurement of the molecular entity.

[0139] 15 shows an embodiment of a device in which the functions of performing pore insertion and mediator regeneration are combined in the same circuit. A combined pore insertion / regeneration circuit 102a may be provided as part of the integrated electronics of the ASIC 108 described above used for membrane channel insertion and molecular entity measurement (e.g. molecular sequencing measurement). Any of the circuit configurations of the embodiments described above may be used for both pore insertion and mediator regeneration purposes, as they are configured as voltage-limited current sources.

[0140] Figure 16 shows a typical operating cycle using the apparatus of Figure 15. The process operates in three distinct phases.

[0141] In the pore insertion phase, which takes place before a measurement of the molecular entity (in this example, a sequencing measurement) is performed, switch S1 is closed, S2 is opened, and the combined pore insertion / regeneration circuit 102a switches I according to the requirements for pore insertion, as described for the previous embodiment. S Or V CLAMP The device is configured to provide:

[0142] In the sequencing measurement phase, switch S1 is open and S2 is closed and the device is configured to measure the sequencing current through the pore, for example using measurement circuit 104. During extended sequencing, the mediator (the buffer that forms the first liquid and / or the second liquid) may become depleted, at which point the sequencing measurement may be interrupted to regenerate the mediator.

[0143] In the mediator regeneration mode, switch S2 is opened, S1 is closed, and the combined pore insertion / regeneration circuit 102a supplies a current I to drive the electrochemical mechanism of mediator regeneration. S2 (Not necessarily I S (not equal to) the current I S2 The sign of should be opposite to the sign of the sequencing current measured in the sequencing measurement. After regeneration of the mediator buffer, a second sequencing measurement phase (not shown) can be used.

[0144] Any of the circuits described above may be used with the pore insertion circuit 102 having either a positive or negative V CLAMP and I S For example, FIG. 17 illustrates such a modification of the circuit of FIG. 7. When one or more of switches S3a and S3b are closed, the current I SN A symmetrical current mirror with T4 and T5 is added, which can sink a second comparator U2 and a negative voltage source -V CLAMP The minimum voltage that appears at OUT is -V CLAMP The exemplary circuit of Figure 17 then operates essentially as described above, with the selection of positive or negative polarity of the output voltage and current being made by selecting whether switch S2 or S3 is closed. Thus, the circuit can operate in one polarity to perform pore insertion, or another polarity for mediator regeneration.

[0145] This implementation may be particularly advantageous when the preferred potential for pore insertion is opposite to the potential for mediator regeneration. CLAMP The preferred indication may depend on several factors, including the pH of the first and second liquids (which may be buffers), and the isoelectric potential of the membrane channel to be inserted.

[0146] Additionally or alternatively, the drive circuitry may be configured to modulate the potential difference applied across the membrane with an AC waveform of smaller amplitude than the average amplitude of the potential difference.

[0147] To promote pore insertion into membrane 4, a potential difference is applied across the membrane, V CLAMP +Va and V CLAMP It may be preferable under certain conditions to superimpose a small AC potential on top of the DC potential, such that it is modulated to or from -Va.

[0148] FIG. 18 shows one exemplary configuration for implementing this. Instead of connecting the first electrode 14 to a fixed DC potential (e.g., 0 volts) as in the previous embodiment, the first electrode is connected to a voltage source VCE capable of providing an AC waveform. Modulation of the potential difference is thereby performed by applying an AC waveform to the first electrode 14. This has the advantage of keeping the AC waveform generation separate from the circuitry of the pore insertion circuit 102. However, this is not necessary and in other embodiments, the AC waveform can be, for example, modulated by V CLAMP may be applied to the second electrode 16 by superimposing it on

[0149] Figure 19 shows an example of an AC waveform applied to the first electrode 14 of the circuit of Figure 18. In this example, the waveform has an amplitude V a Alternatively, the modulation voltage may be any other AC waveform, for example a sawtooth, triangle, or sinusoidal waveform. The AC perturbation may have an amplitude in the range 0-100 mV and / or a frequency in the range 0-10 kHz.

[0150] A typical nanopore sequencing system includes consumable parts, a sequencing flow cell 200 as shown in Figures 20 and 22, and a sequencing instrument as shown in Figures 21 and 23-25.

[0151] The apparatus described above may be provided as part of a sequencing flow cell 200 that includes an application specific integrated circuit 108 as described above. An example of such a flow cell 200 is shown diagrammatically in FIG.

[0152] The flow cell 200 comprises additional electronic components 204, such as a control circuit 50 configured to control the drive circuitry and other circuitry for driving the ASIC 108, such as a voltage reference, or non-volatile memory.

[0153] The flow cell 200 further comprises a sensor array 202, which may be processed on or attached to the ASIC 108. The sensor array 202 may be configured to define the first bath 6 and the second bath 8 and to support the membrane 4, for example, using a support structure to define the fluid wells and to support formation of the membrane. The sensor array 202 may further comprise a fluidic element and one or more first electrodes 14, which may be shared among multiple pixels 106 in the array of the ASIC 108, as mentioned above.

[0154] The flow cell 200 further comprises a housing 206. The housing 206 may be made of any suitable material, for example, plastic, and may cover the entire structure. The flow cell 200 may further comprise one or more fluid loading ports and one or more fluid channels fluidly connecting the fluid loading ports to the sensor array 202 and the first bath 6 and / or the second bath 8. The fluid loading ports may be configured to allow loading of reagents, buffers, mediators, and samples (e.g., molecular entities to be measured) into the flow cell 200 by pipette, "spotting on," or other means. The flow cell 200 may further comprise one or more extraction or waste ports for removing fluids, for example buffers used, from the flow cell 200.

[0155] The flow cell 200 may further comprise an electrical connector for connecting the flow cell 200 to a measurement instrument. In such embodiments, the flow cell 200 may also comprise a docking feature for securely and / or removably mounting the flow cell 200 to a measurement instrument. Alternatively or additionally, the flow cell 200 may be configured to enable wireless operation without requiring a direct electrical connection to the sequencing instrument. For example, the flow cell 200 may include means for wireless communication to the measurement instrument and / or means for wirelessly obtaining power, for example, from the measurement instrument.

[0156] The flow cell 200 may include one or more temperature sensors configured to measure the temperature of all or a portion of the flow cell 200. For example, temperature sensors may be provided to measure the temperature of internal fluids, such as the first liquid and / or the second liquid in the first bath 6 and / or the second bath 8. The flow cell 200 may also include one or more temperature control elements for heating or cooling all or a portion of the flow cell 200. For example, temperature control elements may be provided to heat or cool the temperature of internal fluidic elements, such as the first liquid and / or the second liquid in the first bath 6 and / or the second bath 8.

[0157] An example of a sequencing instrument 208 in which the sequencing flow cell 200 may be used is shown generally in Figure 21. The sequencing instrument 208 may be used with any of the embodiments of the apparatus described above, not just those specifically described as sequencing flow cells.

[0158] The sequencing instrument 208 may include instrumentation electronics 210 configured to interface with and / or control the sequencing flow cell 200. This may be accomplished via electrical connectors 212 or via wireless communication means, as mentioned above. The sequencing instrument 208 may further include docking functionality 214 for mechanically docking the sequencing flow cell 200 to the sequencing instrument 208.

[0159] The sequencing instrument 208 may include integrated computing, such as a processor 216, and / or a means for connecting to external computing 218. The computing (either internal or external) may be configured to control the sequencing instrument 208 and, via the instrumentation electronics 210, the sequencing flow cell 200 to perform functions associated with a sequencing measurement. These functions may include analyzing the measurement data from the flow cell 200 (e.g., calculating the sequence of the nucleotide molecules based on measurements from the device, e.g., based on sequence data from the flow cell 200), controlling the ASIC 108, and in particular the drive circuitry of the ASIC, in response to the measurement data (e.g., controlling set voltages and currents, such as reference voltages and selected currents), controlling the rate at which measurements are taken, setting electronic gains associated with the measurement circuitry 104, controlling the ASIC 108 electronics to perform actions to disconnect, repair, update or maintain the pixels 106 in an optimal state for sequencing (e.g., disconnecting the pixels 106 in the event of a membrane rupture, modifying the applied potential to compensate for mediator concentration loss, reversing the applied potential to cause pore unblocking), or switching between different pixels 106 connected to the pixel circuit.

[0160] The sequencing instrument 208 may comprise a temperature control means 220 for controlling the temperature of the sequencing flow cell 200, specifically the temperature of the first and second liquids. The temperature control means 220 may comprise one or more of a heater element, a cooling element, and control electronics. The control electronics may be based on PID control and may interface with temperature control elements, if present, within the flow cell 200 itself.

[0161] Depending on the application, the sequencing instrument 208 can be configured to connect to a single flow cell 200 or to simultaneously connect to multiple flow cells 200. For example, Oxford Nanopore products include the MinION (1 flow cell per sequencing instrument, FIG. 23), GridION (5 flow cells per sequencing instrument, FIG. 24), and PromethION (24 or 48 flow cells per sequencing instrument, FIG. 25).

[0162] Although embodiments of the invention have been described with reference to the apparatus above, the invention may also be embodied in a method for controlling the insertion of a membrane channel into a membrane.

[0163] A method is provided for controlling insertion of a membrane channel into a membrane using an apparatus comprising a first bath for holding a first liquid in contact with a first surface of the membrane, a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids, a first electrode configured to contact the first liquid, and a second electrode configured to contact the second liquid, the method comprising controlling a drive circuit to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel from the first or second liquid into the membrane, the applied potential difference before insertion of the membrane being defined by a reference voltage and the applied potential difference after insertion of the membrane being defined by a selected current provided by a current source and a resistance between the first and second electrodes, a voltage limit of the current source being independent of the reference voltage, and the applied potential difference after insertion of the membrane channel being lower than the applied potential difference before insertion of the membrane channel.

[0164] The method may further include controlling the drive circuit to apply a gradually increasing potential difference across the membrane. Alternatively or additionally, the method may further include periodically measuring one or more electrical properties of the membrane and shutting off subsequent application of the potential difference across the membrane in response to the one or more electrical properties.

[0165] A method is provided for controlling insertion of a membrane channel into a membrane using an apparatus comprising a first bath for holding a first liquid in contact with a first surface of the membrane, a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids, a first electrode configured to contact the first liquid, and a second electrode configured to contact the second liquid, the method comprising controlling a drive circuit to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel from the first or second liquid into the membrane, the drive circuit being configured to automatically lower the potential difference in response to insertion of the membrane channel into the membrane, the drive circuit comprising a latch arrangement configured to automatically latch the potential difference applied across the membrane after insertion of the membrane channel into the membrane at a lower value than the potential difference applied before insertion of the membrane channel into the membrane.

[0166] A method is provided for controlling insertion of a membrane channel into a membrane using an apparatus comprising a first bath for holding a first liquid in contact with a first surface of the membrane, a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids, a first electrode configured to contact the first liquid, and a second electrode configured to contact the second liquid, the method comprising controlling a drive circuit to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel from the first or second liquid into the membrane, and regenerating a mediator after measuring an interaction between a molecular entity and the membrane channel inserted into the membrane, the regeneration of the mediator being performed by applying a potential difference across the membrane that is opposite in polarity to the potential difference applied during the measurement of the interaction between the molecular entity and the membrane channel.

[0167] A method is provided for controlling insertion of a membrane channel into a membrane using an apparatus comprising a first bath for holding a first liquid in contact with a first surface of the membrane, a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids, a first electrode configured to contact the first liquid, and a second electrode configured to contact the second liquid, the method comprising controlling a drive circuit to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel into the membrane from the first or second liquid, and modulating the potential difference applied to the membrane with an AC waveform having an amplitude smaller than the average amplitude of the potential difference.

[0168] A method is provided for controlling insertion of a membrane channel into a membrane using an apparatus comprising a first bath for holding a first liquid in contact with a first surface of the membrane, a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first and second liquids, a first electrode configured to contact the first liquid, and a second electrode configured to contact the second liquid, the method including controlling a drive circuit to apply a potential difference across the membrane via the first and second electrodes to promote insertion of the membrane channel from the first or second liquid into the membrane, measuring one or more electrical properties of the membrane, and setting or adjusting the potential difference applied across the membrane prior to insertion of the membrane channel into the membrane based on the measured one or more electrical properties.

[0169] The method may be modified using any of the features described above for the apparatus as desired and as would be apparent to one of ordinary skill in the art. [Explanation of symbols]

[0170] 4 membrane 6. First Bath 8. Second Bath 14 First electrode 15 Drive circuit 16 Second electrode 20 Pixel circuit 50 Control circuit 102 Pore insertion circuit 102a regeneration circuit 104 Measurement circuit 106 pixels 108 Integrated Circuits 110 Circuit 112 Circuit 200 Flow Cell 202 Sensor Array 204 Electronic Components 206 Housing 208 Sequencing Instruments 210 Instrumentation Electronic Equipment 212 Electrical Connectors 214 Docking Function 216 processors 218 External calculation 220 Temperature control means

Claims

1. 1. A device for controlling the insertion of a membrane channel into a membrane, comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first liquid and the second liquid; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; and and a drive circuit configured to apply a potential difference across the membrane via the first electrode and the second electrode to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid; The drive circuit is the applied potential difference prior to insertion of the membrane channel is defined by a reference voltage; A device wherein the applied potential difference after insertion of the membrane channel is defined by a selected current provided by a current source and a resistance between the first electrode and the second electrode, the voltage limit of the current source is independent of the reference voltage, and the applied potential difference after insertion of the membrane channel is lower than the applied potential difference before insertion of the membrane channel.

2. 2. The apparatus of claim 1, wherein the drive circuit comprises at least one power rail defining the reference voltage and at least one diode in series between the at least one power rail and the first electrode or the second electrode, the at least one power rail and at least one diode configured to limit the applied potential difference to less than or equal to the reference voltage.

3. 3. The apparatus of claim 2, wherein the at least one power rail and at least one diode are configured to limit a magnitude of a voltage applied to the first electrode or the second electrode to less than or equal to the reference voltage.

4. 2. The apparatus of claim 1, wherein the drive circuit comprises a comparator configured to compare the reference voltage to a voltage at the first electrode or the second electrode, and an output from the comparator determines whether the applied potential difference is defined by the reference voltage or the selected current.

5. An apparatus according to any one of claims 1 to 4, wherein the voltage at the first electrode is held constant and the applied potential difference is controlled by controlling the voltage at the second electrode.

6. 6. The device of any one of claims 1 to 5, wherein the selected current is in the range of 50 to 300 pA, and optionally the resistance of the inserted membrane channel is in the range of 0.1 to 10 GOhm.

7. Apparatus according to any one of the preceding claims, wherein the drive circuitry is configured, preferably by logic control, to enable the selected current to be selected from a plurality of available current values.

8. An apparatus according to any preceding claim, wherein the drive circuit is configured to apply a gradually increasing potential difference across the membrane.

9. 9. The device of any one of claims 1 to 8, wherein the device is configured to periodically measure one or more electrical properties of the membrane, and in response to the one or more electrical properties indicating that a membrane channel has been inserted into the membrane, to interrupt subsequent application of the potential difference across the membrane by the drive circuit.

10. the apparatus comprising a measurement circuit configured to perform the periodic measurements of the one or more electrical properties of the film; 10. The device of claim 9, wherein the device is configured to switch from a drive state to a measurement state for each measurement of the one or more electrical properties of the membrane, the drive state being a state in which the drive circuit is electrically connected to the first electrode or the second electrode in such a manner that the potential difference can be applied across the membrane, and the measurement state being a state in which the drive circuit is not electrically connected to the first electrode or the second electrode in such a manner that the drive circuit can apply the potential difference across the membrane.

11. The apparatus of claim 10 , wherein the apparatus is configured to block the subsequent application of the potential difference by not switching from the measurement state to the drive state.

12. 12. The device of claim 1, wherein the drive circuitry comprises a latch arrangement configured to automatically latch the potential difference applied across the membrane after insertion of a membrane channel into the membrane at a lower value than the potential difference applied before the insertion of the membrane channel into the membrane.

13. 1. A device for controlling the insertion of a membrane channel into a membrane, comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first liquid and the second liquid; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; and and a drive circuit configured to apply a potential difference across the membrane via the first electrode and the second electrode to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid; the drive circuitry is configured to automatically reduce the potential difference in response to insertion of the membrane channel into the membrane; The device comprises a latch arrangement configured to automatically latch the potential difference applied across the membrane after insertion of a membrane channel into the membrane at a lower value than the potential difference applied prior to the insertion of the membrane channel into the membrane.

14. 14. The device of claim 12 or 13, wherein the latch arrangement is configured to be triggered by a fall in the applied potential difference caused by insertion of the membrane channel into the membrane.

15. 15. The device of claim 1, wherein the drive circuit is further configured to regenerate a mediator after measurement of an interaction between a molecular entity and a membrane channel inserted in the membrane, and wherein the regeneration of the mediator is performed by applying a potential difference across the membrane that is opposite in polarity to the potential difference applied during the measurement of the interaction between the molecular entity and the membrane channel.

16. 1. A device for controlling the insertion of a membrane channel into a membrane, comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first liquid and the second liquid; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; and and a drive circuit configured to apply a potential difference across the membrane via the first electrode and the second electrode to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid; The device, wherein the driving circuit is further configured to regenerate a mediator after measuring an interaction between a molecular entity and a membrane channel inserted in the membrane, and the regeneration of the mediator is performed by applying a potential difference across the membrane that is opposite in polarity to the potential difference applied during the measurement of the interaction between the molecular entity and the membrane channel.

17. 17. The apparatus of any one of claims 1 to 16, wherein the drive circuit is configured to modulate the potential difference applied across the membrane with an AC waveform of smaller amplitude than the average amplitude of the potential difference.

18. 1. A device for controlling the insertion of a membrane channel into a membrane, comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first liquid and the second liquid; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; and and a drive circuit configured to apply a potential difference across the membrane via the first electrode and the second electrode to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid; The apparatus, wherein the drive circuitry is configured to modulate the potential difference applied across the membrane with an AC waveform having an amplitude less than the average amplitude of the potential difference.

19. 19. Apparatus according to claim 17 or 18, wherein the AC waveform has an amplitude in the range of 0-100 mV and / or a frequency in the range of 0-10 kHz.

20. 20. Apparatus according to any one of claims 17 to 19, wherein the modulation of the potential difference is performed by applying the AC waveform to the first electrode.

21. measuring one or more electrical properties of the film; 17. The device of claim 1, configured to set or adjust the potential difference applied across the membrane prior to insertion of a membrane channel into the membrane based on the measured one or more electrical properties.

22. 1. A device for controlling the insertion of a membrane channel into a membrane, comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first liquid and the second liquid; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; and and a drive circuit configured to apply a potential difference across the membrane via the first electrode and the second electrode to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid; The apparatus, measuring one or more electrical properties of the film; The apparatus is configured to set or adjust the potential difference applied across the membrane prior to insertion of a membrane channel into the membrane based on the measured one or more electrical properties.

23. 23. The device of any one of claims 1 to 22, wherein the drive circuitry is configured to facilitate simultaneous insertion of membrane channels into the membrane at a number of different pixels, each pixel being associated with a first electrode and a second electrode driven by the drive circuitry.

24. 24. The apparatus of claim 23, wherein the drive circuitry is configured to apply a common potential difference to pixels that do not yet have a membrane channel inserted into the membrane.

25. 25. The apparatus of claim 24, wherein the drive circuitry is configured to incrementally increase the common potential difference and to control the incremental increase based on detection of membrane channel insertion in other pixels.

26. 26. The apparatus of claim 25, wherein the drive circuit is configured to control the gradual increase based on a detected membrane channel insertion rate.

27. 27. The apparatus of claim 26, wherein the gradual increase comprises a series of steps, and the drive circuit is configured to initiate a step to a higher potential difference when the detected membrane channel insertion rate drops below a predetermined threshold.

28. The drive circuit is measuring one or more electrical properties of the film at each of the pixels; 28. The apparatus of claim 23, configured to set or adjust the potential difference applied across the membrane at each pixel prior to insertion of a membrane channel into the membrane of the pixel based on the measured one or more electrical properties.

29. Apparatus according to any one of claims 23 to 28, wherein each pixel is associated with a respective pair of first and second electrodes driven by the drive circuit.

30. An apparatus according to any one of claims 23 to 28, wherein two or more of the plurality of pixels are associated with respective second electrodes and share a common first electrode.

31. 31. The apparatus of claim 30, wherein the two or more of the plurality of pixels are associated with the first bath and a plurality of respective second baths, the common first electrode configured to contact the first liquid in the first bath and the respective second electrodes configured to contact the second liquid in the respective second baths.

32. Apparatus according to any one of claims 23 to 31, wherein the second electrodes are individually addressable by the drive circuitry.

33. 33. The apparatus of claim 32, wherein the pixels are arranged in a two-dimensional grid, the second electrode associated with each pixel being connected to the second electrodes associated with other pixels on the same row or column of the grid such that each second electrode is individually addressable using a combination of rows and columns.

34. 34. An apparatus according to any one of claims 23 to 33, wherein each of the second electrodes is provided at the base of a respective second bath, and the drive circuitry is provided in an integrated circuit integral with the second baths, and the second electrodes are electrically connected to the drive circuitry.

35. An apparatus according to any one of claims 23 to 33, wherein the second electrode is removable from the drive circuit.

36. Apparatus according to any one of the preceding claims, wherein the driving circuitry is provided by an application specific integrated circuit.

37. A sequencing flow cell comprising a device according to any one of claims 1 to 36, and a control circuit configured to control the drive circuit; a sensor array defining the first and second baths and configured to support the membrane; a housing having one or more fluid loading ports; one or more fluid channels fluidly connecting the fluid loading port to the first bath and / or the second bath; and an electrical connector for connecting the flow cell to a measurement instrument.

38. A sequencing apparatus comprising one or more of the devices according to any one of claims 1 to 36, and instrumentation electronics configured to interface with the one or more devices; a processor configured to control the drive circuitry of the one or more devices via the instrumentation electronics and to calculate a sequence of a nucleotide molecule based on measurements from the devices; Optionally, the sequencing instrument further comprises a temperature control means configured to control a temperature of said first liquid and said second liquid in said one or more devices.

39. The one or more devices are provided by a sequencing flow cell according to claim 37, the electrical connector of each of the one or more sequencing flow cells is configured to connect to the instrumentation electronics; 40. The sequencing instrument of claim 38, wherein the processor is configured to control the drive circuitry of the one or more flow cells via the control circuitry of the respective flow cell.

40. 1. A method for controlling the insertion of a membrane channel into a membrane using an apparatus, the apparatus comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first liquid and the second liquid; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; The method further comprising: controlling a drive circuit to apply a potential difference across the membrane via the first electrode and the second electrode to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid; the applied potential difference prior to insertion of the membrane channel is defined by a reference voltage; The method, wherein the applied potential difference after insertion of the membrane channel is defined by a selected current provided by a current source and a resistance between the first electrode and the second electrode, the voltage limit of the current source is independent of the reference voltage, and the applied potential difference after insertion of the membrane channel is lower than the applied potential difference before insertion of the membrane channel.

41. 41. The method of claim 40, further comprising controlling the drive circuit to apply a gradually increasing potential difference across the membrane.

42. periodically measuring one or more electrical properties of the film; 42. The method of claim 40 or 41, further comprising: in response to the one or more electrical properties indicating that a membrane channel has been inserted into the membrane, blocking subsequent application of the potential difference across the membrane.

43. 1. A method for controlling the insertion of a membrane channel into a membrane using an apparatus, the apparatus comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first liquid and the second liquid; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; The method further comprising: controlling a drive circuit to apply a potential difference across the membrane via the first electrode and the second electrode to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid; the drive circuitry is configured to automatically reduce the potential difference in response to insertion of the membrane channel into the membrane; The method, wherein the drive circuitry comprises a latch arrangement configured to automatically latch the potential difference applied across the membrane after insertion of a membrane channel into the membrane at a lower value than the potential difference applied prior to the insertion of the membrane channel into the membrane.

44. 1. A method for controlling the insertion of a membrane channel into a membrane using an apparatus, the apparatus comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first liquid and the second liquid; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; The method includes controlling a drive circuit to: applying a potential difference across the membrane via the first electrode and the second electrode to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid; and regenerating a mediator after measuring an interaction between a molecular entity and a membrane channel inserted into the membrane, wherein the regeneration of the mediator is performed by applying a potential difference across the membrane that is opposite in polarity to the potential difference applied during the measurement of the interaction between the molecular entity and the membrane channel.

45. 1. A method for controlling the insertion of a membrane channel into a membrane using an apparatus, the apparatus comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first liquid and the second liquid; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; The method further comprising: controlling a drive circuit to apply a potential difference across the membrane via the first electrode and the second electrode to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid; and modulating the potential difference applied across the membrane with an AC waveform of amplitude less than the average amplitude of the potential difference.

46. 1. A method for controlling the insertion of a membrane channel into a membrane using an apparatus, the apparatus comprising: a first bath for holding a first liquid in contact with a first surface of the membrane; a second bath for holding a second liquid in contact with a second surface of the membrane, the membrane separating the first liquid and the second liquid; a first electrode configured to contact the first liquid; a second electrode configured to contact the second liquid; The method further comprising: controlling a drive circuit to apply a potential difference across the membrane via the first electrode and the second electrode to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid; measuring one or more electrical properties of the film; and setting or adjusting the potential difference applied across the membrane prior to insertion of a membrane channel into the membrane based on the measured one or more electrical properties.

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