Sensing the interaction between molecular entities and nanopores

Abstract

A biochemical sensing system uses a sensor device to sense an interaction between a molecular entity and a nanopore, the sensor device comprising an array of sensor elements supporting the nanopore. A switch arrangement selectively connects a detection channel for amplifying a sensed electrical signal to a corresponding sensor element. Based on an analysis of the amplified electrical signal output from the detection channel, the completion of the interaction at the sensor element is detected. In response thereto, the switch arrangement is controlled to connect the detection channel connected to the sensor element in which the completion of the interaction has been detected to another sensor element.

Description

Technical Field

[0001] The present invention relates to the sensing of interactions between molecular entities and nanopores. Background Art

[0002] The use of nanopores to sense interactions with molecular entities (e.g., polynucleotides) is a powerful technology that has undergone many developments recently. Sensor devices have been developed that include arrays of sensor elements arranged to support corresponding nanopores, thereby increasing data collection by allowing multiple nanopores to sense interactions, typically from the same sample, in parallel. The present invention is directed to further improving throughput and sensitivity. Summary of the invention

[0003] According to a first aspect of the present invention, there is provided a method for sensing an interaction between a molecular entity and a nanopore, the method comprising: providing a sensor device comprising an array of sensor elements, the array of sensor elements being arranged to support a corresponding nanopore capable of interacting with the molecular entity and comprising a corresponding electrode, each sensor element being arranged to output an electrical signal at the electrode, the electrical signal depending on the interaction of the molecular entity with the nanopore; providing a detection circuit, the detection circuit comprising: a plurality of detection channels, each detection channel being capable of amplifying an electrical signal from one of the sensor elements, the number of sensor elements in the array being greater than the number of detection channels; and a switch arrangement being capable of selectively connecting the detection channels to corresponding sensor elements; controlling the switch arrangement to connect the detection channels to corresponding sensor elements; and detecting the completion of the interaction at the sensor element based on an analysis of the amplified electrical signal output from the detection channel, and in response thereto, controlling the switch arrangement to connect the corresponding detection channel connected to the sensor element in which the completion of the interaction has been detected to another sensor element which is not currently connected to the detection channel.

[0004] Therefore, the present invention applies a detection circuit with multiple detection channels that allow parallel sensing of electrical signals from sensor elements of the array. There are more sensor elements than detection channels, and a switching circuit is used to connect the detection channels to the corresponding sensor elements, thereby providing a multiplexed system.

[0005] The configuration of a sensor element array, a plurality of detection channels and a switch arrangement is known from WO-2010 / 122293, which teaches the selection of sensor elements in which a membrane is formed and an acceptable number of membrane proteins are inserted, in order to increase the utilization of the detection channels when the formation of the membrane and the insertion of the membrane proteins are subject to random processes so that the preparation of sensor elements of acceptable quality is incomplete. However, in the present invention, the switch arrangement is controlled in different ways for different purposes.

[0006] Specifically, in the present invention, based on the analysis of the amplified electrical signal output from the detection channel, the detection of the completion of the interaction at the sensor element is detected, and in response thereto, the switch arrangement is controlled to connect the corresponding detection channel connected to the sensor element in which the completion of the interaction has been detected to another sensor element that is not currently connected to the detection channel, thereby allowing the electrical signal to be detected from the other sensor element. In fact, the average waiting time between consecutive interactions can be significantly reduced, and a higher throughput can be achieved even at saturation concentrations of molecular entities.

[0007] Similarly, sensitivity is improved because a smaller number of samples are required to saturate the sensing time. In particular, in regimes where the average time between interactions is less than the average interaction period, the amount of samples provided to the sensing device can be reduced without significantly affecting the sensing throughput.

[0008] Surprisingly, it has been found that this increases the overall throughput of sensing of molecular entities in a sample provided to an array of sensor elements in a sensor device. This benefit is achieved as follows. When an interaction is completed at any given sensor element, there is a delay waiting for another molecular entity to become available. However, it is understood that the probability of a molecular entity being available at another sensor element is greater, making it possible for the delay before another interaction to be reduced. The availability of molecular entities to sensor elements is generally subject to stochastic processes, so even if there is no guarantee that the delay will be reduced at every switching instance, the delay will be reduced on average.

[0009] By applying the present invention in which means for capturing molecular entities in the vicinity of a nanopore are provided, the overall delay reduction can be improved. This further increases the overall throughput by further increasing the probability that the molecular entity is available at another sensor element, since the molecular entity has been captured during an interaction at a sensor element previously connected to the detection channel.

[0010] Such capture can be realized, for example, by sensor elements and / or molecular entities, which are suitable for capturing molecular entities near the corresponding nanopore. Similarly, such capture can be realized by sensor elements, which also include capture parts arranged to capture molecular entities near the corresponding nanopore. Various examples of such capture parts are known. For example, the capture part can be a tether, which is combined with the molecular entity and can be attached to the nanopore or attached to the film in the case of the nanopore insertion film.

[0011] Such capture may be achieved by other means, for example by applying appropriate bias signals to the electrodes of the sensor element.

[0012] This effect can be enhanced by determining whether the molecular entity is available for interaction at another sensor element based on an analysis of the amplified electrical signal output from the corresponding detection channel, and continuing to connect the corresponding detection channel to another sensor element in response to determining that the molecular entity is available for interaction, or controlling the switch arrangement to connect the corresponding detection channel to another sensor element that is not currently connected to the detection channel in response to determining that the molecular entity is not available for interaction. In this way, multiple additional sensor elements can be tested, thereby increasing the overall probability of finding a molecular entity available for interaction and correspondingly increasing throughput.

[0013] In the case where the molecular entity is a polymer, such as a polynucleotide, the present invention provides a specific benefit, and the sensor element is arranged to support a corresponding nanopore, which can interact with the molecular entity during the translocation of the molecular entity relative to the nanopore, such as passing through the nanopore. In this case, due to the relatively long interaction period, the throughput improvement is particularly obvious. Therefore, improvements are achieved for molecular entities, such as long fragment libraries of polynucleotides. For example, when relying on diffusion delivery to the nanopore, a library with a fragment of the order of 100kb ("kb" stands for kilobases) can take tens of seconds to minutes between interactions. However, such large fragments also require a long time to interact (e.g., 100kb at 500 bases per second requires 200 seconds), and the time is effectively used to make the next fragment available on another unused sensor element.

[0014] According to a second aspect of the present invention, there is provided a biochemical sensing system for sensing the interaction between a molecular entity and a nanopore, the biochemical sensing system comprising: a sensor device comprising an array of sensor elements, the array of sensor elements being arranged to support a corresponding nanopore capable of interacting with the molecular entity and comprising a corresponding electrode, each sensor element being arranged to output an electrical signal at the electrode, the electrical signal depending on the interaction of the molecular entity with the nanopore; and a detection circuit, the detection circuit comprising: a plurality of detection channels, each detection channel being capable of amplifying an electrical signal from one of the sensor elements, the number of sensor elements in the array being greater than the number of detection channels; a switch arrangement being capable of selectively connecting the detection channels to the corresponding sensor elements; and a controller being arranged to control switching of the switch arrangement to connect the detection channels to the corresponding sensor elements, wherein the controller is arranged to detect the completion of the interaction at the sensor element based on an analysis of the amplified electrical signal output from the detection channel, and in response thereto, control the switch arrangement to connect the corresponding detection channel connected to the sensor element in which the completion of the interaction has been detected to another sensor element that is not currently connected to the detection channel. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] For a better understanding, embodiments of the invention will now be described by way of non-limiting examples with reference to the accompanying drawings, in which:

[0016] Figure 1 is a schematic of a biochemical sensing system;

[0017] Figure 2 is a schematic cross-sectional view of a sensor device;

[0018] Figure 3 is a flow chart of a method performed in a data processor of a biochemical sensing system; and

[0019] Figure 4 is a set of three time diagrams on the same time scale, time diagram (A) showing the use of a single sensor element in a comparative example, time diagram (B) showing the use of each of the four sensor elements in an example of the method of the present invention, and time diagram (C) showing the overall use of the detection channels in the example. DETAILED DESCRIPTION

[0020] Figure 1 A biochemical sensing system 1 for sensing the interaction of a molecular entity with a nanopore is shown. The biochemical sensing system 1 comprises a sensing apparatus 2 comprising a sensor device 3 and a detection circuit 4 connected to the sensor device 3.

[0021] The sensor device 3 comprises an array of sensor elements 30, each supporting a respective nanopore capable of interacting with a molecular entity. The sensor elements 30 comprise respective electrodes 31. In use, each sensor element 30 outputs an electrical signal at its electrode 31, said electrical signal being dependent on the interaction of the molecular entity with the nanopore. Figure 1 Schematically shown in FIG. 1 , but may have a variety of configurations, some non-limiting examples of which are shown below.

[0022] In one example, the sensor device 3 may have Figure 2 . Here, the sensor device 2 comprises an array of sensor elements 30, each of which comprises a membrane 32 supported by a well 33 in a substrate 34, wherein a nanopore 35 is inserted into the membrane 32. The membrane 31 can be made of amphiphilic molecules such as lipids as discussed further below. Each membrane 32 seals a corresponding well 33 to be isolated from a sample chamber 36, which extends through the array of sensor elements 30 and is in fluid communication with each nanopore 35. Each well 33 has a sensor electrode 32 arranged therein. A common electrode 37 is disposed in the sample chamber 36 for providing a common reference potential to each sensor element 30. In use, the sample chamber 36 receives a sample containing a molecular entity that interacts with the nanopore 35 of the sensor element 30.

[0023] For clarity, in Figure 2 Two sensor elements 30 are shown in FIG. 1 , but generally any number of sensor elements 30 may be provided. Generally, a large number of sensor elements 30 may be provided to optimize the data collection rate, such as 256, 1024, 4096 or more sensor elements 30.

[0024] The sensor device 3 may have a detailed structure as disclosed in WO-2009 / 077734 or WO-2014 / 064443.

[0025] The nanopore and related elements of the sensor element 30 may be as follows, but are not limited to: Figure 2 The example shown in .

[0026] A nanopore is a pore that generally has dimensions on the order of nanometers. In embodiments where the molecular entity is a polymer that interacts with the nanopore while translocating therethrough, in such instances the nanopore is of a suitable size to allow the polymer to pass therethrough.

[0027] The nanopore can be a protein pore or a solid-state pore. The size of the pore can be such that only one polymer can translocate the pore at a time.

[0028] Where the nanopore is a protein pore, it may have the following properties.

[0029] The nanopore can be a transmembrane protein pore. The transmembrane protein pore for use according to the present invention can be derived from a β-barrel pore or an α-helical bundle pore. The β-barrel pore comprises a barrel or channel formed by a β-strand. Suitable β-barrel pores include, but are not limited to, β-toxins, such as α-hemolysin, anthrax toxin, and leukocidin; and outer membrane proteins / porins of bacteria, such as Mycobacterium smegmatis porins (Msp), such as MspA, MspB, MspC, or MspD, lysin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, and Neisseria autotransporter lipoprotein (NalP). The α-helical bundle pore comprises a barrel or channel formed by an α-helix. Suitable α-helical bundle pores include, but are not limited to inner membrane proteins and α outer membrane proteins, such as WZA and ClyA toxins. The transmembrane pore can be derived from Msp or from α-hemolysin (α-HL). The transmembrane pore can be derived from lysin. Suitable pores derived from lysenin are disclosed in WO 2013 / 153359. Suitable pores derived from MspA are disclosed in WO-2012 / 107778. The pore may be derived from CsgG as disclosed in WO-2016 / 034591. The pore may be a DNA origami pore.

[0030] The protein pore may be a naturally occurring pore or may be a mutant pore. Typical pores are described in WO-2010 / 109197; Stoddart D et al., Proc Natl Acad Sci, 12; 106(19):7702-7; Stoddart et al., Angew Chem Int Ed Engl. 2010; 49(3):556-9; Stoddart D et al., Nano Lett. 2010 Sep 8; 10(9):3633-7; Butler TZ et al., Proc Natl Acad Sci 2008; 105(52):20647-52; and WO-2012 / 107778.

[0031] The protein pore may be one of the type described in WO-2015 / 140535 and may have a sequence disclosed therein.

[0032] In the case where the nanopore is a protein pore, it can be inserted into a membrane supported in the sensor element 30. Such a membrane can be an amphiphilic layer, such as a lipid bilayer. An amphiphilic layer is a layer formed by amphiphilic molecules such as phospholipids, which have both hydrophilicity and lipophilicity. The amphiphilic layer can be a monolayer or a bilayer. The amphiphilic layer can be a coblock polymer, such as disclosed in Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450 or WO-2014 / 064444. Alternatively, the protein pore can be inserted into a hole provided in a solid layer, for example, as disclosed in WO-2012 / 005857.

[0033] A nanopore may include a hole formed in a solid state layer, which may be referred to as a solid state pore. A hole may be a well, gap, channel, groove or slit provided in a solid state layer through which an analyte may pass or enter the solid state layer. Such a solid state layer is not of biological origin. In other words, the solid state layer is not produced from, nor isolated from, a biological environment (such as an organism or cell) or a biologically usable structure in a synthetically manufactured form. The solid state layer may be formed of both organic and inorganic materials, including (but not limited to): microelectronic materials; insulating materials, such as Si3N4, A12O3 and SiO; organic and inorganic polymers, such as polyamides; plastics, such as or an elastomer, such as a two-component addition-cured silicone rubber; and glass. The solid layer may be formed of graphene. Suitable graphene layers are disclosed in WO-2009 / 035647, WO-2011 / 046706 or WO-2012 / 138357. A suitable method for preparing an array of solid pores is disclosed in WO-2016 / 187519.

[0034] Such solid-state pores are typically pores in a solid-state layer. The pores may be modified chemically or otherwise to enhance their properties as nanopores. Solid-state pores may be used in conjunction with additional components that provide alternatives to polymers or additional measurements, such as tunneling electrodes (Ivanov AP et al., Nano Express 2011 Jan 12; 11(1):279-85), or field effect transistor (FET) devices (e.g., as disclosed in WO-2005 / 124888). Solid-state pores may be formed by known processes, including, for example, those described in WO-00 / 79257.

[0035] The molecular entity interacts with the nanopore in the sensor element 30 , so that an electrical signal dependent on the interaction is output at the electrode 31 .

[0036] In one type of sensor device 3, the electrical signal may be an ionic current flowing through the nanopore. Similarly, electrical properties other than ionic current may be measured. Some examples of alternative types of properties include, but are not limited to, ionic current, impedance, tunneling properties, such as tunneling current (e.g., as disclosed in Ivanov AP et al., Nano lett. 2011 January 12; 11(1): 279-85) and FET (field effect transistor) voltage (e.g., as disclosed in WO2005 / 124888). One or more optical properties may be used, optionally in combination with electrical properties (Soni GV et al., Rev Sci Instrum. 2010 January; 81(1): 014301). The property may be a transmembrane current, such as an ionic current flowing through a nanopore. The ionic current may typically be a DC ionic current, but in principle an alternative uses an AC current (i.e., the magnitude of an AC current flowing under an applied AC voltage).

[0037] The interaction may occur during translocation of the molecular entity relative to, eg, through, the nanopore.

[0038] The electrical signal provides a series of measurements of properties associated with the interaction between the molecular entity and the nanopore. Such interactions may occur at the constriction region of the nanopore. For example, in the case where the molecular entity is a polymer comprising a series of polymer units that are translocated relative to the nanopore, the measurements may have properties that depend on the continuous polymer units that are translocated relative to the pore.

[0039] The ionic solution can be provided on either side of the nanopore. A sample containing a molecular entity of interest as a polymer can be added to one side of the nanopore, for example Figure 2 The sample chamber 36 in the sensor device of the membrane and allows the sample to be translocated relative to the nanopore, for example under a potential difference or chemical gradient. An electrical signal can be obtained during the translocation of the polymer relative to the hole, for example during the translocation of the polymer through the nanopore. The polymer can be partially translocated relative to the nanopore.

[0040] In order to allow measurement when the polymer translocation passes through the nanopore, the translocation rate can be controlled by a binding moiety that is bound to the polymer. Typically, the binding moiety can utilize an applied field or resist an applied field to move the polymer through the nanopore. The binding moiety can be a molecular motor, such as when the binding moiety is an enzyme, the molecular motor uses enzyme activity or acts as a molecular brake. In the case where the polymer is a polynucleotide, many methods for controlling the translocation rate are proposed, including the use of polynucleotide binding enzymes. Suitable enzymes for controlling the translocation rate of polynucleotides include but are not limited to polymerases, helicases, exonucleases, single-stranded and double-stranded binding proteins and topoisomerases (such as gyrase). For other polymer types, a binding moiety that interacts with the polymer type can be used. The binding moiety may be any of those disclosed in WO-2010 / 086603, WO-2012 / 107778 and Lieberman KR et al., J Am Chem Soc. 2010; 132(50): 17961-72) and for voltage gating schemes (Luan B et al., Phys Rev Lett. 2010; 104(23): 238103).

[0041] The binding moiety can be used to control the motion of the polymer in a variety of ways. The binding moiety can use an applied field or resist an applied field to move the polymer through the nanopore. The binding moiety can be used as a molecular motor, for example, when the binding moiety is an enzyme, the molecular motor uses enzyme activity, or is used as a molecular brake. The translocation of the polymer can be controlled by a molecular brake that controls the movement of the polymer through the hole. The molecular brake can be a polymer-bound protein. For polynucleotides, the polynucleotide-binding protein is preferably a polynucleotide processing enzyme. The polynucleotide processing enzyme is a polypeptide that can interact with a polynucleotide and modify at least one of its properties. The enzyme can modify the polynucleotide by cutting the polynucleotide to form a single nucleotide or a shorter nucleotide chain such as a dinucleotide or a trinucleotide. The enzyme can modify the polynucleotide by moving the polynucleotide toward or to a specific position. The polynucleotide handling enzyme does not need to show enzymatic activity, as long as it can bind to the target polynucleotide and control its movement through the hole. For example, the enzyme can be modified to remove its enzymatic activity or can be used under conditions that prevent it from acting as an enzyme. Such conditions are discussed in more detail below.

[0042] Preferred polynucleotide handling enzymes are polymerases, exonucleases, helicases and topoisomerases (such as gyrase). The polynucleotide handling enzyme may be, for example, a type of polynucleotide handling enzyme described in WO-2015 / 140535 or WO-2010 / 086603.

[0043] Translocation of the polymer through the nanopore can occur as cis to trans or trans to cis, with or relative to an applied potential. Translocation can occur under an applied potential, which can control the translocation.

[0044] The exonuclease that gradually or stepwise works on double-stranded DNA can be used on the cis side of the hole to supply the remaining single strand under the applied potential or to supply the trans side under the reverse potential. Similarly, the helicase that unwinds the double-stranded DNA can also be used in a similar manner. There is also the possibility of sequencing applications that need to resist the chain translocation of the applied potential, but DNA must first be "captured" by the enzyme under the opposite or no potential. As the potential is subsequently turned back after binding, the chain will pass through the hole in a cis-to-trans manner and be kept in an extended configuration by electric current. Single-stranded DNA exonucleases or single-stranded DNA-dependent polymerases can serve as molecular motors, and the molecular motors pull the recently translocated single strands back into the hole in a stepwise controlled manner (trans-to-cis, relative to the applied potential). Alternatively, single-stranded DNA-dependent polymerases can serve as molecular brakes that slow down the movement of polynucleotides through holes. Any of the moieties, techniques or enzymes described in WO-2012 / 107778 or WO-2012 / 033524 may be used to control polymer motion.

[0045] The sensor element 30 and / or the molecular entity may be adapted to capture the molecular entity near the corresponding nanopore. For example, the sensor element 30 may also include a capture portion arranged to capture the molecular entity near the corresponding nanopore. The capture portion may be any of the above-mentioned binding portions or exonucleases that also have the purpose of controlling translocation, or may be provided separately.

[0046] The capture moiety may be attached to the nanopore of the sensor element. At least one capture moiety may be attached to the nanopore of each sensor element.

[0047] The capture moiety may be a tag or tether that binds to a molecular entity. In this case, the molecular entity may be adapted to effectuate said binding.

[0048] Such tags or tethers can be attached to a nanopore, for example, as disclosed in WO-2018 / 100370 and as further described below.

[0049] Alternatively, where the nanopore is inserted into a membrane, such tags or tethers may be attached to the membrane, for example as disclosed in WO-2012 / 164270.

[0050] The methods described herein may include the use of an adapter, the purpose of which is to make the molecular entity suitable for capture. For example, polynucleotide adapters suitable for nanopore sequencing of polynucleotides are known in the art. The adapters for nanopore sequencing of polynucleotides may include at least one single-stranded polynucleotide or non-polynucleotide region. For example, Y-adapters for nanopore sequencing are known in the art. The Y adapter typically includes (a) a double-stranded region and (b) a single-stranded region or a region that is not complementary at the other end. If the Y adapter includes a single-stranded region, it can be described as having an overhang. Since the two chains do not hybridize to each other usually like the double-stranded portion, the presence of non-complementary regions in the Y adapter makes the adapter have a Y shape. The Y adapter may include one or more anchors.

[0051] The Y adapter preferably comprises a leader sequence, which is preferably screwed into the hole. The leader sequence generally includes a polymer. The polymer is preferably negatively charged. The polymer is preferably a polynucleotide, such as DNA or RNA, a modified polynucleotide (such as abasic DNA), PNA, LNA, polyethylene glycol (PEG) or a polypeptide. The leader preferably comprises a polynucleotide, and more preferably comprises a single-stranded polynucleotide. The single-stranded leader sequence most preferably comprises a single strand of DNA, such as a poly dT segment. The leader sequence preferably comprises one or more spacers. The leader sequence can be any length, but its length is generally 10 to 150 nucleotides, such as 20 to 150 nucleotides. The length of the leader generally depends on the membrane embedded nanopore used in the method. The leader sequence preferentially screws into the transmembrane hole, and thereby promotes the movement of the polynucleotide through the hole. The adapter can be connected to the DNA molecule using any method known in the art.

[0052] The polynucleotide adapter can include a membrane anchor or a transmembrane pore anchor attached to the adapter. For example, a membrane anchor or a transmembrane pore anchor can promote the positioning of the adapter and the coupled polynucleotide near the nanopore. The anchor can be a polypeptide anchor and / or a hydrophobic anchor that can be inserted into the membrane. In one embodiment, the hydrophobic anchor is a lipid, a fatty acid, a sterol, a carbon nanotube, a polypeptide, a protein or an amino acid, such as cholesterol, palmitate or tocopherol. The anchor can include a thiol, biotin or a surfactant. The anchor can be biotin (for binding to streptavidin), amylose (for binding to maltose binding protein or fusion protein), Ni-NTA (for binding to polyhistidine or polyhistidine tagged protein) or a peptide (such as an antigen).

[0053] The anchor may include one linker, or 2, 3, 4 or more linkers. Preferred linkers include, but are not limited to, polymers such as polynucleotides, polyethylene glycol (PEG), polysaccharides and polypeptides. These linkers may be linear, branched or cyclic. For example, the linker may be a cyclic polynucleotide. The adapter may hybridize with a complementary sequence on a cyclic polynucleotide linker. One or more anchors or one or more linkers may include components that may be cut or decomposed, such as restriction sites or photolabile groups. The linker may be functionalized with a maleimide group to attach to a cysteine ​​residue in a protein. Suitable linkers are described in WO 2010 / 086602. The anchor may be cholesterol or a fatty acyl chain. For example, any fatty acyl chain having a length of 6 to 30 carbon atoms, such as hexadecanoic acid, may be used. Examples of suitable anchors and methods of attaching the anchor to the adapter are disclosed in WO 2012 / 164270 and WO 2015 / 150786.

[0054] Examples of tags and tethers attached to nanopores are shown below.

[0055] The nanopore used in the methods described herein can be modified to include one or more binding sites for binding to one or more analytes (e.g., molecular entities) and thereby act as a capture moiety. In some embodiments, the nanopore can be modified to include one or more binding sites to bind to an adaptor connected to the analyte. For example, in some embodiments, the nanopore can bind to a leader sequence of an adaptor connected to the analyte. In some embodiments, the nanopore can bind to a single-stranded sequence in an adaptor connected to the analyte.

[0056] In some embodiments, the nanopore is modified to include one or more tags or tethers, each tag or tether comprising a binding site for an analyte. In some embodiments, the nanopore is modified to include one tag or tether per nanopore, each tag or tether comprising a binding site for an analyte.

[0057] Attachment of short oligonucleotides to the transmembrane pore, which short oligonucleotides contain a sequence complementary to a leader sequence or another single-stranded sequence in the adaptor, can be used to enhance capture of the target polynucleotide.

[0058] In some embodiments, the tag or tether may include or may be an oligonucleotide (e.g., DNA, RNA, LNA, BNA, PNA, or morpholino). The oligonucleotide (e.g., DNA, RNA, LNA, BNA, PNA, or morpholino) may have a length of about 10-30 nucleotides or a length of about 10-20 nucleotides.

[0059] In some embodiments, the oligonucleotides (e.g., DNA, RNA, LNA, BNA, PNA or morpholino) used in tags or tethers may have at least one modified end (e.g., 3'- or 5'-end) for conjugation with other modifications or solid substrate surfaces (including, for example, beads). The terminal modifier may add reactive functional groups that can be used for conjugation. Examples of functional groups that can be added include, but are not limited to, amino, carboxyl, thiol, maleimide, aminooxy and any combination thereof. The functional group can be combined with spacers of different lengths (e.g., C3, C9, C12, spacers 9 and 18) to increase the physical distance between the functional group and the end of the oligonucleotide sequence.

[0060] In some embodiments, the tag or tether may include or be a morpholino oligonucleotide. The morpholino oligonucleotide may have a length of about 10-30 nucleotides or a length of about 10-20 nucleotides. The morpholino oligonucleotide may be modified or unmodified. For example, in some embodiments, the morpholino oligonucleotide may be modified at the 3' and / or 5' ends of the oligonucleotide. The examples of modifications on the 3' and / or 5' ends of the morpholino oligonucleotide include, but are not limited to, 3' affinity tags and functional groups for chemical attachment (including, for example, 3'-biotin, 3'-primary amines, 3'-disulfide amides, 3'-pyridyl disulfide and any combination thereof); 5' end modifications (including, for example, 5'-primary amines and / or 5'-didecyl), click chemistry modifications (including, for example, 3'-azides, 3'-alkynes, 5'-azides, 5'-alkynes), and any combination thereof.

[0061] In some embodiments, the tag or tether may also include a polymer joint, for example, to promote coupling with a nanopore. Exemplary polymer joints include, but are not limited to, polyethylene glycol (PEG). The polymer joint may have a molecular weight of about 500Da to about 10kDa (including end values), or about 1kDa to about 5kDa (including end values). The polymer joint (e.g., PEG) may be functionalized with different functional groups, including, for example, but not limited to, maleimide, NHS ester, dibenzocyclooctyne (DBCO), azide, biotin, amine, alkyne, aldehyde, and any combination thereof. In some embodiments, the tag or tether may also include a 1kDa PEG with a 5'-maleimide group and a 3'-DBCO group. In some embodiments, the tag or tether may also include a 2kDa PEG with a 5'-maleimide group and a 3'-DBCO group. In some embodiments, the tag or tether may also include a 3kDa PEG with a 5'-maleimide group and a 3'-DBCO group. In some embodiments, the tag or tether may also comprise a 5 kDa PEG having a 5'-maleimide group and a 3'-DBCO group.

[0062] Other examples of tags or tethers include, but are not limited to, a His tag, biotin or streptavidin, an antibody that binds to an analyte, an aptamer that binds to an analyte, an analyte binding domain, such as a DNA binding domain (including, for example, a peptide zipper, such as a leucine zipper, a single-stranded DNA binding protein (SSB)), and any combination thereof.

[0063] The tag or tether can be attached to the outer surface of the nanopore using any method known in the art, for example, on the cis side of the membrane. For example, one or more tags or tethers can be connected to the nanopore by one or more cysteines (cysteine ​​bonds), one or more primary amines (such as lysine), one or more non-natural amino acids, one or more histidines (His tags), one or more biotins or streptavidins, one or more antibody-based tags, one or more enzyme modifications of epitopes (including, for example, acetyltransferases) and any combination thereof. Suitable methods for performing such modifications are well known in the art. Suitable non-natural amino acids include, but are not limited to, 4-azido-L-phenylalanine (Faz), and Liu C.C. and Schultz PG, Annu Rev Biochem, 2010, 79, 413-444. Figure 1 Any of the amino acids numbered 1-71 in .

[0064] In some embodiments where one or more tags or tethers are attached to the nanopore via cysteine ​​linkages, the one or more cysteines may be introduced into one or more monomers forming the nanopore by substitution.

[0065] In some embodiments, the tag or tether can be attached directly to the nanopore or through one or more linkers. The tag or tether can be connected to the nanopore using a hybrid linker described in WO 2010 / 086602. Alternatively, a peptide linker can be used. A peptide linker is an amino acid sequence. The length, flexibility and hydrophilicity of the peptide linker are generally designed so that it does not interfere with the function of the monomer and the pore. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16 serines and / or glycines. More preferred flexible linkers include (SG)1, (SG)2, (SG)3, (SG)4, (SG)5 and (SG)8, wherein S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24 prolines. More preferred rigid linkers include (P) 12 , where P is proline.

[0066] The transmembrane pore can be modified to enhance capture of polynucleotides. For example, the pore can be modified to increase the positive charge within the entrance to the pore and / or within the barrel of the pore. Such modifications are known in the art. For example, WO 2010 / 055307 discloses mutations in α-hemolysin that increase the positive charge within the barrel of the pore.

[0067] Modified MspA, lysenin and CsgG pores comprising mutations that enhance polynucleotide capture are disclosed in WO 2012 / 107778, WO 2013 / 153359 and WO 2016 / 034591, respectively. Any modified pore disclosed in these publications may be used herein.

[0068] The arrangement of the detection circuit 4 will now be discussed. The detection circuit 4 is connected to the electrode 31 of each sensor element 30 and has a main function of processing the electrical signal output therefrom. The detection circuit 4 also has a function of controlling application of a bias signal to each sensor element 30.

[0069] The detection circuit 4 includes a plurality of detection channels 40. Each detection channel 40 receives an electrical signal from a single sensor electrode 31 and is arranged to amplify the electrical signal. Therefore, the detection channel 40 is designed to amplify very small currents with sufficient resolution to detect characteristic changes caused by the interaction of interest. The detection channel 40 is also designed to have a sufficiently high bandwidth to provide the time resolution required to detect each such interaction. These constraints require sensitive and therefore expensive components. Each detection channel 40 can be similar to the standard single-channel recording equipment described in Stoddart D et al., Proc Natl Acad Sci, 12; 106 (19): 7702-7, Lieberman KR et al., J Am Chem Soc 2010; 132 (50): 17961-72 and WO-2000 / 28312. Alternatively, each detection channel 40 may be arranged as described in detail in WO-2010 / 122293, WO-2011 / 067559 or WO-2016 / 181118.

[0070] The number of sensor elements 30 in the array is greater than the number of detection channels 40, and the biochemical sensing system can be used to measure polymers from sensor elements 30 selected in a multiplexed manner, in particular in an electrically multiplexed manner. This is achieved by providing a switch arrangement 42 between the sensor electrodes 31 of the sensor elements 30 and the detection channels 40. For clarity, FIG6 shows a simplified example with four sensor elements 30 and two detection channels 40, but the number of sensor elements 30 and detection channels 40 is typically much larger. For example, for some applications, the sensor device 2 may include a total of 4096 sensor elements 30 and 1024 detection channels 40.

[0071] The switch arrangement 42 may be arranged as described in detail in WO-2010 / 122293. For example, the switch arrangement 42 may include a plurality of 1-to-N multiplexers, each multiplexer connected from a detection channel 40 to a group of N sensor elements 30, and may include appropriate hardware such as latches for selecting the switching state.

[0072] The biochemical sensing system 1 can be used to amplify electrical signals from sensor elements 30 selected in an electrically multiplexed manner by switching of the switch arrangement 42. The detection circuit 4 includes a data processor 5 that receives output signals from the detection channels 40. The data processor 5 acts as a controller that controls the switch arrangement 42 to connect the detection channels 40 to the corresponding sensor elements 30, as further described below.

[0073] In addition, the detection circuit 4 includes a bias control circuit 41 to perform the function of controlling the application of a bias signal to each sensor element 30. The bias control circuit 41 is connected to the common electrode 37 and to the sensor electrode 31 of each sensor device 30. The bias signal is selected to bias the sensor electrode 31 relative to the common electrode 37 to control the translocation of the molecular entity relative to the nanopore. In general, the bias signal provided to a given sensor element 30 can be a driving bias signal that causes translocation to occur at the sensor element 30 or an inhibiting bias signal that inhibits translocation to occur at the sensor element 30.

[0074] The bias control circuit 41 is controlled by the data processor 5. The data processor can select two operation modes for the bias control circuit 41.

[0075] In a first mode of operation of the bias control circuit 41, a drive bias signal is provided to all sensor elements 30, thereby causing translocation of the molecular entity relative to the nanopore of each sensor element 30, where the molecular entity is available. In said first mode of operation, translocation may occur at any sensor element 30, regardless of whether an electrical signal from the sensor element 30 is provided to the detection channel 40 by the switch arrangement 42. Thus, the occurrence of translocation depends solely on the stochastic process that makes the molecular entity available to the sensor element 30. In the case where the sensor element 30 comprises a capture moiety as described above, translocation occurs after capture, but no electrical signal is provided to the detection channel.

[0076] In a second operating mode of the bias control circuit 41, the bias control circuit 41 is controlled synchronously with the switching of the switch arrangement 42 so as to provide a driving bias signal to the sensor element when the corresponding sensor element 30 is connected to the detection channel 41, and to provide an inhibition bias signal to the sensor element when the corresponding sensor element 30 is not connected to the detection channel 40. In the second operating mode, when the sensor element 30 is connected to the detection channel 41 to provide an electrical signal, translocation occurs only at the sensor element. However, translocation is inhibited at the sensor element 30 that is not connected to the detection channel 40 by the switch arrangement. However, at such a sensor element 30, capture of molecular entities near the sensor element 30 may still occur. For example, such capture can occur with the aid of a sensor element 30 comprising a capture portion as described above or with the aid of an appropriately selected inhibition bias signal.

[0077] The data processor 5 is arranged as follows. The data processor 5 is connected to the output of the detection channel 40 and provides the amplified electrical signal therefrom. The data processor 5 stores and analyses the amplified electrical signal and controls other elements of the detection circuit based on the analysis, including controlling the bias voltage circuit 41 as described above and controlling the switch arrangement 42 as described below. The data processor 5 forms part of the detection circuit 2 and may be arranged in a common package with the detection circuit, possibly on a common circuit board. The data processor 5 may be implemented in any suitable form, for example as a processor running an appropriate computer program or as an ASIC (Application Specific Integrated Circuit).

[0078] The data processor 5 of the biochemical sensing system 1 is connected to the analysis system 6. The data processor 5 also provides the amplified output signal to the analysis system 6. The analysis system 6 performs further analysis on the amplified electrical signal, which is the raw signal representing the measured value of the property measured at the nanopore. Such an analysis system 6 can, for example, estimate the identity of the molecular entity as a whole, or in the case where the molecular entity is a polymer, the identity of its polymer unit can be estimated. Therefore, the analysis system can be configured as a computer device running an appropriate program. Such a computer device can be connected to the data processor 5 of the biochemical sensing system 1 directly or via a network (e.g., in a cloud-based system).

[0079] The method for controlling the detection circuit 2 executed by the data processor 5 is Figure 3 The same method is performed in parallel for each detection channel 40.

[0080] The method starts in step S1 , when no sensor element 30 is connected to the detection channel 40. In a second operating mode of the bias control circuit 41 , an inhibit bias signal is now provided to each sensor element 30.

[0081] In step S2, the data processor 5 selects which sensor element 30 will be connected to each corresponding detection channel 40. In the first example of performing step S2, since no information about the sensor elements 30 is known, any sensor element 30 may be selected, for example, sensor elements 30 uniformly dispersed around the sensor element array. In the second operation mode of the bias control circuit 41, as mentioned above, the data processor 5 controls the bias control circuit 41 to provide a driving bias signal to the selected sensor element 30, instead of the previously provided suppression bias signal.

[0082] In step S3 , the data processor 5 controls the switch arrangement to connect the sensor element 30 selected in step S2 to the corresponding detection channel 40 .

[0083] In step S4, the amplified electrical signals from the selected sensor elements 30 are received and stored by the data processor for a period of time corresponding to the plurality of measurements. The stored electrical signals provide a "block" of data representing the amplified electrical signals during the period of time.

[0084] In step S5 , the data processor 5 analyzes the data block of each corresponding sensor element 30 and decides whether to continue receiving data from the corresponding sensor element 30 based on the analysis.

[0085] Typically, when step S5 is performed, at each respective sensor element 30, the molecular entity may or may not be available for interaction with the nanopore of the sensor element. The molecular entity may be available for interaction because it has been captured near the nanopore using the above means, for example by a capture moiety or by an appropriate bias signal. This may include situations where the molecular entity has not yet begun to translocate, or where translocation has only just begun. Alternatively, the molecular entity may be available for interaction because it has already been partially translocated.

[0086] In the first mode of operation of the bias control circuit 41, when step S5 is performed for the first time after connecting a selected sensor element 30 to the corresponding detection channel 40, translocation may have previously occurred at any sensor element 30, and thus the molecular entity may have partially translocated through the selected sensor element 30. Therefore, whether this is the case at any given sensor element 30 may vary due to the stochastic processes driving capture and translocation in the sensor device 3.

[0087] In the second mode of operation of the bias control circuit 41, when step S5 is performed for the first time after the selected sensor element 30 is connected to the corresponding detection channel 40, due to the suppression of the provision of the bias signal, no translocation has previously occurred at any sensor element 30, but the molecular entity may have partially translocated through the selected sensor element 30, but the molecular entity is available for interaction because it has been captured near the nanopore using the above-mentioned means. Again, whether this is the case at any given sensor element 30 will vary due to the stochastic process that drives the capture of molecular entities in the sensor device 3.

[0088] The analysis performed in step S5 involves determining whether the amplified electrical signal indicates that the molecular entity is available for interaction with the nanopore of the corresponding sensor element 30. This determination is possible because the electrical signal is a characteristic of whether the molecular entity is available for interaction. For example, in the case where the measured property is the ionic current flowing through the nanopore, when there are no molecular entities available for interaction, the ionic current can have a relatively high level, and when the molecular entity is available for interaction due to the molecular entity blocking the pore, the ionic current can have a relatively low level. Therefore, for example, by low-pass filtering the amplified electrical signal and testing the amplified electrical signal against a threshold that distinguishes between different situations, it can be directly determined whether the molecular entity is available for interaction. In the case of other measured properties, similar characteristics can be used to make a determination in step S5.

[0089] Therefore, in step S5, the data processor 5 determines based on the analysis of the data block whether the molecular entity is available for interaction with the nanopore of the already selected sensor element 30. If yes, step S5 makes it possible to decide to continue receiving data from the corresponding sensor element 30, and the method returns to step S4. Otherwise, step S5 makes it possible to decide not to continue receiving data from the corresponding sensor element 30, and the method therefore returns to step S2.

[0090] Considering that step S5 is performed for the first time after controlling the switch arrangement 42 to connect the detection channel 40 to a new sensor element 30 when no molecular entities are available for interaction at said new sensor element, this causes step S2 to be performed again to select another new sensor element.

[0091] The process is then repeated with one or more new sensor elements 30 selected in successive executions of step S2 until, in step S5, it is detected that the detection channel is connected to a sensor element in which the molecular entity is available.

[0092] When the molecular entity is available for interaction at a new sensor element 30, then a return to step S4 is made so that steps S4 and S5 are repeated for another data block corresponding to the electrical signal. Repeated execution of steps S4 and S5 effectively records the electrical signal during the interaction of the molecular entity until it is determined in step S4 that the molecular entity is no longer available for interaction with the nanopore of the sensor element 30. This is in fact a detection of the completion of the interaction and causes the method to return to step S2 to select another new sensor element 30.

[0093] The overall effect is that whenever completion of interaction at a sensor element 30 is detected, the detection channel is switched to a new sensor element.

[0094] In step S2, the data processor 5 may select new sensor elements 30 to connect to the corresponding detection channel 4 in any suitable manner, taking into account the connections that the switch arrangement 42 is able to make. For example, where the switch arrangement 42 comprises a plurality of 1-to-N multiplexers, the data processor 5 may continuously select sensor elements 30 in each group of N sensor elements 30 for connection to the corresponding detection channel 40 in a regular cycle. In a more sophisticated approach, the selection may take into account other information available to the data processor 5. In one example, the data processor 5 may record the interval since the last known interaction at each sensor element 30, and preferentially select sensor elements 30 with long intervals. In another example, the data processor 5 may record state information about the sensor elements 50, and take the state information into account in the selection.

[0095] In an optional variation, the data processor 5 may additionally analyze the amplified electrical signal to detect sensor elements 30 that do not have acceptable performance qualities because a membrane has not been formed and an acceptable amount of membrane protein has not been incorporated, and record the results. In this case, the data processor 5 may avoid selecting sensor elements 30 in this case, thereby effectively applying techniques similar to those disclosed in WO-2010 / 122293 but in combination with the additional steps disclosed herein.

[0096] Figure 3 Implementation of the control method shown in improves the overall throughput of data collection from the sensor device 3, because even at saturation concentrations of the molecular entity, the average waiting time between consecutive interactions can be significantly reduced. This is because the probability of the molecular entity being available at another sensor element 30 is greater than the probability of being available at the sensor element 30 where the interaction has just been completed. Therefore, on average, the delay between completing one interaction and collecting the electrical signal from a new interaction on the same detection channel 40 is reduced. Due to stochastic processes in the sensor device 3, the molecular entity is available to other sensor elements 30, thereby increasing this probability.

[0097] Viewed another way, the nanopore interacts with nearby molecular entities faster than with distant molecular entities. The occurrence of such interactions is probabilistic, limited by the probability of diffusion of the molecular entity from the bulk sample into the nanopore vicinity. Typically, the nanopore sensing system captures and depletes molecular entities from the vicinity faster than new molecular entities enter the vicinity. In this case, the occurrence of interactions is limited by the rate at which new molecular entities enter the local vicinity from the bulk sample.

[0098] Similarly, sensitivity is improved because a smaller number of samples are required to saturate the sensing time. In particular, in conditions where the average time between interactions is less than the average interaction period, the amount of samples provided to the sensing device 3 can be reduced without significantly affecting the sensing throughput.

[0099] This benefit is similar in both the first and second operating modes of the bias control circuit 54 .

[0100] In the first mode of operation, translocation can occur at any sensor element 30, so switching increases the probability that the detection channel 40 receives an electrical signal from the sensor element where the electrical signal is occurring. In this case, the possibility is that the molecular entity will partially translocate at the new sensor element 30 to which the detection channel 40 is connected. Therefore, electrical signals are obtained from parts of the molecular entity, that is, the length of a single reading can be shorter, but the overall rate of data collection is still increased without significantly compromising subsequent analysis. This may be appropriate in the case where the molecular entity is a fragment of a larger molecule, such as a polynucleotide fragment, in which the data is combined to estimate the identity of the entire molecular entity.

[0101] In the second mode of operation, translocation is inhibited until the sensor element 30 is connected to the detection channel 40. Thus, in this case, an electrical signal is obtained from the entire polymer in the case of translocation of the polymer relative to the nanopore. This may be appropriate in the case of a molecular entity whose full length needs to be analyzed.

[0102] As described above, these benefits are particularly evident where the sensor element 30 includes means for capturing molecular entities in the vicinity of the nanopore, as this further increases the likelihood that other sensor elements 30 not connected to any detection channel 40 have captured the molecular entity while simultaneously capturing electrical signals from translocation at the sensor element 30 connected to the detection channel 40. Through such capture, many molecular entities may be retained in the vicinity of the nanopore without applying conditions that consume or deplete the molecular entities.

[0103] Where a sensor element 30 is provided with a capture moiety, even if the capture moiety is effective in capturing a molecular entity, it may be difficult to load another molecular entity onto a given sensor element 30 while sensing an interaction with a previously loaded molecular entity, e.g. even using sidearm hybridization. This may be due to steric or electrostatic repulsion effects of the molecular entity being sequenced preventing another molecular entity from getting close enough to bind. In particular, in such cases, throughput and sensitivity may be achieved by stacking the next molecular entity onto an unused sensor element 30.

[0104] Figure 4 shows three time diagrams (A) to (C) each on the same time scale, for example, illustrating an example of the increased throughput provided by the present method. The time diagrams show an occupied period t-occ when the sensor element 30 has a molecular entity actively translocated relative to its nanopore, an unoccupied period t-unocc when the sensor element 30 does not have a molecular entity actively translocated relative to the nanopore, a waiting period t-wait when the sensor element 30 has a molecular entity near the nanopore but no translocation occurs (this can be achieved using the above-mentioned capture means), and a switching period ts when the switch arrangement 30 switches. Thus, the example corresponds to the second operating mode of the bias control circuit 41, but similar advantages are achieved using the first operating mode of the bias control circuit 41.

[0105] The time diagram (A) shows a single nanopore using no switching as a comparative example. After each occupied period t-occ when an interaction has been completed, there is an unoccupied period t-unocc while waiting for a new interaction to occur. Therefore, the throughput is reduced by the ratio between these periods.

[0106] Depending on the length of the polymer being sensed, the occupied period t-occ can be any time from 0.1 seconds to 60+ minutes. The unoccupied period t-unocc depends on both the analyte concentration and the polymer length (for polymer analytes). The unoccupied period t-unocc increases with the decrease in analyte concentration. The relationship is complicated by the polymer length of the polymer analyte, but roughly speaking, a longer polymer increases the unoccupied period t-unocc compared to a shorter polymer because the increased polynucleotide length makes it more difficult for the nanopore to see the free chain end.

[0107] The time diagram (B) shows the use of each of the four sensor elements 30 when the method of the present invention is applied to cyclically switch between the four sensor elements 30, and the second time diagram (C) shows the overall use of the detection channel connected to the four sensor elements in the time diagram (B). Since this is an example: the unoccupied period t-unocc is short enough compared to the occupied period t-occ so that when cyclically switching between the four sensor elements 30, there is always a situation where the molecular entity can be used for interaction at the new sensor element. Therefore, except during the switching period ts, the detection channel 40 always receives an electrical signal. Therefore, since the switching period ts is shorter than the unoccupied period t-unocc, the throughput is greatly reduced. The switching period ts is the time required for the detection circuit 5 itself, which is independent of the pore / analyte chemical properties and can be up to about 1 second.

[0108] Thus, the methods of the invention are particularly beneficial for sensing long polymers (eg, polynucleotides of at least 10, 20, 30, 40 or 50 kb in length) and / or for sensing at relatively low analyte concentrations.

Claims

1. A method for sensing an interaction between a molecular entity and a nanopore, the method comprising: providing a sensor device comprising an array of sensor elements arranged to support a respective nanopore capable of interacting with a molecular entity and comprising respective electrodes, each sensor element being arranged to output an electrical signal at said electrode, said electrical signal being dependent on the interaction of the molecular entity with said nanopore; A detection circuit is provided, the detection circuit comprising: a plurality of detection channels, each detection channel being capable of amplifying an electrical signal from one of the sensor elements, the number of sensor elements in the array being greater than the number of detection channels; as well as a switch arrangement capable of selectively connecting the detection channels to corresponding sensor elements; controlling the switch arrangement to connect the detection channels to the corresponding sensor elements; as well as Completion of the interaction at the sensor element is detected based on analysis of the amplified electrical signal output from the detection channel, and in response thereto, the switch arrangement is controlled to connect the corresponding detection channel connected to the sensor element in which completion of the interaction has been detected to a new sensor element that is not currently connected to the detection channel.

2. The method according to claim 1, wherein the method further comprises the following operations after the step of controlling the switch arrangement to connect the detection channel to a sensor element that is not currently connected to a detection channel: determining whether a molecular entity is available for interaction at the new sensor element based on an analysis of the amplified electrical signal output from the corresponding detection channel, and In response to determining that the molecular entity is available for interaction, continue connecting the corresponding detection channel to the new sensor element, or in response to determining that the molecular entity is not available for interaction, control the switch arrangement to connect the corresponding detection channel to another new sensor element not currently connected to a detection channel.

3. A method according to claim 1 or 2, wherein the molecular entity is a polymer and the sensor element is arranged to support a corresponding nanopore capable of interacting with the molecular entity during translocation of the molecular entity relative to the nanopore.

4. The method of claim 3, wherein the detection circuit further comprises a bias circuit arrangement capable of providing a bias signal to the sensor element, the bias signal capable of controlling the translocation of the molecular entity relative to the nanopore of the corresponding sensor element.

5. A method according to claim 4, wherein the method further comprises controlling the bias circuit arrangement to apply a bias signal arranged to cause the translocation of the molecular entity relative to the nanopore of all the sensor elements.

6. The method of claim 4, wherein the method further comprises causing the bias circuit arrangement to perform the following operations: applying a drive bias signal to the corresponding sensor element when the corresponding sensor element is connected to a detection channel, the drive bias signal being arranged to cause translocation of the molecular entity relative to the nanopore of the corresponding sensor element; and applying an inhibition bias signal to the corresponding sensor element when the corresponding sensor element is not connected to a detection channel, the inhibition drive signal being arranged to inhibit translocation of the molecular entity relative to the nanopore of the corresponding sensor element.

7. The method of claim 6, wherein the inhibition drive signal is selected to inhibit translocation of the molecular entity relative to the nanopore of the corresponding sensor element and to capture the molecular entity near the nanopore.

8. The method according to claim 1 or 2, wherein the sensor element and / or molecular entity is adapted to capture a molecular entity in the vicinity of the corresponding nanopore.

9. The method according to claim 1 or 2, wherein the sensor element further comprises a capture moiety arranged to capture a molecular entity in the vicinity of the corresponding nanopore.

10. The method of claim 8, wherein the sensor elements each comprise at least one capture moiety attached to the corresponding nanopore of the sensor element.

11. The method of claim 1 or 2, wherein the molecular entity is a polynucleotide.

12. The method of claim 1 or 2, wherein the nanopore is a protein pore.

13. The method according to claim 1 or 2, wherein the sensor element is arranged as a supported membrane, wherein the nanopore is inserted into the membrane.

14. A biochemical sensing system for sensing an interaction between a molecular entity and a nanopore, the biochemical sensing system comprising: a sensor device comprising an array of sensor elements arranged to support a respective nanopore capable of interacting with a molecular entity and comprising respective electrodes, each sensor element being arranged to output an electrical signal at said electrode, said electrical signal being dependent on the interaction of the molecular entity with said nanopore; as well as A detection circuit, the detection circuit comprising: a plurality of detection channels, each detection channel being capable of amplifying an electrical signal from one of the sensor elements, the number of sensor elements in the array being greater than the number of detection channels; a switch arrangement capable of selectively connecting the detection channels to corresponding sensor elements; as well as a controller arranged to control switching of the switch arrangement to connect the detection channels to the respective sensor elements, The controller is arranged to detect the completion of the interaction at the sensor element based on the analysis of the amplified electrical signal output from the detection channel, and in response thereto, control the switch arrangement to connect the corresponding detection channel connected to the sensor element in which the completion of the interaction has been detected to another sensor element which is not currently connected to the detection channel.

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