Calibration and Profiling of Nanopore Array Devices

Abstract

A method for calibrating a nanopore array device is described. The nanopore array device comprises an array of nanopore channels, each nanopore channel being formed in a membrane that separates two ionic solutions. The nanopore channels connect the ionic solutions, and the device further comprises a temperature control component for regulating the temperature of the array of nanopore channels. The method includes measuring a signal indicative of an ion current through the nanopore channels, analyzing the measured signal and comparing it to a reference value, and based on the comparison, adjusting the temperature control component to control the temperature of the array of nanopore channels.

Description

Technical Field

[0001] The present invention relates to a method for calibrating a nanopore device. More specifically, the present invention relates to a method for calibrating a nanopore array device. Most specifically, the present invention relates to a method for calibrating a nanopore array device used for sensing the molecular entity of an analyte.

Background Art

[0002] The use of nanopores for sensing interactions with molecular entities such as polynucleotides, for example, has been a powerful technology that has made great progress in recent years. Nanopore devices have been developed that include arrays of nanopore sensing elements, increasing data collection by typically sensing interactions from the same sample in parallel across multiple nanopores.

[0003] Nanopore devices can typically use an electrical signal across the nanopore channel to generate a measurement signal, which is decoded to sense and / or characterize when a molecular entity interacts with the nanopore. Typically, the electrical signal is applied across the array of nanopore channels as a potential difference or current, providing a meaningful measurement signal to be decoded. Examples of measurements can include one of ion current flow, electrical resistance, or voltage.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Typically, electrical signals for an array device are applied to the system at a predetermined value(s). Subsequently, changes in the measured signal over time can be decoded to determine the molecular entities present in the analyte. However, in an array device, due to fluctuations in external factors (i.e., reference conditions) across the entire array, the measured signal may vary across the entire array. Additionally, in a device having multiple flow cells with an array of nanopore channels, more significant fluctuations from the reference conditions can occur. In such devices, it may be more difficult to establish, stabilize, and control the reference conditions for each flow cell during the analysis cycle.

Means for Solving the Problem

[0005] In a first embodiment, the present invention relates to a method for calibrating a nanopore array device, the nanopore array device comprising an array of nanopore channels, each nanopore channel being formed in a membrane separating two ionic solutions, the nanopore channel connecting the ionic solutions, the device further comprising a temperature control component for adjusting the temperature of the array of nanopore channels, the method comprising measuring a signal indicative of an ionic current through the nanopore channel, analyzing the measured signal and comparing it with a reference value, and based on the comparison, adjusting the temperature control component to control the temperature of the array of nanopore channels.

[0006] Due to differences in controllable conditions such as temperature across the array, fluctuations in the measurement signal may occur, which has been found to be harmful to the reliability of the measured values and thus the overall results generated from the device. When used to measure the translocation of a polymeric analyte through a nanopore channel, a nanopore array device generally generates a complex measurement signal with a generally small range, depending on multiple polymer units. For example, the measured current value is generally in the range of 60 - 120 pA for the translocation of a polynucleotide through the protein CsgG nanopore in the presence of 0.5 M salt. Additionally, there may be changes in the polymer units that can cause even more subtle modulation of the nanopore measurement signal compared to the unchanging (canonical) polymer units. An example of such is the methylation of the nucleobase cytosine that forms 5-methylcytosine in DNA. To analyze the signal and accurately determine the sequence order of the polymer units, various probabilistic and machine learning algorithms have been developed. However, as described above, due to the sensitivity of the device and the scale of the measurements taken by the device, even a slight variation from the reference conditions across the device can affect the measurement signal and thus the reliability of the device's measured values for accurately predicting, for example, the sequence order of the polymer units.

[0007] It has been found that the temperature across the array of nanopore channels in a nanopore array device can vary for several reasons. For example, there may be differences in the ambient temperature of the room or across the device as a whole. More specifically, in the case of a device that includes a plurality of flow cells each comprising an array of nanopore channels, there may be a temperature difference between individual flow cells. The temperature difference in a nanopore array device can also be due to the temperature of the analyte or the proximity of the flow cell to components within the device that generate heat during operation (e.g., ASICs and electronic components). Temperature variations can also occur due to the different intrinsic heat capacities of components within the device. In many cases, the flow cells are in close proximity to each other within the device but are not connected. This means that there is no direct heat conduction between the flow cells, which can lead to the flow cells experiencing different temperatures.

[0008] Calibration of the temperature across the nanopore array device enables improvement of the normalized signal output and enhancement of the reliability of the measurements for determining the molecular entity of the analyte. In an example, the reference value may be a value determined by the user, a value held on the device and read out as needed. In this example, the reference value may refer to an ideal value for the analysis of a particular analyte of interest, and the nanopore array device can be calibrated accordingly. Alternatively, the reference value may be a value determined from measurements of the ion current through each of a plurality of nanopores (e.g., an average value, a mode value, or a median value). In this example, the reference value can be used to calibrate and normalize the speed of the ion current through each of the plurality of nanopores to reduce the variation in measurements across the device.

[0009] By using an array of nanopores, it is possible to perform analyte characterization with higher accuracy and speed by combining measurement signals across the array or multiple arrays. The analyte can include, for example, fragments of a target analyte, such as fragments of a target polynucleotide. Alternatively, the analyte being measured may be the target. Combining data generated from a nanopore array for the measurement of an analyte can increase the accuracy and reliability of results such as the estimation of the sequence of the analyte or target. In such cases, it is advantageous to use a single algorithm to analyze the measurement signals generated by multiple nanopores, and thus it is desirable to normalize factors that cause variations in the measurement signals in order to optimize the data analysis capabilities of the algorithm.

[0010] The temperature control component can include an active heat source such as a heater or Peltier heater, a cooler, or a heat pump. It can also include a passive source such as a heat sink, a fan, or an impeller.

[0011] Optionally, the method can contain one or more feedback loops for calibration, where the method is performed two or more times to adjust the temperature experienced in each flow cell or nanopore channel of the device. The method can also include an optional step that allows the temperature control component to set an initial temperature as part of the calibration operation. In this example, the temperature of the device is increased or decreased to match an initial state with a predetermined temperature. Subsequently, the calibration method is performed until the device reaches the desired temperature, which may or may not include two or more repetitions of the method of the present invention.

[0012] A signal indicating an ion current can be measured during the translocation of an analyte through a nanopore channel, where the measurement signal in this case indicates at least a partial translocation of a polymer through the nanopore channel. Additionally, each measurement signal can be analyzed to determine the translocation rate of the analyte through the nanopore channel.

[0013] The analyte may be a species of interest, or the analyte may be a test analyte of known composition for calibrating the device to subsequently measure the analyte of interest. It will be appreciated that relying on the translocation of the analyte through the nanopore channel for calibration gives more meaning to the measurement signals generated across the array as compared to, for example, measurement signals of ionic currents based on open pore currents (i.e., the flow of electrolytes or ions from an ionic solution through the nanopore).

[0014] During translocation of the analyte through the nanopore, the ionic current decreases from an initial value (which may be referred to as the open pore current), and when the analyte exits the nanopore, it returns to its initial value. The measurement signals can be analyzed to determine the time and rate of translocation of the analyte through the nanopore. It has been found that analysis based on the rate of translocation of the analyte through the nanopore can roughly correlate with the local temperature of the nanopore system. More specifically, the analyte can be a polymer comprising an array of polymer units, and each measurement signal can be analyzed to determine the number of polymer units in the array and thus the rate of translocation of the polymer units per unit time.

[0015] There are various methods for determining the rate of translocation (i.e., the rate of translocation of the polymer through the nanopore channel). For example, a polymer having polymer units of a known sequence length (i.e., the number of polymer units in the polymer is known) can pass through the nanopore channel and provide a measurement signal for determining the translocation time. The rate of translocation can be compared to a reference rate of translocation, and the temperature of the device can be changed to provide either an increase or a decrease in the rate of translocation. The user can easily identify from the nanopore channel measurements the time interval between two signals of the open-source current and thus the time it takes for a polymer of known length to translocate through the nanopore channel.

[0016] In a further embodiment, the translocation rate can be determined using a polymer having an unknown sequence of polymer units, such as an analyte of interest, where the number of bases, and thus the translocation rate / polymer unit, is determined by analyzing the measurement signal.

[0017] In yet a further embodiment, a marker can be provided at a known position to generate a distinct measurement signal from the polymer unit. Suitable embodiments are the provision of a polynucleotide analyte or a non-basic moiety in one or more non-nucleotides, such as a hexaethylene glycol phosphate spacer. The marker can advantageously be provided within a leader sequence during sample preparation of the polynucleotide analyte of interest. This has the advantage that the translocation rate of an analyte of unknown sequence can be determined without the need to determine the sequence of the polynucleotide analyte (e.g., by measuring the time taken to observe the signal from the open pore signal value to the measurement signal due to the marker). Suitable sample preparation methods for providing a leader sequence for nanopore measurements are disclosed in WO 2015 / 110813.

[0018] The device can be calibrated one or more times during measurement of the continuous translocation of the analyte through the nanopore. Preferably, the calibration step is performed at the start of the measurement run.

[0019] The translocation rate of the analyte through the nanopore channel can be controlled by an enzyme - molecular motor. In an embodiment, the polymer can be a polynucleotide and the enzyme can be a polynucleotide binding protein. Since the function of the enzyme is temperature-dependent, the translocation rate of the analyte under the control of the enzyme will be affected by changes in the local temperature at the nanopore. Generally, an increase in temperature will result in an increase in the translocation rate. Thus, the signal can be measured to determine the translocation rate as an accurate reflection of the temperature experienced by the enzyme - molecular motor during translocation of the polymer analyte through the nanopore channel.

[0020] As already described, there are many factors that can affect the temperature of the nanopore array device, and thus the translocation speed, such as environmental factors, the temperature of the fluid sample, the heat generated by electronic devices, and the different specific heat capacities of the individual components. By measuring the translocation speed and comparing it to a reference value, a method for calibrating a device is advantageously provided that can automatically adjust the thermal energy provided to the nanopore array without directly calculating the local temperature in the nanopore and without taking into account the variable factors that affect the local temperature.

[0021] The signal indicating the ion current can be the measured current under the potential difference provided across the entire membrane. The potential difference can be maintained at a stable value to ensure that the fluctuations in the measured signal can be more easily correlated with the fluctuations in temperature across the device. Alternatively, the voltage can be varied during calibration to ensure that higher reliability can be given to the fluctuations in the measured signal from the device.

[0022] The device can comprise a common chamber with a common electrode in contact with an ionic solution provided on one side of the membrane, and an array of wells, each well containing an electrode and an ionic solution, with each membrane and nanopore channel isolating the ionic solution in the common chamber from the ionic solution contained in each corresponding well. In this regard, the array of nanopore channels can be provided within a removable flow cell. The flow cell is generally removable from the device, the device can have a plurality of flow cells spaced apart from each other within the device, and the device can have one or more temperature control components.

[0023] The potential difference across the nanopore can be maintained by reference electrodes provided in a common chamber and in each well, respectively. A preferred embodiment is a soluble redox pair in contact with an inert electrode or an Ag / AgCl reference electrode. The potential difference is temperature-dependent, and a small temperature change, such as 1 °C, can generally have an impact of 2% on the electrode potential. The change in the potential difference gives rise to an ion flow and, consequently, a current. Further, in the case of a polymer having a charge such as a polynucleotide, the change in the potential difference alters the migration rate through the nanopore and, consequently, the measurement signal. If the migration is controlled, for example, by an enzyme-molecular motor, the change in the potential difference can affect the measurement signal, for example, by changing the force applied to the bound enzyme-polynucleotide complex.

[0024] Each flow cell can have its own dedicated temperature control component. In a further embodiment, the device can include a combination of an active temperature control component and a passive temperature control component. For example, the device can have its own temperature control component (i.e., a global or large-scale temperature control component such as a fan), as well as a temperature control component for each group of flow cells (i.e., a local temperature control component such as a Peltier heat pump) and even a temperature control component for each flow cell (such as a heat sink). The temperature control component can be arranged very close to the flow cell.

[0025] The first temperature of the device can be measured, and after comparison, the heat source is adjusted to provide a second device temperature. However, knowledge of the temperature is not necessarily required, and the heat source can be adjusted from a first level to a second level. The adjustment is preferably automated and implemented as a result of knowledge of the relative migration rate. The execution instructions can be implemented, for example, in software or programmed in hardware such as an FPGA.

[0026] In this method, subsequently, a signal indicating the ion flow during the migration of the polymer analyte through the nanopore array can be measured, and the measurement signal can be analyzed to determine the sequence of polymer units.

[0027] In another aspect, the present invention provides a nanopore array device configured to implement the method of the present invention, wherein the steps of the method can be stored in a memory and implemented on a hardware device or a computer device. The device may include the above-mentioned hardware and / or software devices.

[0028] The device may include an array of nanopore channels as part of a removable flow cell. The device may include a plurality of removable flow cells spaced apart from each other. The device may include an array of flow cells that are not in direct contact with each other (i.e., spaced apart), which affects the thermal energy moving between the flow cells. This poses a problem when attempting to control the temperature experienced by the entire array of flow cells and the array of nanopore channels in each flow cell. The method enables calibration of the temperature experienced by these flow cells as part of the measurement signal derived from each nanopore channel.

[0029] For better understanding, embodiments of the present invention will now be described as non-limiting examples with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Embodiments for Carrying Out the Invention

[0031] A nanopore array device 1 for sensing the interaction of molecular entities is shown in FIG. 1. The nanopore array device 1 includes a sensing device 2, and the sensing device 2 includes a sensor device 3 and a detection circuit 4 connected to the sensor device 3.

[0032] The sensor device 3 includes an array of sensing elements 30 that each support a corresponding nanopore channel capable of interacting with a molecular entity. The sensing element 30 includes a corresponding electrode 31. In use, each sensing element 30 outputs an electrical measurement value that changes in response to the interaction between the molecular entity and the nanopore at its electrode 31. The sensor device 3 is schematically illustrated in FIG. 1 but may have various configurations, and non-limiting examples thereof are as follows.

[0033] In one embodiment, the sensor device 3 may have the form shown in FIG. 2. In this specification, the sensor device 2 includes an array of sensing elements 30, and each sensing element 30 includes a membrane 32 supported across a well 33 of a substrate 34, with a nanopore 35 inserted into the membrane 32. The membrane 31 may include an amphiphilic molecule such as a lipid or a polymer, which will be described in more detail below. Each membrane 32 seals the corresponding well 33 from a sample chamber 36 that extends across the array of sensing elements 30 and is in fluid communication with each nanopore 35. Each well 33 has a sensor electrode 32 disposed therein. A common electrode 37 is provided within the sample chamber 36 to provide a reference signal (generally a potential or voltage) to each sensor element 30. During use, the sample chamber 36 receives a sample containing a molecular entity that interacts with the nanopores 35 of the sensing elements 30.

[0034] For clarity, two sensing elements 30 are shown in FIG. 2, but generally any number of sensing elements 30 may be provided. Generally, in order to optimize the data collection rate, a large number of sensing elements 30, such as 256, 1024, 4096, or more sensing elements 30 may be provided.

[0035] The sensor device 3 may have a detailed structure as disclosed in International Publication No. WO 2009 / 077734 or WO 2014 / 064443, which are hereby incorporated by reference in their entirety.

[0036] FIGS. 3-5 show an example of a nanopore array device 1 including a plurality of flow cells 38. Each flow cell 38 includes a plurality of nanopore channels 25 supported on a substrate 34 and forming at least a portion of the flow cell 38.

[0037] As shown in FIG. 4, the flow cell 38 can be an individual component of the nanopore array device 1, and thus can be replaced if damage or problems occur in a particular flow cell 38. In the particular devices shown in FIGS. 4-5, the flow cells 38 are not formed from a single piece of material and thus can be said not to be in direct thermal communication with each other. Thus, the thermal energy experienced by one flow cell 38 may not be related to the thermal energy of another flow cell 38 within the nanopore array device 1, even if they are adjacent or very close to each other.

[0038] FIG. 4 shows a temperature control component 39 associated with an assembly of flow cells 38. In this case, the temperature control component 39 is a fan coupled to the assembly of flow cells 38 to assist in the dissipation and control of the thermal energy experienced by the assembly of flow cells 38 in the device. A temperature control component that can be coupled to each flow cell 38 of the assembly is not shown. In addition, a global thermal control couple for the entire device may also exist.

[0039] The elements of the nanopore channel 35 and the related sensing element 30 can be as follows, without being limited to the example shown in FIG. 2.

[0040] The nanopore channel 35 is generally a pore having a size on the order of nanometers. In an embodiment where the molecular entity is a polymer that interacts with the nanopore channel 35 while migrating through the nanopore channel 35, in this case, the nanopore channel 35 is of a size suitable for allowing the polymer to pass through it.

[0041] The nanopore can be a protein pore or a solid-state pore. The pore can be sized such that only one polymer at a time can migrate through the pore.

[0042] When the nanopore is a protein pore, it can have the following characteristics.

[0043] The nanopore can be a transmembrane protein pore. Transmembrane protein pores for use in accordance with the present invention include, but are not limited to, β-toxins such as α-hemolysin, anthrax toxin, and leukocidin, and bacterial outer membrane proteins / polysins such as Mycobacterium smegmatis polysin (Msp) such as MspA, lysenin, outer membrane polysin F (OmpF), outer membrane polysin G (OmpG), outer membrane phospholipase A, and Neisseria autotransporter lipoprotein (NalP). The α-helix bundle pore includes a barrel or channel formed from α-helices. Suitable α-helix 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 lysenin. The pore can be derived from CsgG as disclosed in International Application No. 2016 / 034591, which is hereby incorporated by reference in its entirety. The pore can be a DNA origami pore.

[0044] The protein pore can be a natural pore, a mutant pore, or a completely synthetic one.

[0045] When the nanopore is a protein pore, it may be inserted into a membrane supported within the sensor element 30. Such a membrane may be an amphiphilic layer, such as a lipid bilayer. The amphiphilic layer is a layer formed from amphiphilic molecules such as phospholipids having both hydrophilic and lipophilic properties. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer may be a co-block polymer as disclosed in WO 2014 / 064444. Alternatively, the protein pore may be inserted into a pore provided in a solid state layer, for example, as disclosed in WO 2012 / 005857.

[0046] The nanopore may include a pore formed in a solid state layer, which may be referred to as a solid state pore. The pore may be a well, gap, channel, groove, or slit provided in the solid state layer, and the analyte can pass along or through it. The solid state layer can be formed from 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 Teflon (registered trademark), or elastomers such as two-component addition-curing silicone rubber, as well as glass. The solid state layer may be formed from graphene.

[0047] The molecular entity interacts with the nanopore within the sensing element 30 to output an electrical signal at the electrode 31 that depends on the interaction.

[0048] In one type of sensor device 3, the electrical signal can be an ionic current flowing through a nanopore. Similarly, electrical properties other than the ionic current can also 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 Jan 12;11(1):279-85, which is hereby incorporated by reference in its entirety), and FET (field effect transistor) voltage (e.g., as disclosed in International Publication No. WO 2005 / 124888, which is hereby incorporated by reference in its entirety). One or more optical properties may optionally be used in combination with the electrical properties (Soni GV et al., Rev Sci Instrum. 2010 Jan;81(1):014301, which is hereby incorporated by reference in its entirety). The property can be a transmembrane current, such as an ionic current flowing through a nanopore. The ionic current can typically be a DC ionic current, but in principle there are alternative means using the flow of an AC current (i.e., the magnitude of the AC current that flows when an AC voltage is applied).

[0049] During the translocation of the molecular entity, an interaction with the nanopore, e.g., via the nanopore, can occur.

[0050] The electrical signal is provided as a measurement of a set of properties related to the interaction between the molecular entity and the nanopore. Such interactions can occur in the constricted region of the nanopore. For example, if the molecular entity is a polymer containing a series of polymer units that translocate with respect to the nanopore, the measurement can be of a property that varies according to the successive polymer units translocating with respect to the pore.

[0051] An ionic solution may be provided on either side of the nanopore. A sample containing the molecular entity of interest that is a polymer is added to one side of the nanopore, for example, in sample chamber 36 of the sensor device of the membrane of FIG. 2, and can be translocated relative to the nanopore, for example, under a potential difference or chemical gradient. An electrical signal can be induced during the translocation of the polymer relative to the pore (e.g., can be taken during the translocation of the polymer through the nanopore). The polymer may partially translocate relative to the nanopore.

[0052] To enable measurement when the polymer translocates through the nanopore, the translocation rate can be controlled by a binding moiety that binds to the polymer. Generally, the binding moiety can move the polymer through the nanopore either with or against the applied electric field. The binding moiety can be, for example, a molecular motor using enzyme activity if the binding moiety is an enzyme, or can be a molecular brake. When the polymer is a polynucleotide, several methods for controlling the translocation rate including the use of polynucleotide-binding enzymes have been proposed. 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 that polymer type can be used. The binding moiety can be any of those disclosed in International Publication Nos. 2010 / 086603, 2012 / 107778, and Lieberman KR et al, J Am Chem Soc. 2010;132(50):17961-72), and those disclosed with respect to the potential gating scheme (Luan B et al., Phys Rev Lett. 2010;104(23):238103) (all of which are hereby incorporated by reference in their entirety).

[0053] The binding moiety can be used in several ways to control the movement of the polymer. The binding moiety can move the polymer through the nanopore with or against the applied electric field. The binding moiety can be used, for example, as a molecular motor using enzymatic activity if the binding moiety is an enzyme, or can also be used as a molecular brake. The translocation of the polymer can be controlled by a molecular ratchet that controls the movement of the polymer through the pore. The molecular ratchet can be a polymer-binding protein.

[0054] The polynucleotide handling enzyme can be, for example, one of the types of polynucleotide handling enzymes described in International Publication Nos. 2015 / 140535, 2015 / 055981, or 2010 / 086603.

[0055] The translocation of the polymer through the nanopore can occur either from cis to trans or from trans to cis, both with or against the applied potential. The translocation can occur under an applied potential that can control the translocation.

[0056] An exonuclease that acts processively or progressively on double-stranded DNA can be used on the cis side of the pore to feed the single strand remaining under the applied potential into it, or on the trans side under reverse potential. Similarly, a helicase that unwinds double-stranded DNA can also be used in a similar manner. There is also a possibility of applying sequencing that requires translocation of a strand against the applied potential, but the DNA must first be "captured" by the enzyme under reverse potential or zero potential. Then, when the potential is switched after binding, the strand passes through the pore from cis to trans and is held in the extended conformation by the flow of current. A single-stranded DNA exonuclease or a single-stranded DNA-dependent polymerase can act as a molecular motor and pull back the single strand that has just translocated from trans to cis through the pore in a controlled stepwise manner against the applied potential. Alternatively, a single-stranded DNA-dependent polymerase can act as a molecular brake that slows down the movement of the polynucleotide through the pore. Either can use any of the parts, techniques, or enzymes described in International Publication No. WO 2012 / 107778 or WO 2012 / 033524, which are hereby incorporated by reference in their entirety, to control the movement of the polymer.

[0057] The sensing element 30 and / or the molecular entity can be adapted to capture the molecular entity in the vicinity of the corresponding nanopore. For example, the sensing element 30 can further include a capture portion arranged to capture the molecular entity in the vicinity of the corresponding nanopore. The capture portion may be either the above-described binding portion or exonuclease that also has the purpose of further controlling translocation, or may be provided separately.

[0058] The capture portion can be attached to the nanopore of the sensing element. At least one capture portion can be attached to the nanopore of each sensor element.

[0059] The capture portion may be a tag or tether that binds to the molecular entity. In that case, the molecular entity can be adapted to achieve its binding.

[0060] Such tags or tethers can be attached to the nanopore as disclosed, for example, in International Publication No. WO 2018 / 100370, which is incorporated herein by reference in its entirety, and described in more detail below.

[0061] Alternatively, when the nanopore is inserted into a membrane, such tags or tethers can be attached to the membrane as disclosed, for example, in International Publication No. WO 2012 / 164270, which is incorporated herein by reference in its entirety.

[0062] The methods described herein may include the use of an adapter that adapts a molecular entity for the purpose of capturing the molecular entity. As an example, polynucleotide adapters suitable for use in nanopore sequencing of polynucleotides are known in the art. An adapter for use in nanopore sequencing of a polynucleotide may include at least one single-stranded polynucleotide or non-polynucleotide region. For example, Y-adapters for use in nanopore sequencing are known in the art. A Y-adapter typically includes (a) a double-stranded region and (b) a single-stranded region or a region that is non-complementary at the other end. When a Y-adapter includes a single-stranded region, it can be described as having an overhang. Due to the presence of the non-complementary region in the Y-adapter, the two strands generally do not hybridize to each other, unlike the double-stranded portion, causing the adapter to adopt a Y-shape. A Y-adapter may include one or more anchors.

[0063] The Y-adapter preferably includes a leader sequence that is preferentially threaded into the pore. The leader sequence typically 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 includes a polynucleotide, more preferably a single-stranded polynucleotide. The adapter can be ligated to a DNA molecule using any method known in the art.

[0064] The polynucleotide adapter may include a membrane anchor or a transmembrane pore anchor attached to the adapter. For example, the membrane anchor or transmembrane pore anchor may facilitate the localization of the adapter and the binding polynucleotide near the nanopore. The anchor may 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, fatty acid, sterol, carbon nanotube, polypeptide, protein, or amino acid, such as cholesterol, palmitate, or tocopherol.

[0065] The anchor may include one linker, or two, three, four, or more than four 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. Suitable linkers are described in International Publication No. WO 2010 / 086602. Examples of suitable anchors and methods for attaching the anchor to the adapter are disclosed in International Publication Nos. WO 2012 / 164270 and WO 2015 / 150786, which are hereby incorporated by reference in their entirety.

[0066] Examples of tags and tethers attached to the nanopore are as follows.

[0067] The nanopores for use in the methods described herein may be modified to include one or more binding sites for binding to one or more analytes (e.g., molecular entities) and thereby acting as a capture moiety. In some embodiments, the nanopore may be modified to include one or more binding sites for binding to an adapter attached to the analyte. For example, in some embodiments, the nanopore may bind to the leader sequence of an adapter attached to the analyte. In some embodiments, the nanopore may bind to a single-stranded sequence within an adapter attached to the analyte.

[0068] In some embodiments, the nanopore is modified to include one or more tags or tethers, and each tag or tether includes a binding site for an analyte. In some embodiments, the nanopore is modified to include one tag or tether per nanopore, and each tag or tether includes a binding site for an analyte.

[0069] In some embodiments, the tag or tether may include an oligonucleotide or may be an oligonucleotide.

[0070] Other examples of tags or tethers include, but are not limited to, His tags, biotin or streptavidin, antibodies that bind an analyte, aptamers that bind an analyte, analyte binding domains such as DNA binding domains (e.g., peptide zippers such as leucine zippers, single-stranded DNA binding protein (SSB)), and any combination thereof.

[0071] Any method known in the art can be used to attach the tag or tether to the outer surface of the nanopore, e.g., the cis side of the membrane. For example, one or more tags or tethers can be attached to the nanopore via one or more cysteines (cysteine binding), primary amines such as one or more lysines, 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 an epitope (e.g., including acetyltransferase), and any combination thereof. Suitable methods for performing such modifications are well known in the art. Suitable non-natural amino acids include 4-azido-L-phenylalanine (Faz), and any one of the amino acids numbered 1-71 in FIG. 1 of Liu C.C. and Schultz P.G., Annu. Rev. Biochem., 2010, 79, 413-444, which is hereby incorporated by reference in its entirety, but are not limited thereto.

[0072] In some embodiments where one or more tags or tethers are attached to the nanopore via cysteine linkage(s), one or more cysteines can be introduced by substitution into one or more monomers that form the nanopore.

[0073] 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 of α-hemolysin that increase the positive charge within the barrel of the pore.

[0074] Modified MspA, lysenin, and CsgG pores containing mutations that enhance polynucleotide capture are disclosed in WO 2012 / 107778, WO 2013 / 153359, and WO 2016 / 034591, respectively, which are hereby incorporated by reference in their entirety. Any of the modified pores disclosed in these publications can be used herein.

[0075] Next, the arrangement of the detection circuit 4 will be described. The detection circuit 4 is connected to the electrodes 31 of each sensor element 30 and has a main function of processing the electrical signals output therefrom. The detection circuit 4 also has a function of controlling the application of a bias signal to each sensor element 30.

[0076] The detection circuit 4 includes a plurality of detection channels 40. Each detection channel 40 is arranged to receive an electrical signal from a single sensor electrode 31 and amplify the electrical signal. Thus, the detection channel 40 is designed to amplify very small currents with a resolution sufficient to detect characteristic changes caused by the interaction of interest. The detection channel 40 is also designed with a sufficiently high bandwidth to provide the time resolution necessary to detect such interactions respectively. Due to such constraints, sensitive and expensive components are required. Each detection channel 40 may be similar to a standard single-channel recording device as 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 International Publication No. WO 2000 / 28312. Alternatively, each detection channel 40 may be arranged as described in detail in International Publication Nos. WO 2010 / 122293, WO 2011 / 067559, or WO 2016 / 181118.

[0077] The analyte of interest to be detected by the nanopore can be a polynucleotide such as DNA or RNA. The analyte may be a polypeptide or a polysaccharide.

[0078] The number of sensing elements 30 in the array is greater than the number of detection channels 40, and the nanopore array device is operable to take measurements of the polymer from the sensing elements 30 selected in a multiplexed manner, particularly an electrically multiplexed manner. This is achieved by providing a switching device 42 between the sensor electrode 31 of the sensing element 30 and the detection channel 40. For clarity, FIG. 1 shows a simplified example with four sensing elements 30 and two detection channels 40, but typically the number of sensor cells 30 and detection channels 40 is much larger. For example, in some applications, the sensor device 2 may include a total of 4096 sensing elements 30 and 1024 detection channels 40.

[0079] The switch device 42 can be arranged as described in detail in International Publication No. WO2010 / 122293. For example, the switch device 42 can include a plurality of 1-to-N multiplexers each connected from the detection channel 40 to a group of N sensing elements 30, and can also include appropriate hardware such as a latch for selecting the switching state.

[0080] By switching the switch device 42, the nanopore array device 1 can be operated to amplify the electrical signal from the selected sensing element 30 in an electrically multiplexed manner. The detection circuit 4 includes a data processing device 5 that receives the output signal from the detection channel 40. The data processing device 5 functions as a controller that controls the switch device 42 to connect the detection channel 40 to the corresponding sensing element 30, as will be described in more detail below.

[0081] Furthermore, the detection circuit 4 includes a bias control circuit 41 that performs 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 the sensor electrode 31 of each sensor device 30. The bias signal is selected to bias the sensor electrode 31 with respect to the common electrode 37 in order to control the translocation of molecular entities through the nanopore. Generally, the bias signal supplied to a given sensor element 30 could be a drive bias signal that causes translocation in the sensor element 30, or a blocking bias signal that prevents translocation from occurring in the sensor element 30.

[0082] The bias control circuit 41 is controlled by the data processing device 5. The data processing device has an operating mode for the bias control circuit 41. That is, three independent test bias signals are supplied to all the sensing elements 30, thereby causing a flow of ionic current through the nanopores of each sensing element 30. The flow of current corresponding to each test signal is recorded in the data processing device 5 as an amplified electrical signal.

[0083] The data processing device 5 is arranged as follows. The data processing device 5 is connected to the output section of the detection channel 40 and supplied with the amplified electrical signal therefrom. The data processing device 5 stores and analyzes the amplified electrical signal from the test bias signal to generate a calibration signal. The data processing device 5 controls other elements of the detection circuit, including the control of the bias voltage circuit 41 described above and the control of the switch device 42 described later. The data processing device 5 forms part of the detection circuit 2 and can be provided in a common package, and in some cases, on a common circuit board, together with it. The data processing device 5 can be implemented in any suitable form, for example, as a processor that executes an appropriate computer program, or as an ASIC (application-specific integrated circuit).

[0084] The data processing device 5 of the nanopore array device 1 is connected to the analysis system 6. The data processing device 5 also supplies the amplified output signal to the analysis system 6. The analysis system 6 performs further analysis of the amplified electrical signal, which is the original signal representing the measured value of the characteristics measured by the nanopore. Such an analysis system 6 can, for example, also estimate the identity of the entire molecular entity, or, if the molecular entity is a polymer, estimate the identity of its polymer units. Therefore, the analysis system can be configured as computer equipment that executes an appropriate program. Such computer equipment can be directly connected to the data processing device 5 of the nanopore array device 1, or can be connected via a network, for example, within a cloud-based system.

[0085] As can be understood, the temperature of the device does not vary only due to fluctuations from the reference conditions experienced throughout the nanopore channel 35. The data processing device 5 and other electrical components may generate heat that is not evenly distributed between the nanopore channels 35 on the flow cell 38, or between multiple flow cells 38 within the nanopore array device 1 described above.

[0086] A calibration method of the nanopore array device 1 executed by the data processing device 5 is shown in FIG. 6, and this is executed as follows.

[0087] This method starts by performing an initial calibration of the temperature of the flow cell of the nanopore array device 1. In the method described herein, the initial calibration is performed by the global temperature control component. However, as shown in FIG. 4, other temperature control components such as the fan 39 connected to the assembly of the flow cell 38 can also be relied upon. Further, if the device is already in a ready state, this initial heating / cooling step can be completely bypassed.

[0088] In the embodiments described herein, a polymer is transferred through the nanopore channel 35 to generate a measurement signal related to the array data. An estimated value is provided from the basecall program provided in the data processing device 5 that converts the measurement signal into an estimated value of the polymer transfer rate when passing through the nanopore channel 35.

[0089] The estimated transfer rate generated by decoding the measurement signal from the nanopore channel 35 is averaged over the entire array of nanopore channels 35 per flow cell 38. In the device 1 including a plurality of flow cells 38, the average values of the transfer rates of each flow cell 38 within the device 1 can be compared, and the temperature control component 39 can be adjusted to ensure that the temperatures experienced by each flow cell 38 are balanced.

[0090] The average translocation speed is calculated by statistical analysis of the population of speed values for each nanopore channel 35. For example, an average value can be determined. This average may be a standard average or a weighted average based on the magnitude of the speed values. Using a standard average is not preferred because it has little effect on the calculated global speed value of the flow cell for any data skewness or abnormal speed value(s). Further, it is time-consuming to assign the correct weights to the speed values to record the true weighted average for each calibration. Alternatively, the mode speed value can be selected as the global offset value. However, this is not preferred because there may be more than two modes in the speed value data. Another alternative is to assign the median speed value as the global speed value of the flow cell. This is most preferred because the median speed value is not affected by extreme outliers or the asymmetric distribution of speed values.

[0091] The average speed per flow cell is considered and compared to a reference value. The reference value can be a value programmed by the user. It can also be the average value of the average translocation speeds provided from each flow cell 38. In this embodiment, the reference value is the latter. The average speed from each flow cell 38 is compared to the average speed generated by analyzing the average speeds from all other flow cells within the device.

[0092] If it is determined that all flow cells 38 are operating at the required speed (or within a specific deviation range from that speed), the device is considered calibrated and analysis can begin. However, if the speed from one or more flow cells 38 is unacceptable, recalibration can be performed and the device 1 can adjust one or more of the temperature control components, returning to the first step of the calibration to allow the device to stabilize in this new state before the speed is determined.

[0093] The calibration or profiling method of the present invention can be employed before the nanopore array device 1 is first used. Additionally, or alternatively, the calibration or method of the present invention can also be used after the nanopore array device 1 has been used to sense or detect an analyte of interest.

Example

[0094] The following experiments detail a method for calibrating a nanopore array device in accordance with the claimed invention.

[0095] Eight R9.4.1 PromethION flow cells were installed within the device. A sample containing a polynucleotide (polyT) with a leader was loaded into the flow cells. In this experiment, a 24-hour live-based call (high-speed mode) was required to measure the translocation speed.

[0096] Analysis was performed without calibration using the same reagents and chemical reactions of the kit through the same nanopore array device, and an initial distribution of translocation speeds across the nanopores of the eight flow cells was observed. There was a correlation with the position of the flow cells within the nanopore array device and the variation between devices, which was considered to be due to the variation in temperature within the fluid chambers of the flow cells and the variation in temperature between the flow cells (see the difference in translocation speed from the first 20 minutes as shown in Figure 7).

[0097] To normalize the translocation speed at the start of the sequencing run, the measured translocation speed was compared with a reference temperature value and adjusted as appropriate (by calculating the conversion ratio from temperature to speed) to affect the speed.

[0098] A model was generated using a ratio of translocation speed of bases to temperature of 30 bases = 1 °C. This model measures the temperature and translocation speed (median) at each position and uses it to generate the increase or decrease in temperature required to achieve a target normalized translocation speed of 400 bps (presumed to correspond to 34.5 °C) within a specified tolerance of ±5 bps.

[0099] This model was used to adjust and normalize the migration rate by increasing or decreasing the temperature by adjusting the temperature control component so that the same migration rate could be obtained in each flow cell.

[0100] Within the device, the temperature of each flow cell was established by the data recorded from the temperature of the heat sink and ASIC during the execution period. A temperature gun using an infrared signal for measuring the surface temperature of each flow cell was installed to determine the temperature change of the flow cell before and after calibration. The flow cell was calibrated more uniformly towards the target temperature of 34.5. The results are shown in the following table.

Table 1

[0101] The migration rate from each flow cell was continuously measured and it was observed that it converged after the implementation of the method. After about 1 hour (63 minutes - see Figure 7), all flow cells reached the same migration rate.

[0102] The results shown in Figure 7 suggest that when the temperature of the device stabilizes after calibration, the median migration rate of the flow cells converges. The median current was evaluated for eight flow cells and it was confirmed that the convergence of the migration rate was due to the temperature change experienced by the flow cells.

Explanation of Symbols

[0103] 1 Nanopore array device 2 Sensing device 3 Sensor device 4 Detection circuit 5 Data processing device 6 Analysis system 25 Nanopore channels 30 Sensing elements 31 Electrodes 32 Membrane 33 Well 34 Substrate 35 Nanopore channels 36 Sample chamber 37 Electrode 38 Flow cell 39 Temperature control component, fan 40 Detection channel 41 Bias control circuit 42 Switch device

Claims

1. 1. A method of calibrating a nanopore array device, comprising: the nanopore array device comprises an array of nanopore channels, each nanopore channel formed in a membrane separating two ionic solutions, the nanopore channels connecting the ionic solutions; the device further comprising a temperature control component for regulating a temperature of the array of nanopore channels; The method comprises: measuring a signal indicative of ion flow through the nanopore channel; analyzing said measurement signals and comparing them with reference values; and adjusting the temperature control component to control the temperature of the array of nanopore channels based on the comparison. method.

2. The method of claim 1 , wherein a signal indicative of ion flow is measured during translocation of an analyte through the nanopore channel.

3. The method of claim 2 , wherein each measurement signal is analyzed to determine a translocation rate of the analyte through the nanopore channel.

4. The method of claim 2 or 3, wherein the analyte is a polymer comprising a sequence of polymer units and the measurement signal indicates at least partial translocation of the polymer through the nanopore channel.

5. 5. The method of claim 4, wherein each measurement signal is analyzed to determine the number of polymer units in the sequence, thereby determining an average migration rate of polymer units per unit time and comparing it with the reference value.

6. The method of claim 4 , wherein the polymer is a polynucleotide.

7. The method of claim 4 , wherein the translocation rate of the polymer through the nanopore channel is controlled by an enzymatic molecular motor.

8. The method of claim 5 , wherein the migration rate of a polymer unit per unit time comprises determining a sequence length of the polymer unit.

9. The method of claim 7 , wherein the enzyme is a polynucleotide binding protein.

10. The method of any one of claims 1 to 3, wherein the signal indicative of ion flow through the nanopore channel is a current measurement under a potential difference applied across each membrane.

11. The method of any one of claims 1 to 3, wherein the device comprises a common chamber comprising a common electrode in contact with an ionic solution provided on one side of the array of nanopore channels and corresponding membranes, and an array of wells, each well containing an electrode and an ionic solution, each nanopore channel and membrane isolating the ionic solution in the common chamber from the ionic solution contained in each corresponding well.

12. The method of any one of claims 11, wherein the array of nanopore channels and the array of wells are provided on a removable flow cell.

13. 4. The method of claim 1, wherein a first temperature of the device is measured and the temperature control component is adjusted from a first level to a second level after the comparison to provide a second device temperature.

14. A method for determining a polymer sequence, comprising the steps of calibrating a nanopore device described in any one of claims 1 to 3, followed by measuring a signal indicative of ion flow during translocation of a polymer analyte through the array of nanopore channels, and analyzing the measured signal to determine the sequence of polymer units.

15. A nanopore array device configured to perform the method according to any one of claims 1 to 3, wherein the steps of the method are stored in a memory and implemented in a hardware or computer device.

16. The device of claim 15 , wherein the array of nanopore channels is included as part of a removable flow cell.

17. The device of claim 16 , wherein the removable flow cell is included as part of a plurality of spaced-apart removable flow cells.

Images