Patent Application
20240409993
Some polynucleotide sequencing techniques involve performing a large number of controlled reactions on support surfaces or within predefined reaction chambers. The controlled reactions may then be observed or detected, and subsequent analysis may help identify properties of the polynucleotide involved in the reaction. Examples of such sequencing techniques include next-generation sequencing or massive parallel sequencing involving sequencing-by-ligation, sequencing-by-synthesis, reversible terminator chemistry, or pyrosequencing approaches.
Some polynucleotide sequencing techniques utilize a nanopore, which can provide a path for an ionic electrical current. For example, as the polynucleotide traverses through the nanopore, it influences the electrical current through the nanopore. Each passing nucleotide, or series of nucleotides, that passes through the nanopore yields a characteristic electrical current. These characteristic electrical currents of the traversing polynucleotide can be recorded to determine the sequence of the polynucleotide.
Provided in examples herein are methods for sequencing biopolymers, particularly polynucleotides, and systems and kits for performing the methods.
The systems, devices, kits, and methods disclosed herein each have several aspects, no single one of which is solely responsible for their desirable attributes. Without limiting the scope of the claims, some prominent features will now be discussed briefly. Numerous other examples are also contemplated, including examples that have fewer, additional, and/or different components, steps, features, objects, benefits, and advantages. The components, aspects, and steps may also be arranged and ordered differently. After considering this discussion, and particularly after reading the section entitled “Detailed Description,” one will understand how the features of the devices and methods disclosed herein provide advantages over other known devices and methods.
In some embodiments of a nanopore nucleic acid sequencing system, the system includes an array of nanopore unit cells/sequencing cells (or “pixels”) and uses an array of amplifiers to measure small signals (e.g., currents) in the array of nanopore unit cells. In some cases, low pixel-to-pixel variation of the voltage bias across the nanopore (or “sensor”) may be needed to ensure accurate actuation of the nucleic acid and/or measurement of the nanopore signal due to the chemistry used in the nanopore sequencing reaction. However, due to process variations in integrated circuit manufacturing, it is challenging to fabricate identical amplifiers in an integrated circuit, resulting in an undesired variation of the voltage bias across the nanopore. Therefore, a method for minimizing pixel-to-pixel voltage bias variation on a nanopore/amplifier array may be needed for efficient and accurate nanopore sequencing.
In some embodiments, disclosed is an analog mechanism and a circuit design to compensate for the variation of the voltage bias on a nanopore/amplifier array. In some embodiments, the disclosed technology comprises using an offset capacitor to measure and store the offset voltage of each pixel, wherein the offset voltage would cancel out any offset in the amplifier array.
Additional details of exemplary nanopore sequencing devices and methods of operating the devices that can be used in conjunction with the present disclosure can be found in U.S. Provisional Patent Application Nos. 63/200,868 and 63/169,041 (International Patent Application Numbers PCT/US2021/038125 and PCT/US2022/020395), the entirety of each of the disclosures is incorporated herein by reference.
In some examples, a device for sequencing polynucleotides is disclosed. The device comprises: a nanopore for sensing a polynucleotide; an amplifier configured to measure an electrical response associated with the nanopore, wherein the amplifier has a bias offset voltage between a first input terminal and a second input terminal; and a bias compensation circuit coupled to the nanopore and the amplifier. The bias compensation circuit is configured to: store a voltage potential indicative of the bias offset voltage; and compensate the bias offset voltage using the voltage potential.
In some embodiments, the bias compensation circuit comprises an offset capacitor, and wherein the offset capacitor stores the voltage potential indicative of the bias offset voltage during a first operation mode. In some examples, the first operation mode is a calibration mode of the bias compensation circuit. In some embodiments, the offset capacitor compensates the bias offset voltage using the voltage potential during a second operation mode. In some examples, the second operation mode is a measurement mode of the bias compensation circuit. In some embodiments, during the second operation mode, the amplifier measures the electrical response associated with the nanopore. In some embodiments, the nanopore has a first side and a second side, and wherein the offset capacitor has a first terminal and a second terminal, and wherein during the first operation mode: the first terminal is operably connected to the first input terminal through a switching circuit; and the second terminal is operably connected to a common electrode through the switching circuit. In some embodiments, the common electrode is tied to a ground potential during the first operation mode.
In some embodiments, the nanopore is an opening in a protein or nucleic acid structure, which is deposited in a lipid or polymer membrane. In some embodiments, the nanopore is an opening in a solid-state structure. For example, the nanopore may be a polypeptide nanopore that forms an opening in a lipid, polymer, or solid-state membrane. In some embodiments, the electrical response associated with the nanopore is an ionic current through the nanopore, and wherein the ionic current is modulated by: nucleotides in the polynucleotide near a sensing zone of the nanopore, labels on nucleotides in the polynucleotide near the sensing zone of the nanopore, nucleotides being incorporated to the polynucleotide, labels on nucleotides being incorporated to the polynucleotide, or any combination thereof. In some embodiments, the device for sequencing polynucleotides further comprises an analog-to-digital-converter, wherein an output of the amplifier is fed to the analog-to-digital converter for generating a digital signal representative of the output of the amplifier.
In some examples, a method for sequencing polynucleotides is disclosed. The method comprises: providing a polynucleotide to a sequencing cell comprising a nanopore, an amplifier for measuring an electrical response associated with the nanopore, and a bias compensation circuit between the nanopore and the amplifier; storing, by the bias compensation circuit, a voltage potential indicative of a bias offset voltage between a first input terminal and a second input terminal of the amplifier; and measuring the electrical response, wherein the bias offset voltage is compensated for in the electrical response using the voltage potential.
In some embodiments, the voltage potential is stored in an offset capacitor of the bias compensation circuit. In some embodiments, storing the voltage potential comprises: connecting a first terminal of the offset capacitor to the first input terminal of the amplifier through a switching circuit; and connecting a second terminal of the offset capacitor to a common electrode through the switching circuit. In some embodiments, the common electrode and the second input terminal of the amplifier are set to a ground potential, thereby inversely charging the offset capacitor to the voltage potential equal to a voltage at the first input terminal of the amplifier. In some embodiments, the common electrode and the second input terminal of the amplifier are set to a reference potential, thereby inversely charging the offset capacitor to the voltage potential equal to a difference between voltages at the first input terminal and the second input terminal of the amplifier.
In some embodiments, measuring the electrical response comprises: connecting a first terminal of the offset capacitor to a first side of the nanopore through a switching circuit, while a second side of the nanopore is connected to a common electrode; and connecting a second terminal of the offset capacitor to first input terminal of the amplifier through the switching circuit.
In some examples, a system for sequencing polynucleotides is disclosed. The system comprises a common cis well associated with a common cis electrode and a plurality of sequencing cells. Each of the plurality of sequencing cells comprises: (1) a trans well associated with a trans electrode; (2) a nanopore for sensing a polynucleotide, the nanopore fluidically connecting the trans well to the common cis well; (3) an amplifier configured to measure an electrical response in the nanopore, the amplifier having a bias offset voltage between a first input terminal and a second input terminal; and (4) a bias compensation circuit coupled to the nanopore and the amplifier, the bias compensation circuit configured to: store a voltage potential indicative of the input offset voltage; and compensate the input offset voltage using the voltage potential.
It is to be understood that any features of the device and/or of the array disclosed herein may be combined together in any desirable manner and/or configuration. Further, it is to be understood that any features of the method of using the device may be combined together in any desirable manner. Moreover, it is to be understood that any combination of features of this method and/or of the device and/or of the array may be used together, and/or may be combined with any of the examples disclosed herein. Still further, it is to be understood that any feature or combination of features of any of the devices and/or of the arrays and/or of any of the methods may be combined together in any desirable manner, and/or may be combined with any of the examples disclosed herein.
It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits and advantages described herein.
Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear.
All patents, applications, published applications and other publications referred to herein are incorporated herein by reference to the referenced material and in their entireties. If a term or phrase is used herein in a way that is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the use herein prevails over the definition that is incorporated herein by reference.
All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.
As used herein, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a sequence” may include a plurality of such sequences, and so forth.
The terms comprising, including, containing and various forms of these terms are synonymous with each other and are meant to be equally broad. Moreover, unless explicitly stated to the contrary, examples comprising, including, or having an element or a plurality of elements having a particular property may include additional elements, whether or not the additional elements have that property.
As used herein, the term “operably connected” refers to a configuration of elements, wherein an action or reaction of one element affects another element, but in a manner that preserves each element's functionality.
As used herein, the term “membrane” refers to a non-permeable or semi-permeable barrier or other sheet that separates two liquid/gel chambers (e.g., a cis well and a fluidic cavity) which can contain the same compositions or different compositions therein. The permeability of the membrane to any given species depends upon the nature of the membrane. In some examples, the membrane may be non-permeable to ions, to electric current, and/or to fluids. For example, a lipid membrane may be impermeable to ions (i.e., does not allow any ion transport therethrough), but may be at least partially permeable to water (e.g., water diffusivity ranges from about 40 μm/s to about 100 μm/s). For another example, a synthetic/solid-state membrane, one example of which is silicon nitride, may be impermeable to ions, electric charge, and fluids (i.e., the diffusion of all of these species is zero). Any membrane may be used in accordance with the present disclosure, as long as the membrane can include a transmembrane nanoscale opening and can maintain a potential difference across the membrane. The membrane may be a monolayer or a multilayer membrane. A multilayer membrane includes two or more layers, each of which is a non-permeable or semi-permeable material.
The membrane may be formed of materials of biological or non-biological origin. A material that is of biological origin refers to material derived from or isolated from a biological environment such as an organism or cell, or a synthetically manufactured version of a biologically available structure (e.g., a biomimetic material).
An example membrane that is made from the material of biological origin includes a monolayer formed by a bolalipid. Another example membrane that is made from the material of biological origin includes a lipid bilayer. Suitable lipid bilayers include, for example, a membrane of a cell, a membrane of an organelle, a liposome, a planar lipid bilayer, and a supported lipid bilayer. A lipid bilayer can be formed, for example, from two opposing layers of phospholipids, which are arranged such that their hydrophobic tail groups face towards each other to form a hydrophobic interior, whereas the hydrophilic head groups of the lipids face outwards towards the aqueous environment on each side of the bilayer. Lipid bilayers also can be formed, for example, by a method in which a lipid monolayer is carried on an aqueous solution/air interface past either side of an aperture that is substantially perpendicular to that interface. The lipid is normally added to the surface of an aqueous electrolyte solution by first dissolving it in an organic solvent and then allowing a drop of the solvent to evaporate on the surface of the aqueous solution on either side of the aperture. Once the organic solvent has at least partially evaporated, the solution/air interfaces on either side of the aperture are physically moved up and down past the aperture until a bilayer is formed. Other suitable methods of bilayer formation include tip-dipping, painting bilayers, and patch-clamping of liposome bilayers. Any other methods for obtaining or generating lipid bilayers may also be used.
A material that is not of biological origin may also be used as the membrane. Some of these materials are solid-state materials and can form a solid-state membrane, and others of these materials can form a thin liquid film or membrane. The solid-state membrane can be a monolayer, such as a coating or film on a supporting substrate (i.e., a solid support), or a freestanding element. The solid-state membrane can also be a composite of multilayered materials in a sandwich configuration. Any material not of biological origin may be used, as long as the resulting membrane can include a transmembrane nanoscale opening and can maintain a potential difference across the membrane. The membranes may include organic materials, inorganic materials, or both. Examples of suitable solid-state materials include, for example, microelectronic materials, insulating materials (e.g., silicon nitride (Si3N4), aluminum oxide (Al2O3), hafnium oxide (HfO2), tantalum pentoxide (Ta2O5), silicon oxide (SiO2), etc.), some organic and inorganic polymers (e.g., polyamide, plastics, such as polytetrafluoroethylene (PTFE), or elastomers, such as two-component addition-cure silicone rubber), and glasses. In addition, the solid-state membrane can be made from a monolayer of graphene, which is an atomically thin sheet of carbon atoms densely packed into a two-dimensional honeycomb lattice, a multilayer of graphene, or one or more layers of graphene mixed with one or more layers of other solid-state materials. A graphene-containing solid-state membrane can include at least one graphene layer that is a graphene nanoribbon or graphene nanogap, which can be used as an electrical sensor to characterize the target polynucleotide. It is to be understood that the solid-state membrane can be made by any suitable method, for example, chemical vapor deposition (CVD). In an example, a graphene membrane can be prepared through either CVD or exfoliation from graphite. Examples of suitable thin liquid film materials that may be used include diblock copolymers or triblock copolymers, such as amphiphilic PMOXA-PDMS-PMOXA ABA triblock copolymers.
As used herein, the term “nanopore” is intended to mean a hollow structure discrete from, or defined in, and extending across the membrane. The nanopore permits ions, electric current, and/or fluids to cross from one side of the membrane to the other side of the membrane. For example, a membrane that inhibits the passage of ions or water-soluble molecules can include a nanopore structure that extends across the membrane to permit the passage (through a nanoscale opening extending through the nanopore structure) of the ions or water-soluble molecules from one side of the membrane to the other side of the membrane. The diameter of the nanoscale opening extending through the nanopore structure can vary along its length (i.e., from one side of the membrane to the other side of the membrane), but at any point is on the nanoscale (i.e., from about 1 nm to about 100 nm, or to less than 1000 nm). Examples of the nanopore include, for example, biological nanopores, solid-state nanopores, and biological and solid-state hybrid nanopores. In some embodiments, a nanopore refers to a pore having an opening with a diameter at its most narrow point of about 0.3 nm to about 2 nm. For example, a nanopore may be a solid-state nanopore, a graphene nanopore, an elastomer nanopore, or may be a naturally-occurring or recombinant protein that forms a tunnel upon insertion into a bilayer, thin film, membrane, or solid-state aperture, also referred to as a protein pore or protein nanopore herein (e.g., a transmembrane pore). If the protein inserts into the membrane, then the protein is a tunnel-forming protein.
As used herein, the term “biological nanopore” is intended to mean a nanopore whose structure portion is made from materials of biological origin. Biological origin refers to a material derived from or isolated from a biological environment such as an organism or cell, or a synthetically manufactured version of a biologically available structure. Biological nanopores include, for example, polypeptide nanopores and polynucleotide nanopores.
As used herein, the term “polypeptide nanopore” is intended to mean a protein/polypeptide that extends across the membrane, and permits ions, electric current, polymers such as DNA or peptides, or other molecules of appropriate dimension and charge, and/or fluids to flow therethrough from one side of the membrane to the other side of the membrane. A polypeptide nanopore can be a monomer, a homopolymer, or a heteropolymer. Structures of polypeptide nanopores include, for example, an α-helix bundle nanopore and a β-barrel nanopore. Example polypeptide nanopores include α-hemolysin, Mycobacterium smegmatis porin A (MspA), gramicidin A, maltoporin, OmpF, OmpC, PhoE, Tsx, F-pilus, acrolysin, etc. The protein α-hemolysin is found naturally in cell membranes, where it acts as a pore for ions or molecules to be transported in and out of cells. Mycobacterium smegmatis porin A (MspA) is a membrane porin produced by Mycobacteria, which allows hydrophilic molecules to enter the bacterium. MspA forms a tightly interconnected octamer and transmembrane beta-barrel that resembles a goblet and contains a central pore.
A polypeptide nanopore can be synthetic. A synthetic polypeptide nanopore includes a protein-like amino acid sequence that does not occur in nature. The protein-like amino acid sequence may include some of the amino acids that are known to exist but do not form the basis of proteins (i.e., non-proteinogenic amino acids). The protein-like amino acid sequence may be artificially synthesized rather than expressed in an organism and then purified/isolated.
As used herein, the term “polynucleotide” refers to a molecule that includes a sequence of nucleotides that are bonded to one another. A polynucleotide is one nonlimiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogues thereof such as locked nucleic acids (LNA) and peptide nucleic acids (PNA). A polynucleotide may be a single stranded sequence of nucleotides, such as RNA or single stranded DNA, a double stranded sequence of nucleotides, such as double stranded DNA, or may include a mixture of a single stranded and double stranded sequences of nucleotides. Double stranded DNA (dsDNA) includes genomic DNA, and PCR and amplification products. Single stranded DNA (ssDNA) can be converted to dsDNA and vice-versa. Polynucleotides may include non-naturally occurring DNA, such as enantiomeric DNA, LNA, or PNA. The precise sequence of nucleotides in a polynucleotide may be known or unknown. The following are examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, expressed sequence tag (EST) or serial analysis of gene expression (SAGE) tag), genomic DNA, genomic DNA fragment, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, synthetic polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, primer or amplified copy of any of the foregoing.
As used herein, the term “solid-state nanopore” is intended to mean a nanopore whose structure portion is defined by a solid-state membrane and includes materials of non-biological origin (i.e., not of biological origin). A solid-state nanopore can be formed of an inorganic or organic material. Solid-state nanopores include, for example, silicon nitride nanopores, silicon dioxide nanopores, and graphene nanopores.
The nanopores disclosed herein may be hybrid nanopores. A “hybrid nanopore” refers to a nanopore including materials of both biological and non-biological origins. An example of a hybrid nanopore includes a polypeptide-solid-state hybrid nanopore and a polynucleotide-solid-state nanopore.
The application of the potential difference across a nanopore may force the translocation of a nucleic acid through the nanopore. One or more signals are generated that correspond to the translocation of the nucleotide through the nanopore. Accordingly, as a target polynucleotide, or as a mononucleotide or a probe derived from the target polynucleotide or mononucleotide, transits through the nanopore, the current across the membrane changes due to base-dependent (or probe dependent) blockage of the nanopore constriction, for example. The signal from that change in current can be measured using any of a variety of methods. Each signal is unique to the species of nucleotide(s) (or probe) in the nanopore, such that the resultant signal can be used to determine a characteristic of the polynucleotide. For example, the identity of one or more species of nucleotide(s) (or probe) that produces a characteristic signal can be determined.
As used herein, the term “nanopore sequencer” refers to any of the devices disclosed herein that can be used for nanopore sequencing. In the examples disclosed herein, during nanopore sequencing, the nanopore is immersed in examples of the electrolyte disclosed herein and a potential difference is applied across the membrane. In an example, the potential difference is an electric potential difference or an electrochemical potential difference. An electrical potential difference can be imposed across the membrane via a voltage source that injects or administers current to at least one of the ions of the electrolyte contained in the cis well or one or more of the trans wells. An electrochemical potential difference can be established by a difference in ionic composition of the cis and trans wells in combination with an electrical potential. The different ionic composition can be, for example, different ions in each well or different concentrations of the same ions in each well.
As used herein, a “nucleotide” includes a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are monomeric units of a nucleic acid sequence. Examples of nucleotides include, for example, ribonucleotides or deoxyribonucleotides. In ribonucleotides (RNA), the sugar is a ribose, and in deoxyribonucleotides (DNA), the sugar is a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present at the 2′ position in ribose. The nitrogen containing heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine. The phosphate groups may be in the mono-, di-, or tri-phosphate form. These nucleotides are natural nucleotides, but it is to be further understood that non-natural nucleotides, modified nucleotides or analogs of the aforementioned nucleotides can also be used.
As used herein, the term “signal” is intended to mean an indicator that represents information. Signals include, for example, an electrical signal and an optical signal. The term “electrical signal” refers to an indicator of an electrical quality that represents information. The indicator can be, for example, current, voltage, tunneling, resistance, potential, voltage, conductance, or a transverse electrical effect. An “electronic current” or “electric current” refers to a flow of electric charge. In an example, an electrical signal may be an electric current passing through a nanopore, and the electric current may flow when an electric potential difference is applied across the nanopore.
As used herein, the term “bias offset voltage” and “input offset voltage” are intended to mean bias voltage difference between input terminals of an active circuitry (e.g., an amplifier) due to semiconductor process variation and/or non-ideality effect. For example, both “bias offset voltage” and “input offset voltage” of an amplifier refer to the voltage potential difference between input terminals of the amplifier. Specifically, if the amplifier is a differential amplifier, both “bias offset voltage” and “input offset voltage” refer to the voltage potential difference between two input terminals of the differential amplifier.
The aspects and examples set forth herein and recited in the claims can be understood in view of the above definitions.
Polynucleotides may be sequenced using a nanopore unit cell, or a nanopore sensor, based on electrical responses. In some embodiments, such unit cell may include a nanopore, a flow chamber containing a liquid, one or more electrodes, and an electronic circuit for measurement. In some cases, the nanopore may be a solid-state nanopore as illustrated in
In some cases, the nanopore may be a biological nanopore formed of peptides or polynucleotides and deposited in a lipid bilayer or a polymer membrane, e.g., a synthetic polymeric membrane. In an example shown in
In other embodiments, the DNA 110 may not pass through the nanopore 120. A unique tag or label for a nucleotide in the DNA 110 may pass through the nanopore 120. In one example, a tag or label of the nucleotide may be a particular sequence combination of nucleotides. When the tag or label is in or near the nanopore, it may result in a unique ionic current blockade at the nanopore, and therefore a unique nanopore resistance depending on the identity of the molecule of interest. By measuring the ionic current or the nanopore resistance, the tag or label at or near the nanopore, and therefore the corresponding nucleotide, can be identified.
Although embodiments herein describe determining a signal level by determining the ionic current through the nanopore, embodiments also include, alone or in combination, determining the signal level by measuring other electrical characteristics of the cis/trans nanopore cell. For example, in some embodiments, a signal level is determined by the voltage potential at a specified area or component of the cis/trans nanopore cell. For example, in some embodiments, a signal level is determined by the electrical impedance at a specified area or component of the cis/trans nanopore cell. For example, in some embodiments, a signal level is determined by the conductivity/resistance of the nanopore membrane.
In some embodiments, sequencing of a target polynucleotide may involve nanopore sensing of (1) a single-stranded portion of the target polynucleotide; (2) a nucleic acid duplex of a portion of the target polynucleotide; (3) a label or tag that can be tethered or untethered to the target polynucleotide; or any combination thereof.
In some embodiments, multiple such nanopore unit cells may be arranged in an array, and each unit cell or each nanopore sensor may be individually accessed by a logic circuit.
In some embodiments, a nanopore array is formed of an array of biochemical sensors, e.g., an array of nanopore unit cells described above. In some embodiments, a nanopore array can be used to perform long read DNA sequencing. A characteristic feature of a nanopore array is G-base per second per square centimeter of a chip. In some embodiments, to achieve higher data rate, the density of nanopores in a 2D array is increased. In some embodiments, a 2D readout circuit is used to take measurements from a 2D nanopore array.
As illustrated in
As illustrated in
As mentioned previously, the amplifier array 209 can be used to measure electrical currents flowing through the nanopore array 201 for decoding the nucleic acid sequences. For example, when different nucleotide (e.g., the nucleotide of the DNA 110 in
As shown in
However, due to process variations and limitations, the amplifier array 209 may not be fabricated ideally. One non-ideality effect associated with the amplifier array 209 is the presence of bias offset between the two inputs for each of the amplifier 209-1, 209-2 through 209-N. For example, referring to
As illustrated in
In some embodiments, the bias compensation circuit 317 may include an analog mechanism to compensate the bias offset voltage between the second input terminal 213 and the first input terminal 215-1 of the amplifier 209-1. The bias compensation circuit 317 may include an offset capacitor to store the bias offset of the amplifier 209-1. In some embodiments, the bias compensation circuit 317 may have a calibration mode and a measurement mode. For example, in the calibration mode, the offset capacitor may inversely connect to the first input terminal 215-1 of the amplifier 209-1. By setting the voltage of the second input terminal 213 of the amplifier 209-1 to 0V, the bias offset between the second input terminal 213 and the first input terminal 215-1 is stored in the offset capacitor. Subsequently, in the measurement mode, the offset capacitor and the nanopore resistance Rnp are connected to the second input terminal 213 of the amplifier 209-1. The bias offset of the amplifier 209-1 then is canceled out by the voltage potential (equals to the additive inverse of the bias offset) stored in the offset capacitor. Thus, the bias voltage on the nanopore resistance Rnp can be the same as the voltage of the second input terminal 213 of the amplifier 209-1 and would be independent of the bias offset Voffset. The operation of the bias compensation circuit 317 will be better understood by referring to detailed embodiments of the bias compensation circuit 317 as described below.
As shown in
In some embodiments, during the calibration mode, the first switch S1 is connected to the floating node N1, the second switch S2 connects the second terminal 403 of the offset capacitor C1 to the common electrode 211, and the third switch S3 connects the first terminal 401 of the offset capacitor C1 to the first input terminal 215-1 of the amplifier 209-1. During the measurement mode, the first terminal 401 of the offset capacitor C1 is connected to a first side 405 of the nanopore (represented by a nanopore resistance Rnp) through the first switch S1; the second terminal 403 of the offset capacitor C1 is connected to the first input terminal 215-1 of the amplifier 209-1 through the third switch S3; and the second switch S2 is connected to the floating node N2. The second side 407 of the nanopore resistance Rnp is connected to the common electrode 211.
As shown in
As mentioned previously, the bias compensation circuit 317 may utilize a calibration mode and a measurement mode to cancel out the bias offset of the amplifier 209-1. In some examples, during the calibration mode, the first switch S1 is connected to the floating node N1, the second switch S2 connects the second terminal 403 of the offset capacitor C1 to the common electrode 211, and the third switch S3 connects the first terminal 401 of the offset capacitor C1 to the first input terminal 215-1 of the amplifier 209-1. During the measurement mode, the first terminal 401 of the offset capacitor C1 is connected to the first side 405 of the nanopore resistance Rnp through the first switch S1; the second terminal 403 of the offset capacitor C1 is connected to the first input terminal 215-1 of the amplifier 209-1 through the third switch S3; and the second switch S2 is connected to the floating node N2.
As shown in
As such, the voltage across the nanopore resistance Rnp is independent from the bias offset of the amplifier 209-1. Advantageously, the cancellation of the bias offset may lead to better measurement for nanopore sequence, in contrast to the scenario illustrated in
Block 802 includes providing a polynucleotide to a sequencing cell that has a nanopore sensor. The sequencing cell include a nanopore sensor shown in
At block 804, a bias compensation circuit stores a voltage potential that is indicative of an input offset voltage between a first input terminal and a second input terminal of an amplifier 209-1. For example, the bias compensation circuit may store the input offset voltage of the amplifier 209-1.
At block 806, the electrical response of the nanopore sensor is measured, where the bias offset voltage of the amplifier is compensated using the stored voltage potential. For example, the amplifier 209-1 measures the electrical response associated with the sequencing cell that includes the nanopore sensor 201-1 to provide information about the identity of the polynucleotide that is provided with the nanopore sensor 201-1. Although not shown in
To independently control or address each unit cell (i.e., sequence cell), in some embodiments, each of the nanopore unit cells in a nanopore array may have its own trans electrode but may share a common cis electrode. In some embodiments, each of the nanopore unit cells in a nanopore array may have its own cis electrode but may share a common trans electrode. In some embodiments, each of the nanopore unit cells in a nanopore array may have its own cis electrode and trans electrode. In some embodiments, each of the nanopore unit cells in a nanopore array may share a common cis electrode and a common trans electrode.
The array may have any suitable number of nanopore unit cells. In some instances, the array comprises about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 3000, about 4000, about 5000, about 10000, about 15000, about 20000, about 40000, about 60000, about 80000, about 100000, about 200000, about 400000, about 600000, about 800000, about 1000000, about 10000000 or more nanopore unit cells. In some instances, the array comprises at least 200, at least 400, at least 600, at least 800, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, at least 10000, at least 15000, at least 20000, at least 40000, at least 60000, at least 80000, at least 100000, at least 200000, at least 400000, at least 600000, at least 800000, at least 1000000 or at least 10000000 nanopore unit cells. In some cases, the array can include individually addressable nanopore unit cells at a density of at least about 500, 600, 700, 800, 900, 1000, 10,000, 100,000 or 1,000,000 unit cells per mm2.
It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be appreciated that terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein.
Reference throughout the specification to “one example”, “another example”, “an example”, and so forth, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise.
It is to be understood that the ranges provided herein include the stated range and any value or sub-range within the stated range, as if such value or sub-range were explicitly recited. For example, a range from about 2 nm to about 20 nm should be interpreted to include not only the explicitly recited limits of from about 2 nm to about 20 nm, but also to include individual values, such as about 3.5 nm, about 8 nm, about 18.2 nm, etc., and sub-ranges, such as from about 5 nm to about 10 nm, etc. Furthermore, when “about” and/or “substantially” are/is utilized to describe a value, this is meant to encompass minor variations (up to +/−10%) from the stated value.
While several examples have been described in detail, it is to be understood that the disclosed examples may be modified. Therefore, the foregoing description is to be considered non-limiting.
While certain examples have been described, these examples have been presented by way of example only, and are not intended to limit the scope of the disclosure. Indeed, the novel methods and systems described herein may be embodied in a variety of other forms. Furthermore, various omissions, substitutions and changes in the systems and methods described herein may be made without departing from the spirit of the disclosure. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the disclosure.
Features, materials, characteristics, or groups described in conjunction with a particular aspect, or example are to be understood to be applicable to any other aspect or example described in this section or elsewhere in this specification unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The protection is not restricted to the details of any foregoing examples. The protection extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
Furthermore, certain features that are described in this disclosure in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable sub-combination. Moreover, although features may be described above as acting in certain combinations, one or more features from a claimed combination can, in some cases, be excised from the combination, and the combination may be claimed as a sub-combination or variation of a sub-combination.
Moreover, while operations may be depicted in the drawings or described in the specification in a particular order, such operations need not be performed in the particular order shown or in sequential order, or that all operations be performed, to achieve desirable results. Other operations that are not depicted or described can be incorporated in the example methods and processes. For example, one or more additional operations can be performed before, after, simultaneously, or between any of the described operations. Further, the operations may be rearranged or reordered in other implementations. Those skilled in the art will appreciate that in some examples, the actual steps taken in the processes illustrated and/or disclosed may differ from those shown in the figures. Depending on the example, certain of the steps described above may be removed or others may be added. Furthermore, the features and attributes of the specific examples disclosed above may be combined in different ways to form additional examples, all of which fall within the scope of the present disclosure. Also, the separation of various system components in the implementations described above should not be understood as requiring such separation in all implementations, and it should be understood that the described components and systems can generally be integrated together in a single product or packaged into multiple products. For example, any of the components for an energy storage system described herein can be provided separately, or integrated together (e.g., packaged together, or attached together) to form an energy storage system.
For purposes of this disclosure, certain aspects, advantages, and novel features are described herein. Not necessarily all such advantages may be achieved in accordance with any particular example. Thus, for example, those skilled in the art will recognize that the disclosure may be embodied or carried out in a manner that achieves one advantage or a group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.
Conditional language, such as “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain examples include, while other examples do not include, certain features, elements, and/or steps. Thus, such conditional language is not generally intended to imply that features, elements, and/or steps are in any way required for one or more examples or that one or more examples necessarily include logic for deciding, with or without user input or prompting, whether these features, elements, and/or steps are included or are to be performed in any particular example.
Conjunctive language such as the phrase “at least one of X, Y, and Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to convey that an item, term, etc. may be either X, Y, or Z. Thus, such conjunctive language is not generally intended to imply that certain examples require the presence of at least one of X, at least one of Y, and at least one of Z.
Language of degree used herein, such as the terms “approximately,” “about,” “generally,” and “substantially” represent a value, amount, or characteristic close to the stated value, amount, or characteristic that still performs a desired function or achieves a desired result.
The scope of the present disclosure is not intended to be limited by the specific disclosures of preferred examples in this section or elsewhere in this specification, and may be defined by claims as presented in this section or elsewhere in this specification or as presented in the future. The language of the claims is to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive.
This application claims priority to U.S. Provisional Application No. 63/268,728, filed Mar. 1, 2022, the content of which is incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/013810 | 2/24/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63268728 | Mar 2022 | US |
