阅读说明：本技术 相控纳米孔阵列 (Phased nanopore array ) 是由 S.费南德斯-戈梅斯 J.W.曼尼 于 2018-04-18 设计创作，主要内容包括：本文中描述的技术可以将具有不同相位的AC信号施加到纳米孔传感器芯片中的纳米孔单元的不同群组。当纳米孔单元的第一群组处于暗时段并且没有被模数转换器(ADC)采样或被最小采样来捕获有用数据时,纳米孔单元的第二群组处于亮时段,在此期间来自纳米孔的单元的第二群组的输出信号由模数转换器采样。ADC的参考电平设置基于施加的AC信号动态改变以充分利用ADC的动态范围。(When the th group of nanopore cells are in a dark period and are not sampled or minimally sampled by an analog-to-digital converter (ADC) to capture useful data, the second group of nanopore cells are in a bright period during which output signals from the second group of nanopore cells are sampled by the ADC.)

1, A sensor chip for nucleic acid sequencing, the sensor chip comprising:

sets of cells organized into N groups, N being two or more integers, each cell having a cell electrode configured to provide an AC signal to the cell for use in characterizing the nucleotides of a nucleic acid molecule, and

at least N circuits, each circuit of the at least N circuits configured to provide an individually configurable AC signal to or more cell electrodes of a respective group of or more cells of the N groups.

2. The sensor chip of claim 1, wherein each cell of said th set of cells comprises:

a common electrode configured to provide a second signal to a cell, the common electrode shared by two or more cells in the th group of cells.

3. The sensor chip of claim 2, wherein each cell of the th set of cells further comprises a nanopore positioned between the cell electrode and the common electrode, wherein the nanopore is configured to receive a label connected to a nucleotide and act as a variable resistor between a cell electrode and a common electrode.

4. The sensor chip of claim 2, wherein:

each cell in said th group of cells includes an integrating capacitor coupled to a cell electrode, an

Each circuit of the at least N circuits is configured to precharge the integration capacitors from a respective group of or more cells of the N groups with an AC signal.

5. The sensor chip of claim 4, wherein a precharged integration capacitor of a group of or more cells is configured to be charged or discharged by the second signal.

6. The sensor chip of claim 2, further comprising a sampling circuit coupled to the th set of cells, wherein:

the N circuits are configured to provide different AC signals to N groups; and

the sampling circuit is configured to selectively sample or more groups of voltage signals from or more cells of the N groups based on the AC signals provided to the N groups.

7. The sensor chip of claim 1, wherein said N circuits are configured to provide AC signals having different phases to different groups of or more cells of said N groups.

8. The sensor chip of claim 1, further comprising:

a second set of cells organized into N groups, each cell in the second set of cells having a cell electrode configured to provide an AC signal to the cell for characterizing a nucleotide of a nucleic acid molecule,

wherein each of the at least N circuits is configured to provide an individually configurable AC signal to or more cell electrodes of a respective group of the N groups of th group of cells and to or more cell electrodes of a respective group of the N groups of the second group of cells.

9. The sensor chip of claim 1, further comprising two or more fluidic channels, wherein cells in different fluidic channels are organized into different ones of the N groups.

10. The sensor chip of claim 1, wherein:

each of the at least N circuits includes a switch configured to alternately connect a cell electrode to two voltage levels; and

each switch is controlled by an AC control signal.

11. The sensor chip of claim 1, wherein:

each cell in the th group of cells includes a switch configured to alternately connect a cell electrode to two voltage levels;

each switch is controlled by an AC control signal;

each of the at least N circuits including or more switches of or more cells in a respective group, and

or more switches of or more cells in a respective group receive the same AC control signal.

12, A sensor chip for nucleic acid sequencing, comprising sets of cells, wherein each cell in the set of cells comprises:

a nanopore configured to receive a label linked to a nucleotide;

a membrane within which the nanopore resides, wherein the membrane functions as a capacitor and the nanopore functions as a resistor in the circuit;

a electrode electrically coupled to the circuit at the th end of the cell;

a second electrode electrically coupled to the circuit at a second end of the cell and shared by two or more cells in the set of cells; and

a control circuit configured to:

applying a AC signal through the th electrode to precharge the capacitor, and

a second signal is applied through the second electrode to charge or discharge the pre-charged capacitor via the nanopore.

A sensor chip for nucleic acid sequencing of the species , comprising:

sets of cells organized into N groups, N being two or more integers, each cell having a cell electrode configured to provide an AC signal to the cell for use in characterizing a nucleotide of a nucleic acid molecule;

an analog-to-digital converter (ADC) coupled to the th group of cells and configured to convert output signals from the th group of cells, an

A control circuit configured to:

applying an th AC signal to a th group of cells of the N groups of cells;

applying a second AC signal to a second group of cells in the N groups of cells, wherein the th AC signal and the second AC signal have different phases, and

during the th portion of the th AC signal, the ADC is caused to convert output signals from the th group of cells, while not converting output signals from the second group of cells.

14, a method of nucleic acid sequencing using a sensor chip comprising sets of cells, the method comprising:

applying an th AC signal to a th group of cells in the set of cells;

applying a second AC signal to a second group of cells in the set of cells, wherein the th AC signal and the second AC signal have different phases;

sampling output signals from the group of cells using an analog-to-digital converter (ADC) during the portion of the AC signal without sampling output signals from the second group of cells, and

during a second portion of the th AC signal, output signals from the second group of cells are sampled using the ADC, while output signals from the th group of cells are not sampled.

15, computer product comprising a computer readable medium storing a plurality of instructions for controlling a computer system to perform the operations of any of the above methods.

Background

Nanomembrane devices having a pore size of about nanometers in internal diameter show promise in rapid nucleotide sequencing.

As an alternative to a DNA molecule (or other nucleic acid molecule to be sequenced) moving through a nanopore, the molecule (e.g., the nucleotide added to the DNA strand) may include a specific label of a specific size and/or structure. The ionic current or voltage (e.g., at an integrating capacitor) in a circuit including the nanopore can be measured as a way of measuring the nanopore resistance corresponding to the molecule, thereby allowing detection of a particular molecule in the nanopore as well as a particular nucleotide at a particular location of the nucleic acid.

Nanopore-based sequencing sensor chips can be used for DNA sequencing. Nanopore-based sequencing sensor chips may incorporate a large number of sensor units configured as an array for parallel DNA sequencing. For example, a nanopore based sequencing sensor chip may include 100,000 or more cells arranged in a two-dimensional array for sequencing 100,000 or more DNA molecules in parallel. It may be difficult to fit so many cells into the sensor chip without compromising the measurement. It may also be difficult to efficiently operate the circuitry on such sensor chips.

Disclosure of Invention

The techniques described herein may also be applied to systems that use a periodically changing DC bias, which may also have "dark" periods when the electrodes are recharged.

According to examples, for nucleic acids: (For exampleDNA) sequencing sensor chip includes a th set of cells organized into N groups, where N is two or more integers, each cell including a cell electrode configured to provide an AC signal to the cell for characterizing nucleotides of a nucleic acid molecule.

According to another embodiments, a sensor chip for nucleic acid sequencing includes sets of cells each cell of the set of cells may include a nanopore configured to receive a tag connected to a nucleotide, a membrane in which the nanopore resides, wherein the membrane functions as a capacitor in a circuit and the nanopore functions as a resistor, a electrode electrically coupled to the circuit on a th end of the cell, and a second electrode coupled to the circuit on a second end of the cell and shared by two or more cells of the set of cells.

According to another embodiments, the method of nucleic acid sequencing using a sensor chip comprising sets of cells can comprise applying a st AC signal to a th group of cells in the set of cells and applying a second AC signal to a second group of cells in the set of cells, wherein the th AC signal and the second AC signal have different phases the method can further comprise sampling output signals from the th group of cells using an analog-to-digital converter (ADC) and not sampling output signals from the second group of cells during the th portion of the st AC signal the method can further comprise sampling output signals from the second group of cells using an ADC and not sampling output signals from the th group of cells during the second portion of the th AC signal.

These and other embodiments of the invention are described in detail below. For example, other embodiments are directed to systems, devices, and computer-readable media associated with the methods described herein.

In other words, the present invention provides sensor chips for nucleic acid sequencing, the sensor chip including a th set of cells organized into N groups, N being two or more integers, each cell having a cell electrode configured to provide an AC signal to the cell for characterizing a nucleotide of a nucleic acid molecule, and at least N circuits, each circuit of the at least N circuits configured to provide an individually configurable AC signal to or more cell electrodes of a respective group of or more cells of the N groups.

Each cell of the th set of cells may further steps include a nanopore positioned between the cell electrode and the common electrode, wherein the nanopore is configured to receive a marker connected to a nucleotide and act as a variable resistor between the cell electrode and the common electrode, each cell of the th set of cells may further steps include an integration capacitor coupled to the cell electrode, and each circuit of the at least N circuits may be configured to precharge the integration capacitor of a respective group of or more cells from the N groups with an AC signal.

The sensor chip may further include a sampling circuit coupled to the second group of cells, wherein the N circuits are configured to provide different AC signals to the N groups, and the sampling circuit is configured to selectively sample voltage signals from 1 or more groups of 0 or more cells of the N groups based on the AC signals provided to the N groups, during a 2 nd time period, the voltage level of the AC signal provided to the 5 or more cell electrodes of the 4 th group of 3 or more cells of the N groups may be higher than the voltage level of the second signal provided to the common electrode of the 7 th group of 6 or more cells, the voltage level of the AC signal provided to the 9 or more cell electrodes of the second group of 8 or more cells of the N groups may be lower than the voltage level of the second signal provided to the common electrode of the second group of the one or more cells, and the sampling circuit may be configured to dynamically sample voltage signals from the second group of 1 or more cells of the 0 or 2 or more cells at a sampling rate that the voltage level of the second group of 3 or more cells is lower than the voltage level of the second signal provided to the second group of the 6 or more cells, and the sampling circuit is configured to dynamically sample voltage levels of the second group of the second sampling cells provided to the second group of the N or more cells at a sampling rate that the voltage level of the second sampling circuit and the second sampling circuit is lower than the voltage level of the second sampling circuit, wherein the sampling circuit is configured to sample voltage level of the second sampling unit.

The second signal may be a second AC signal having a frequency lower than a frequency of the AC signal, or may be a DC signal, the N circuits may be configured to provide the AC signals having different phases to different groups of or more cells of the N groups.

The sensor chip may further include a second set of cells organized into N groups, each cell in the second set of cells having a cell electrode configured to provide an AC signal to the cell for characterizing nucleotides of the nucleic acid molecule, wherein each circuit of the at least N circuits is configured to provide an individually configurable AC signal to a respective group of or more cell electrodes in the N groups of the set of cells and a respective group of or more cell electrodes in the N groups of the second set of cells, the sensor chip may further include a a sampling circuit coupled to the set of cells and configured to selectively sample voltage signals from or more groups of the N groups of the set of cells, and a second sampling circuit coupled to the second set of cells and configured to selectively sample voltage signals from or more groups of the N groups of the second set of cells.

The sensor chip may also include two or more fluidic channels, wherein cells in different fluidic channels are organized into different ones of N groups, each of the at least N circuits may include a switch configured to alternately connect a cell electrode to two voltage levels, and each switch is controlled by an AC control signal, each of the N groups may include at least cells.

Each cell in the th set of cells can include a switch configured to alternately connect the cell electrodes to two voltage levels, each switch controlled by an AC control signal, wherein each circuit of the at least N circuits includes or more switches of or more cells in a respective group, and or more switches of or more cells in the respective group receive the same AC control signal.

The present invention also provides sensor chips for nucleic acid sequencing, including a set of cells, wherein each cell of the set of cells includes a nanopore configured to receive a label connected to a nucleotide, a membrane within which the nanopore resides, wherein the membrane functions as a capacitor and the nanopore functions as a resistor in a circuit, a electrode electrically coupled to the circuit on a end of the cell, a second electrode coupled to the circuit on a second end of the cell and shared by two or more cells of the set of cells, and a control circuit configured to apply a AC signal through the electrode to precharge the capacitor, and a second signal through the second electrode to charge or discharge the precharged capacitor via the nanopore.

In this case, the electrode of each cell may be independent of the electrodes of the other cells in the set of cells each cell in the set of cells may further include an integrating capacitor, and the control circuit may be further configured to apply a AC signal through the electrode to precharge the integrating capacitor, and a second signal through the second electrode to charge or discharge the precharged integrating capacitor via the nanopore.

The present invention also provides a sensor chip for nucleic acid sequencing comprising sets of cells organized into N groups, N being two or more integers, each cell having a cell electrode configured to provide an AC signal to the cell for characterizing a nucleotide of a nucleic acid molecule, an analog-to-digital converter (ADC) coupled to the set of cells and configured to convert an output signal from the set of cells, and a control circuit configured to apply a th AC signal to the th group of cells of the N groups of cells, apply a second AC signal to a second group of cells of the N groups of cells, wherein the th AC signal and the second AC signal have different phases, and during the th portion of the AC signal, cause the ADC to convert the output signal from the th group of cells without converting the output signal from the second group of cells.

The present invention also provides methods of nucleic acid sequencing using a sensor chip comprising sets of cells, the method comprising applying a th AC signal to a th group of cells in the set of cells, applying a second AC signal to a second group of cells in the set of cells, wherein the th AC signal and the second AC signal have different phases, sampling output signals from the th group of cells using an analog-to-digital converter (ADC) without sampling output signals from the second group of cells during a th portion of the th AC signal, and sampling output signals from the second group of cells using the ADC without sampling output signals from the th group of cells during a second portion of the th AC signal.

Applying the AC signal to the group of cells in the set of cells can include applying the AC signal to the cell electrode of each cell in the group of cells, and applying the second AC signal to the second group of cells in the set of cells can include applying the second AC signal to the cell electrode of each cell in the second group of cells.

The method may further comprise the step of applying a common signal to a common electrode shared by the set of cell groups, wherein the common signal is a DC signal or a third AC signal having a frequency less than the frequency of the th AC signal and the frequency of the second AC signal, and the method may further comprise the step of sampling output signals from both the second group of cells and the th group of cells using the ADC during a third portion of the th AC signal, and the reference level setting for the ADC based on the th AC signal and the second AC signal may be changed.

The present invention also provides a computer product comprising a computer readable medium storing a plurality of instructions for controlling a computer system to perform the operations of any of the above methods.

The invention further provides systems that include the disclosed computer product and or more processors for executing instructions stored on a computer readable medium.

Drawings

Fig. 1 is a top view of an embodiment of a nanopore sensor chip having an array of nanopore cells.

Figure 2 illustrates an embodiment of a nanopore cell in a nanopore sensor chip that may be used to characterize a polynucleotide or polypeptide.

Fig. 3 illustrates an embodiment of a nanopore unit performing nucleotide sequencing by using a nanopore-based sequencing-by-synthesis (nano-SBS) technique.

Fig. 4 illustrates an embodiment of a circuit in a nanopore cell.

Fig. 5 shows example data points captured from a nanopore cell during the light and dark periods of an AC cycle.

Fig. 6 is a cross-sectional view illustrating an array of nanopore cells in a nanopore sensor chip.

Fig. 7 is a top view of an array of example nanopore cells comprising a two-dimensional array of nanopore cells.

Fig. 8 is a schematic diagram of an array of example nanopore cells including a two-dimensional array of nanopore cells.

Fig. 9 illustrates an example data sample captured from a nanopore cell in a column of a nanopore cell array.

Fig. 10 is a schematic diagram of an example nanopore cell array comprising a two-dimensional array of nanopore cells, according to certain aspects of the present disclosure.

Fig. 11 illustrates example control signals for an array of nanopore cells, in accordance with certain aspects of the present disclosure.

Fig. 12 illustrates an example data sample captured from a nanopore cell in a column of a nanopore cell array, in accordance with certain aspects of the present disclosure.

Fig. 13 illustrates example control signals for a nanopore cell array, in accordance with certain aspects of the present disclosure.

Fig. 14A illustrates a fixed reference level for an ADC in a nanopore sensor chip according to certain aspects of the present disclosure.

Fig. 14B illustrates a variable reference level for an ADC in a nanopore sensor chip according to certain aspects of the present disclosure.

Fig. 15 is a flow diagram illustrating an example method of nucleic acid sequencing using a sensor comprising sets of cells, according to certain aspects of the present disclosure.

Fig. 16 is a block diagram of an example computer system that may be used with the system and method according to certain aspects of the present disclosure.

Definition of

Nucleic acid "It may refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term may include nucleic acids that comprise known nucleotide analogs or modified stem residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, that have similar binding properties as the reference nucleic acid, and that are metabolized in a manner similar to the reference nucleotide. Examples of such analogs can include, without limitation, phosphorothioates, phosphoramidites, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, Peptide Nucleic Acids (PNAs). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

The term "template" may refer to a single-stranded nucleic acid molecule that is copied into the complementary strand of DNA nucleotides for DNA synthesis in cases, a template may refer to the sequence of DNA that is copied during synthesis of mRNA.

The term "primer" may refer to a short nucleic acid sequence that provides a point of initiation for DNA synthesis. Enzymes that catalyze DNA synthesis (such as DNA polymerases) can add new nucleotides to primers for DNA replication.

As used herein, the term "Column(s) of"may generally refer to a nanopore cell in an array of nanopore cells sharing sampling and conversion circuitry. The nanopore cells in a column may or may not be physically fabricated in a column on the nanopore sensor chip.

As used herein, the term "light period" may refer generally to to a time period when a label of a labeled nucleotide is forced into a nanopore by an electric field applied via an AC signal the term "dark period" may refer generally to to a time period when a label of a labeled nucleotide is pushed out of a nanopore by an electric field applied via an AC signal.

Detailed Description

The technology disclosed herein relates to nanopore-based nucleic acid sequencing, and more particularly to increasing data sampling rates by a nanopore-based sequencing sensor chip that includes a large number of parallel sequencing nanopore cells.

Examples of such events may include unbound nucleotide labels entering the nanopore briefly, nucleotides bound briefly but not catalyzed, and nucleotides catalyzed quickly (potentially following the same nucleotides catalyzed at the next positions), however, there is an upper limit on the possible sampling rate due to, for example, limited sampling and conversion speed of the analog-to-digital converter and/or limited bandwidth of the bus, data storage device, or data processing circuitry.

The AC signal may be used in Nano-SBS to improve the lifetime of a nanopore sensor chip comprising an array of nanopore cells. For example, a constant level may be applied to the working electrode of each nanopore cell in the nanopore sensor chip, and a common AC signal may be applied to the shared counter electrode of the nanopore cell. In this example, each nanopore cell undergoes an AC cycle with substantially the same phase. Each AC cycle may include a light period (labels may be pushed into the nanopore for identifying nucleotides) and a dark period, where the duty cycle may be low (i.e., the dark period may be much longer than the light period). Thus, all nanopore cells of the nanopore sensor chip will be in either a light period or a dark period at approximately the same time.

During the light period, the data sampling and conversion circuitry associated with the nanopore cells in the column may sample and convert the output voltage signal from each nanopore cell in the column sequentially, as part identifying the label and thus incorporating the nucleotide, the AC signal draws (passes) the bound nucleotide label into the nanopore during the light period, and the signal thus measured provides information about which label (and therefore which nucleotide) is currently bound.

Furthermore, for nanopore sensor chips with high cell density, a single sampling and conversion circuit may serve multiple cells. Thus, each cell may be sampled at a rate much lower than the full sampling rate of the sampling and conversion circuit.

The technology disclosed herein solves the above problem by applying AC signals having different phases to different nanopore cells in a column, such that when nanopore cells in a column are in a dark period, the same other nanopore cells in column are in a light period.

In this manner, at any given time, the data sampling and conversion circuitry may sample and convert output voltage signals from portions of the nanopore cells in a column that are in a bright period, with the dark period being minimally sampled, for example, only for the purpose of return .

Nanopore-based sequencing chip

FIG. 1 is a top view of an embodiment of a nanopore sensor chip 100 having an array 140 of nanopore cells 150, each nanopore cell 150 including control circuitry integrated on a silicon substrate of the nanopore sensor chip 100 in embodiments , sidewalls 136 may be included in the array 140 to separate groups of nanopore cells 150 so that each group may receive a different sample for characterization.

In embodiments, nanopore sensor chip 100 may comprise multiple chips in the same package, such as, for example, a multi-chip module (MCM) or a System In Package (SiP), which may include, for example, a memory, a processor, a field programmable array (FPGA), an Application Specific Integrated Circuit (ASIC), a data converter, a high speed I/O interface, and so forth.

In some embodiments, nanopore sensor chip 100 may be coupled to (e.g., docked to) a nanochip workstation 120, which nanochip workstation 120 may include various components for implementing (e.g., automatically implementing) various embodiments of the processes disclosed herein, including, for example, an analyte delivery mechanism (such as a pipette for delivering a lipid suspension or other membrane structure suspension, an analyte solution, and/or other liquids, suspensions, or solids), a robotic arm, a computer processor, and/or a memory a plurality of polynucleotides may be detected on array 140 of nanopore cells 150. in some embodiments, each nanopore cell 150 may be individually addressable.

Nanopore sequencing unit

Nanopore cells 150 in nanopore sensor chip 100 may be implemented in many different ways, for example, in embodiments labels of different sizes and/or chemical structures may be attached to different nucleotides in a nucleic acid molecule to be sequenced in embodiments complementary strands of a template of a nucleic acid molecule to be sequenced may be synthesized by hybridizing differently polymer labeled nucleotides with the template.

A.Nano meterPore sequencing unit structure

Fig. 2 illustrates an embodiment of an example nanopore cell 200 in a nanopore sensor chip that may be used to characterize a polynucleotide or polypeptide, such as nanopore cell 150 in nanopore sensor chip 100 of fig. 1. Nanopore cell 200 may include a well 205 formed by dielectric layers 201 and 204; a membrane, such as a lipid bilayer 214 formed over the well 205; and a sample chamber 215 on the lipid bilayer 214 and separated from the well 205 by the lipid bilayer 214. The well 205 may contain an electrolyte volume 206, and the sample chamber 215 may hold a bulk electrolyte 208 containing a nanopore, such as a soluble Protein Nanopore Transmembrane Molecule Complex (PNTMC), and an analyte of interest, such as a nucleic acid molecule to be sequenced.

Nanopore cell 200 may include a working electrode 202 at the bottom of a well 205, and a counter electrode 210 disposed in a sample chamber 215 signal source 228 may apply a voltage signal between working electrode 202 and counter electrode 210 individual nanopores (e.g., PNTMC) may be inserted into lipid bilayer 214 through an electrical pass-through process caused by the voltage signal, forming nanopores 216 in lipid bilayer 214 the individual membranes (e.g., lipid bilayer 214 or other membrane structures) in the array may be neither chemically nor electrically connected to each other, thus, each nanopore cell in the array may be a separate sequencing machine that produces data unique to for individual polymer molecules associated with the nanopore that operates on an analyte of interest and modulates ion current through an otherwise impermeable lipid bilayer.

As shown in FIG. 2, nanopore cell 200 may be formed on a substrate 230 (such as a silicon substrate). dielectric layer 201 may be formed on substrate 230. dielectric materials used to form dielectric layer 201 may include, for example, glass, oxides, nitrides, and the like. circuitry 222 for controlling electrical excitation and for processing signals detected from nanopore cell 200 may be formed on substrate 230 and/or within dielectric layer 201. for example, a plurality of patterned metal layers (e.g., metal 1 through metal 6) may be formed in dielectric layer 201, and a plurality of active devices (e.g., transistors) may be fabricated on substrate 230. in embodiments, signal source 228 is included as part of of circuitry 222. circuitry 222 may include, for example, amplifiers, integrators, analog to digital converters, noise filters, feedback control logic, and/or various other components. circuitry 222 may additionally be coupled to processor 224, which processor 224 is coupled to memory 226, where processor 224 may analyze sequencing data to determine the sequence of polymer molecules that have been sequenced in an array.

Working electrode 202 may be formed on dielectric layer 201 and may form at least portions of the bottom of well 205. in embodiments, working electrode 202 is a metal electrode for non-faradaic conduction, working electrode 202 may be made of metal or other materials that are resistant to corrosion and oxidation (such as, for example, platinum, gold, titanium nitride, and graphite). for example, working electrode 202 may be a platinum electrode with electroplated platinum.

Dielectric layer 204 may be formed over dielectric layer 201 dielectric layer 204 forms walls surrounding well 205 dielectric material used to form dielectric layer 204 may include, for example, glass, oxide, silicon nitride (SiN), polyimide, or other suitable hydrophobic insulating material the top surface of dielectric layer 204 may be silanized the silanization may form hydrophobic layer 220 over the top surface of dielectric layer 204 in embodiments, hydrophobic layer 220 has a thickness of approximately 1.5 nanometers (nm).

The well 205 formed by the dielectric layer wall 204 includes a volume of electrolyte 206 above the working electrode 202 the volume of electrolyte 206 may be buffered and may include or more of lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate, sodium glutamate, glutamatePotassium, lithium, sodium, potassium, calcium chloride (CaCl)₂) Strontium chloride (SrCl)₂) Manganese chloride (MnCl)₂) And magnesium chloride (MgCl)₂) In embodiments, the volume of electrolyte 206 has a thickness of approximately three micrometers (μm).

As also shown in FIG. 2, a membrane may be formed on top of the dielectric layer 204 and across the well 205 in embodiments the membrane may comprise a lipid monolayer 218 formed on top of the hydrophobic layer 220. when the membrane reaches the opening of the well 205, the lipid monolayer 208 may be transformed into a lipid bilayer 214, the lipid bilayer 214 spanning the opening of the well 205. the lipid bilayer may comprise or consist of a phospholipid selected, for example, from the group consisting of diphytanoyl-phosphatidylcholine (DPhPC), 1, 2-diphytanoyl-sn-glycero-3-phosphocholine (DoPhPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidyl-methyl ester (DOPME), Dipalmitoylphosphatidylcholine (DPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, lysophosphatidic acid, phosphatidylinositol, phosphatidylglycerol, phospholipids, 1, 2-di-O-sn-glycero-glycerol-ethanolamine, 1, 2-dipalmitoyl-sn-glycero-sn-phosphatidylethanolamine (DPPC), 1, 2-dipalmitoyl-sn-glycero-sn-phosphatidylethanolamine, phosphatidylethanolamine (DPME), phosphatidylethanolamine, 1, 2-dipalmitoyl-sn-glycero-sn-3-phosphoethanolamine (DPME), phosphatidylethanolamine, DPMA, DP.

As shown, the lipid bilayer 214 is embedded with a single nanopore 216, formed, for example, from a single PNTMC. As described above, the nanopore 216 may be formed by: a single PNTMC is inserted into lipid bilayer 214 by electrical crossing. Nanopore 216 may be large enough for passing an analyte of interest between two sides of lipid bilayer 214 and/or small enoughSeed (e.g. Na)⁺、K⁺、Ca²⁺、CI^-) At least portions.

The sample chamber 215 is above the lipid bilayer 214 and may hold a solution of the analyte of interest for characterization the solution may be an aqueous solution that contains the bulk electrolyte 208 and is buffered to an optimal ion concentration and maintained at an optimal pH to keep the nanopore 216 open the nanopore 216 spans the lipid bilayer 214 and provides the only path for ion flow from the bulk electrolyte 208 to the working electrode 202 the bulk electrolyte 208 may further include or more of lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCl), in addition to the nanopore (e.g., PNTMC) and the analyte of interest₂) Strontium chloride (SrCl)₂) Manganese chloride (MnCl)₂) And magnesium chloride (MgCl)₂）。

Counter Electrode (CE) 210 may be an electrochemical potential sensor in embodiments, counter electrode 210 may be shared between a plurality of nanopore cells, and may therefore be referred to as a common electrode in cases, the common potential and common electrode may be common to all nanopore cells, or at least all nanopore cells within a particular grouping, the common electrode may be configured to apply the common potential to bulk electrolyte 208 in contact with nanopore 216, counter electrode 210 and working electrode 202 may be coupled to signal source 228 for providing electrical stimulation (e.g., bias) across lipid bilayer 214, and may be used for sensing electrical characteristics (e.g., resistance, capacitance, and ionic current flow) of lipid bilayer 214 in embodiments, nanopore cell 200 may further include reference electrode 212.

in some embodiments, various checks may be made during creation of the nanopore cell as part of of the calibration once the nanopore cell is created, additional calibration steps may be performed, such as for identifying nanopore cells that perform as desired (e.g., nanopores in the cell).

B.Detection signal of nanopore sequencing unit

Nanopore cells in a nanopore sensor chip, such as nanopore cell 150 in nanopore sensor chip 100, may enable parallel sequencing using single molecule nanopore based sequencing by synthetic (nano-SBS) techniques.

FIG. 3 illustrates an embodiment of a nanopore cell 300 that performs nucleotide sequencing by using a nano-SBS technique in which a template 332 to be sequenced (e.g., a nucleotide molecule or another analyte of interest) and a primer may be introduced into a bulk electrolyte 308 in a sample chamber of the nanopore cell 300.

In embodiments, an enzyme (e.g., polymerase 334, such as a DNA polymerase) may be associated with nanopore 316 for use in synthesizing a complementary strand into template 332. for example, polymerase 334 may be covalently attached to nanopore 316. polymerase 334 may catalyze incorporation of nucleotide 338 onto a primer by using a single-stranded nucleic acid molecule as a template. nucleotide 338 may include a label species ("multiple labels"), where the nucleotide is of four different types A, T, G or C.

As used herein, a "loaded" or "passed through" tag can be that are positioned in and/or remain in or near a nanopore for a considerable amount of time (e.g., 0.1 milliseconds (ms) to 10,000 ms.) in cases, the tag is loaded into the nanopore prior to release from the nucleotide in instances, the probability that the loaded tag passes through (and/or is detected by) the nanopore upon a nucleotide incorporation event after being released is suitably high, e.g., 90% to 99%.

In embodiments, the conductance of the nanopore 316 may be high before the polymerase 334 is attached to the nanopore 316, such as, for example, approximately 300 pico seeds (300 pS) — when the tag is loaded into the nanopore, a conductance signal of only (e.g., signal 340) is generated due to the unique chemical structure and/or size of the tag-for example, the conductance of the nanopore may be approximately 60 pS, 80 pS, 100 pS, or 120 pS, each corresponding to of the four types of tagged nucleotides-the polymerase may then undergo isomerization and transphosphorylation reactions to incorporate the nucleotides into the growing nucleic acid molecule and release the tagged molecule.

In cases, of the labeled nucleotides may not match (complementary base) the current location of the nucleic acid molecule (template). labeled nucleotides that do not base pair with the nucleic acid molecule may also pass through the nanopore.these unpaired nucleotides may be rejected by the polymerase on a time scale that is shorter than the time scale at which the properly paired nucleotides remain associated with the polymerase.

In some embodiments, a Direct Current (DC) signal may be applied to the nanopore cell (e.g., such that the direction in which the label moves through the nanopore is not reversed). however, operating the nanopore sensor for a long period of time by using direct current may change the composition of the electrodes, unbalance the concentration of ions across the nanopore, and have other undesirable effects that can affect the lifetime of the nanopore cell.

The ability to recharge the electrode during an AC detection cycle can be advantageous when using sacrificial electrodes, electrodes that change molecular properties in a current carrying reaction (e.g., electrodes comprising silver), or electrodes that change molecular properties in a current carrying reaction.

Suitable conditions for measuring ionic current through a nanopore are known in the art, and examples are provided herein. measurements can be performed Using voltages applied across the membrane and the pore in some embodiments, the voltages used may range from-400 mV to +400 mV. are preferably in a range having a lower limit selected from-400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20 mV, and 0mV, and an upper limit independently selected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV, and +400 mV. the voltages used may more preferably be in the range of 100mV to 240mV, and most preferably in the range of 160mV to 240 mV. it is possible to increase the distinction between different nucleotides by nanopore Using an increased applied potential by Using an AC waveform and labeled nucleotides to sequence Nucleic acids on day 6 of 2013, entitled "guanine",.

C.Circuit of nanopore sequencing unit

FIG. 4 illustrates an embodiment of a nanopore cell, such as circuit 400 in nanopore cell 200 (which may include part of circuit 222 in FIG. 2.) As described above, in embodiments, circuit 400 includes a counter electrode 210, which counter electrode 210 may be shared among a plurality or all of the nanopore cells in a nanopore sensor chip, and may therefore also be referred to as a common electrode_LIQ420 to apply a common potential to the bulk electrolyte (e.g., bulk electrolyte 208) in contact with the lipid bilayer (e.g., lipid bilayer 214) in the nanopore cell in some embodiments, an AC non-faraday mode may be used to modulate the voltage V with an AC signal (e.g., a square wave)_LIQAnd it is applied to the bulk electrolyte in contact with the lipid bilayer in the nanopore cell in examples, V_LIQIs a square wave having a magnitude of 200-250 mV and a frequency of, for example, between 25 and 400 Hz. The bulk electrolyte between the counter electrode 210 and the lipid bilayer (e.g., lipid bilayer 214) may be modeled by a large capacitor (not shown), such as, for example, 100 μ F or more。

Fig. 4 also shows an electrical model 422, which represents the electrical properties of the working electrode (e.g., working electrode 202) and the lipid bilayer (e.g., lipid bilayer 214). Electrical model 422 includes a capacitor 426 (C) that models a capacitance associated with the lipid bilayer_Bilayer) And a resistor 428 (R) that models a variable resistance associated with the nanopore_PORE) Which may vary based on the presence of a particular label in the nanopore. Electrical model 422 also includes a capacitor 424, capacitor 424 having a double layer capacitance (C)_{Double Layer}) And represents the electrical properties of working electrode 202 and well 205. Working electrode 202 may be configured to apply a different potential independent of the working electrodes in other nanopore cells.

The pass-through device 406 is an switch that may be used to connect or disconnect the lipid bilayer and the working electrode from the circuit 400 the pass-through device 406 may be controlled by a memory bit to enable or disable the voltage excitation to be applied across the lipid bilayer in the nanopore cell.

The circuit 400 may further include an on-chip integrating capacitor 408 (n)_cap). The integrating capacitor 408 may be pre-charged by closing the switch 401 using the preset signal 403 such that the integrating capacitor 408 is connected to the voltage source V_PRE405. In embodiments, voltage source V_PRE405 provides a constant positive voltage having a magnitude of, for example, 900 mV. When switch 401 is closed, integrating capacitor 408 may be precharged to voltage source V_PRE405.

After the integration capacitor 408 is precharged, the preset signal 403 may be used to open the switch 401 such that the integration capacitor 408 is driven from the voltage source V_PRE405 is disconnected. At this point, depending on the voltage source V_liqTo the electrode 210May be at a higher level than the potential of the working electrode 202 (and the integrating capacitor 408), or vice versa. For example, in the case of a voltage from a voltage source V_LIQDuring a positive phase of the square wave (e.g., a light or dark period of an AC voltage source signal cycle), the potential of the counter electrode 210 is at a higher level than the potential of the working electrode 202. At a voltage from a voltage source V_LIQDuring the negative phase of the square wave (e.g., the dark or light period of the AC voltage source signal cycle), the potential of the counter electrode 210 is at a lower level than the potential of the working electrode 202. thus, in embodiments, the integrating capacitor 408 may additionally be at a potential from the voltage source V_PRE405 is charged to a higher level during a bright period of the pre-charged voltage level and discharged to a lower level during a dark period due to the potential difference between the counter electrode 210 and the working electrode 202. In other embodiments, the charging and discharging may occur in the dark and light periods, respectively.

The integrating capacitor 408 may be charged or discharged for a fixed period of time depending on the sampling rate of the analog-to-digital converter (ADC) 410, which may be higher than 1 kHz, 5 kHz, 10 kHz, 100 kHz or more. For example, with a sampling rate of 1 kHz, the integration capacitor 408 may be charged/discharged for a period of about 1ms, and then the voltage level may be sampled and converted by the ADC 410 at the end of the integration period. A particular voltage level will correspond to a particular label species in the nanopore and thus to the nucleotide at the current location on the template.

After being sampled by the ADC 410, the integrating capacitor 408 may be precharged again by closing the switch 401 using the preset signal 403, so that the integrating capacitor 408 is again connected to the voltage source V_PRE405. The following steps may be repeated in cycles throughout the sequencing process: to precharge the integration capacitor 408, a fixed period of time is waited for the integration capacitor 408 to charge or discharge, and the voltage level of the integration capacitor is sampled and converted by the ADC 410.

The digital processor 430 may process the ADC output data, for example, for binning , data buffering, data filtering, data compression, data reduction, event extraction, or assembly of ADC output data from an array of nanopore cells into various data frames in embodiments, the digital processor 430 may perform downstream processing of steps, such as base determination.

Another four possible states of the nanopore each correspond to a state when of four different types of label-attached polyphosphate nucleotides (A, T, G or C) are held in the nanopore barrel tube, another possible state of the nanopore is when the phospholipid bilayer is ruptured.

When the voltage level on the integrating capacitor 408 is measured after a fixed period of time, different states of the nanopore may result in measurement of different voltage levels. This is because the rate at which the voltage across the integration capacitor 408 decays (either decreases through discharge or increases through charge), i.e., the steepness of the slope of the voltage versus time curve across the integration capacitor 408, depends on the nanopore resistance (e.g., resistor R)_PORE428 resistance). More specifically, where the resistances associated with nanopores in different states differ due to the different chemical structures of the molecules (of the labels), different corresponding rates of voltage decay may be observed and used to identify the different states of the nanopores. The voltage decay curve may be an exponential curve having an RC time constant τ = RC, where R is a resistance associated with the nanopore (i.e., R_PORE428) And C is in parallel with R and the membrane (i.e., capacitor 426 (C)_Bilayer) Associated capacitance). The time constant of the nanopore cell may be, for example, about 200 and 500 ms. The decay curve may not fit exactly to the exponential curve due to the detailed implementation of the bilayer, but the decay curve may resemble an exponential curve and be monotonic in thatAllowing detection of the label.

In some embodiments, the resistance associated with the nanopore in the open channel state may be in the range of 100 MOhm to 20 GOhm in some embodiments, the resistance associated with the nanopore in the state where it is labeled inside the nanopore barrel may be in the range of 200 MOhm to 40 GOhm in other embodiments, the integrating capacitor 408 may be omitted because the voltage to the ADC 410 will still vary due to voltage decay in the electrical model 422.

The decay rate of the voltage across the integrating capacitor 408 can be determined in different ways. As explained above, the rate of voltage decay may be determined by measuring the voltage decay during a fixed time interval. For example, the voltage on the integrating capacitor 408 may be measured first by the ADC 410 at time t1, and then the voltage is measured again by the ADC 410 at time t 2. The voltage difference is greater when the slope of the voltage versus time curve over the integration capacitor 408 is steeper and the voltage difference is less when the slope of the voltage curve is less steep. Thus, the voltage difference may be used as a metric for determining the rate of voltage decay across the integrating capacitor 408 and thus the state of the nanopore cell.

For example, the time required for the voltage to drop or increase from the th voltage level V1 to the second voltage level V2 may be measured_cap408 and thus a measure of the state of the nanopore cell. Those skilled in the art will appreciate various circuits that may be used to measure nanopore resistance, including, for example, amperometric techniques.

In embodiments, circuit 400 may not include pass-through devices fabricated on a chip (e.g., such asPass device 406) and an additional capacitor (e.g., integrating capacitor 408 (n)_cap) To facilitate a reduction in the size of nanopore-based sequencing chips. Due to the thin nature of the membrane (lipid bilayer), compared to a separate membrane (e.g., capacitor 426 (C)_Bilayer) Associated capacitance) may be sufficient to create the required RC time constant without requiring additional on-chip capacitance. Thus, the capacitor 426 may be used as an integrating capacitor and may pass the voltage signal V_PREIs pre-charged and then passes a voltage signal V_LIQIs discharged or charged. Eliminating additional capacitors and pass-through devices that would otherwise be fabricated on the chip in the circuit can significantly reduce the footprint of a single nanopore cell in a nanopore sequencing chip, facilitating scaling of the nanopore sequencing chip to include more and more cells (e.g., millions of cells in a nanopore sequencing chip).

D.Data sampling in nanopore cells

To perform nucleic acid sequencing, an integrating capacitor (e.g., integrating capacitor 408 (n)_cap) Or capacitor 426 (C)_Bilayer) Voltage levels may be sampled and converted by an ADC (e.g., ADC 410) while labeled nucleotides are added to the nucleic acid. The label of the nucleotide may be pushed into the nanopore barrel by an electric field across the nanopore applied via the counter and working electrodes, for example when the applied voltage is such that V_LIQBelow V_PREThat time.

Through the hole

The pass event is when a labeled nucleotide is attached to a template (e.g., a nucleic acid fragment) and labeled into and out of the barrel tube of the nanopore. This may occur multiple times during a pass through event. When the tag is in a barrel of the nanopore, the resistance of the nanopore may be higher and a lower current may flow through the nanopore.

During sequencing, the tag may not be in the nanopore (referred to as an open channel state) in AC cycles, where the current is highest due to the lower resistance of the nanopore.

Light and dark periods

For example, in embodiments, the AC voltage signal may be applied across the system at, for example, about 100Hz, and the acquisition rate of the ADC may be about 2000 Hz. per cell so there may be about 20 data points (voltage measurements) captured per AC cycle (cycle of the AC waveform.) the data points corresponding to cycles of the AC waveform may be referred to as a set_LIQBelow V_PREA subset captured at the time, which may correspond to a bright mode (period) in which the marker is forced into the barrel of the nanopore, another subset may correspond to a dark mode (period) in which when, for example, V_LIQHigher than V_PREAt this time, the tag is pushed out of the nanopore barrel by the applied electric field.

Measured voltage

For each data point, when switch 401 is open, at the integrating capacitor (e.g., integrating capacitor 408 (n)_cap) Or capacitor 426 (C)_Bilayer) Voltage at) will be taken as the pass V_LIQAs a result of charging/discharging, in a decaying manner, e.g. as when V_liqHigher than V_PREFrom V at the time_PRETo V_LIQIncrease of (2), or when V_LIQBelow V_PREFrom V at the time_PRETo V_liqIs reduced. The final voltage value may be from V when the working electrode is charged_LIQAnd (4) deviating. The rate of change of the voltage level on the integrating capacitor may be governed by the resistance value of the bilayer, which may include a nanopore, which in turn may include molecules (e.g., labels of labeled nucleotides) in the nanopore. The voltage level may be measured at a predetermined time after the switch 401 is turned off.

The switch 401 may operate at the data acquisition rate. The switch 401 may be closed for a relatively short period of time between two data acquisitions, typically just after a measurement by the ADC. The switch allows for multiple data points to be collected for each cycle. If the switch 401 remains open, the voltage level on the integrating capacitor, and thus the output value of the ADC, will completely decay and stay there. Such multiple measurements may allow for higher resolution with fixed ADCs (e.g., the measurements may be averaged due to a larger number of 8-14 bits of measurements). The plurality of measurements may also provide kinetic information about the molecules passing into the nanopore. The timing information may allow a determination of how long the crossing occurred. This can also be used to help determine whether a plurality of nucleotides added to a nucleic acid strand are being sequenced.

Fig. 5 shows example data points captured from a nanopore cell during the light and dark periods of an AC cycle. In fig. 5, changes in data points are exaggerated for illustrative purposes. Voltage (V) applied to working electrode or integrating capacitor_PRE) At a constant level, such as for example 900 mV. A voltage signal 510 (V) applied to the counter electrode of the nanopore cell_LIQ) Is an AC signal shown as a rectangular wave, where the duty cycle may be any suitable value, such as less than or equal to 50%, for example about 40%.

During the bright period 520, the voltage signal 510 (V) applied to the counter electrode_LIQ) Lower than the voltage V applied to the working electrode_PRESuch that the label can be forced into the nanopore barrel by an electric field caused by different voltage levels applied at the working and counter electrodes (e.g., due to charge on the label and/or ion flow). When switch 401 is open, the voltage at the node before the ADC (e.g., at the integrating capacitor) will decrease. After the voltage data point is captured (e.g., after a specified time period), switch 401 may be closed and the voltage at the measurement node will again increase back to V_PRE. The process may be repeated to measure multiple voltage data points. In this manner, multiple data points may be captured during the bright period.

As shown in fig. 5, at V_LIQThe th data point 522 (which is also referred to as the th point increment (FPD)) in the bright period after the change in sign of the signal may be lower than the subsequent data point 524 this may be because there is no marker in the nanopore (open channel) and thus it has low resistance and a high discharge rate in examples, the th data point 522 may exceed V as shown in fig. 5_LIQAnd (4) horizontal. This may be caused by the capacitance of the double layer coupling the signal to the on-chip capacitor. Data points 524 may be captured after a pass through event has occurred (i.e., the tag is forced into the nanopore's barrel tube), where the resistance of the nanopore, and thus the rate of discharge of the integrating capacitor, depends on the particular type of tag being forced into the nanopore's barrel tube. Due to the fact that in C_{Double Layer}The accumulated charge at 424, data point 524 may decrease slightly for each measurement, as mentioned below.

During the dark period 530, the voltage signal 510 (V) applied to the counter electrode_LIQ) Higher than the voltage (V) applied to the working electrode_PRE) So that any labels will be pushed out of the nanopore barrel. When switch 401 is open, the voltage at the measurement node increases because of voltage signal 510 (V)_LIQ) At a voltage level higher than V_PRE. After the voltage data point is captured (e.g., after a specified time period), switch 401 may be closed and the voltage at the measurement node will again decrease back to V_PREThus, multiple data points may be captured during the dark period, including th point increment 532 and subsequent data points 534. As described above, during the dark period, any nucleotide tag is pushed out of the nanopore, and thus minimal information related to any nucleotide tag is obtained except for use in binning .

Fig. 5 also shows the voltage signal 510 (V) applied to the counter electrode during the bright period 540, although_LIQ) Is lower than being applied to the workVoltage (V) of the electrode_PRE) And no pass event occurs (open channel) — thus, the resistance of the nanopore is low and the discharge rate of the integrating capacitor is high as a result, the captured data points (including the data point 542 and the subsequent data point 544) show low voltage levels.

It may be expected that the measured voltage during the light or dark period is about the same for each measurement of the constant resistance of the nanopore (e.g., made during the light mode of a given AC cycle when markers are in the nanopore), but when the charge is on the double layer capacitor 424 (C)_{Double Layer}) This may not be the case, as the charge buildup may cause the time constant of the nanopore cell to become longer, as a result, the voltage level may shift such that the measured value decreases for each data point in the cycle, thus, the data point may change slightly from one data point to another data point within the cycle, as shown in FIG. 5.

Determination of base

After return , embodiments may determine voltage clusters for the channels traversed, where each cluster corresponds to a different label species, and thus to a different nucleotide.

Additional details regarding Sequencing operations can be found, for example, in U.S. patent publication No. 2016/0178577 entitled "Nano-Based Sequencing With Varying Voltage Stimum", U.S. patent publication No. 2016/0178554 entitled "Nano-Based Sequencing With Varying Voltage Stimum", U.S. patent application No. 15/085,700 entitled "Non-structural Bilayer Monitoring Using measuring of Bilayer Response To electric Stimum", and U.S. patent application No. 15/085,713 entitled "Electrical engineering of Bilayer Format".

Nanopore cell array

Each cell may have dedicated circuits (e.g., integrating capacitors), but may also share circuits, such as ADCs, signal sources, electrodes, or control circuits.

FIG. 6 is a cross-sectional view of an array 600 of nanopore cells in an example nanopore sensor chip, such as an array of nanopore cells 150 in nanopore sensor chip 140, viewed along line A-A shown in FIG. 1 FIG. 6 shows a plurality of nanopore cells in a row or column of the array 600 of nanopore cells FIG. 2, each nanopore cell includes circuitry 622 integrated on a silicon substrate 630 and/or a dielectric layer 601 of the nanopore sensor chip, each nanopore cell includes a respective well 605 formed by dielectric layers 601 and 604 and a working electrode 602 at the bottom of the well 605, as described above with respect to FIG. 2. the well 605 may hold a volume of electrolyte 606. a lipid bilayer 614 may be formed on the dielectric layer 604 and cover each well 605. the lipid bilayer 614 includes a nanopore 616 at the top of each well 605. a sample chamber 615 on top of the lipid bilayer 614 may be configured to hold a bulk electrolyte 608 that may include molecules to be analyzed and nucleotides labeled with a polymer, or primers as described above may include a nanopore 616 between each other primer, 615, a sample chamber 615 may be configured to hold a plurality of molecules in a sample chamber such as a group of nanopores 636, such as a nanopore group 636, a nanopore group of cells, similar to be characterized by the interaction of the sample cells, such as a nanopore group of cells, such as a nanopore group 636, similar to be received on a substrate, e.g. a substrate, similar to be characterized by a substrate, such as a substrate, similar to a substrate, such as a substrate.

Counter electrodes 610 from different nanopore cells may be disposed in sample chamber 615, and may be connected to voltage source 628 for coupling a common V_LIQThe counter electrodes 610 for different nanopore cells may be physically connected to each other to form a common electrode the working electrodes 602 of different nanopore cells may be connected to a common voltage source, or may be independently connected to different voltage sources in embodiments, the circuits 622 of different nanopore cells may be connected to a bus 660, and the voltage levels on the integrating capacitors of different nanopore cells may be sequentially read out through the bus 660 by sequentially selecting different nanopore cells, as described in detail below.

Fig. 7 is a top view of an example nanopore cell array 700 including a two-dimensional array of nanopore cells 708, the nanopore cell array 700 may include thousands or even millions of nanopore cells, for example, in embodiments, the nanopore cell array 700 may include 512 × 512 nanopore cells arranged in 512 rows and 512 columns, in embodiments, the nanopore cell array 700 may be grouped into different banks (banks) 706, where each bank may include a subset of nanopore cells in the nanopore cell array 700, in embodiments, nanopore cells in each column of the nanopore cell array 700 may be grouped together, and voltage levels at the integrating capacitors of nanopore cells in each column may be sampled and converted by the ADC 712.

Row driver and precharge circuit 718 may be used to selectively precharge nanopore cells in one or more rows (e.g., by closing switch 401 of fig. 4 to connect nanopore cells in one or more rows to V using row select line (or word line) 714_PRE) The row driver and precharge circuitry 718 may also be used to sequentially select each rows using a row select line (i.e., word line) 714 the integration capacitors of the nanopore cells on a selected row may be connected to a corresponding column line 716 (e.g., by integrating the capacitors 408 (n) on-chip_cap) And ADC 410, or if on-chip integrating capacitor 408 (n) is not used (not shown)_cap) Then the device 406 is communicated). FromThe voltage signals of the nanopore cells on the selected row may be optionally processed (e.g., sensed and amplified) by a corresponding column amplifier 720 and converted to a digital output by a corresponding ADC 712 in embodiments, the same column amplifier and ADC may serve multiple columns.

Fig. 8 is a schematic diagram of an example nanopore cell array 800, the example nanopore cell array 800 including a two-dimensional array of nanopore cells 802. The nanopore cell array 800 may include all nanopore cells of the nanopore sensor chip, or may include only a subset (e.g., a reservoir) of nanopore cells of the nanopore sensor chip. The working electrode of each nanopore cell 802 may be connected to a voltage source (e.g., V of fig. 4)_PRE) (not shown) and the counter electrode of each nanopore cell 802 may be connected to a common signal V_LIQ Nanopore cell array 800 includes a plurality of column lines 820, each column line 820 coupled to a nanopore cell 802 in the same column and to an ADC 840. nanopore cell array 800 includes M rows of nanopore cells 802, where each of the M rows is selectable by a row select line 810-0 to 810-M-1.

During the sequencing process, the integration capacitor of each nanopore cell 802 may first be driven by a voltage source V applied to the working electrode (e.g., via switch 401 as shown in fig. 4)_PREPrecharged and common signal V_LIQAfter the integration capacitors on the nanopore cells 802 have been charged/discharged, each of the M rows may be sequentially selected to connect the integration capacitors of the nanopore cells on the row to the corresponding column line and the corresponding ADC.

In this manner, sets of data samples may be captured from the nanopore cells in a column of the nanopore cell array after each precharge and charge/discharge operation when multiple precharge and charge/discharge operations are performed in the light and dark periods of the AC cycle, multiple sets of data samples may be captured, where each set of data samples includes data samples from each of the nanopore cells in the column.

Fig. 9 illustrates an example data sample captured from a nanopore cell in an array of nanopore cells, e.g., a column such as nanopore cell array 800, during an AC cycle. In fig. 9, the horizontal axis represents time during the sequencing process. FIG. 9 shows that at time T of the bright period of the AC cycle_BDuring this time, a total of K samples can be captured from each nanopore cell by the ADC serving the column.

As described above with respect to FIG. 8, a column of an array of nanopore cells may include M nanopore cells, each on a different row.the ADC may capture data samples for the nanopore cells in row 0, data samples … … for the nanopore cells in row 1, and data samples for the nanopore cells in row M-1_PREPrecharge the nanopore cells in a column and pass a common signal V_LIQCharging/discharging is performed. Thereafter, a second data sample may be captured from each of the M nanopore cells in the column, either sequentially or in a pipeline. The sequencing process may be repeated to capture K samples from each of the M nanopore cells in the column during the bright period. As a result, it is possible to be at the time T of the bright period_BDuring which the total number of M x K samples are captured from the M nanopore cells in each column. Thus, the sampling rate of the ADC may be at least M K/T_BSo as to be at time T of the bright period_BDuring which M × K samples are captured. Data samples may be captured in a similar manner during the dark period.

Phased nanopore cell array

As discussed above, there is an upper limit on the possible sampling rate due to, for example, limited sampling and conversion speed of the analog-to-digital converter and/or limited bandwidth of the bus, data storage device, or data processing circuitAll nanopore cells of (2) are subject to a common V_LIQIn signal control, nearly all of the useful data is captured during the common light period, while little or no useful data may be captured during the dark period, therefore, a significant portion of the bandwidth of the data sampling and conversion circuitry may not be utilized to capture useful data, at least during the dark period.

The techniques disclosed herein address the above issues and increase the effective sampling rate per cell of the sampling and conversion circuit by applying AC signals having different phases to different nanopore cells in columns As a result, when nanopore cells are in the dark period, other nanopore cells are in the bright period and are sampled by a shared sampling and conversion circuit, for example, in embodiments, nanopore cells in columns may be organized into two or more groups_LIQAn AC signal V applied to the counter electrodes of all nanopore cells and to the working electrodes of the nanopore cells in each group of nanopore cells_PREIn this manner, at any given time, the data sampling and conversion circuitry may sample and convert output voltage signals from a portion of the columns of nanopore cells in a light period, with the dark period being minimally sampled, e.g., for return purposes only.

A.Framework

FIG. 10 is a schematic diagram of an example nanopore cell array 1000 including a two-dimensional array of nanopore cells 1002 in accordance with certain aspects of the present disclosure similar to nanopore cell array 800 of FIG. 8, nanopore cell array 1000 may include all nanopore cells of a nanopore sensor chip, or may include only a subset of nanopore cells of a nanopore sensor chip, nanopore cell array 1000 includes a plurality of column lines 1020, each column line 1020 coupled to nanopore cells 1002 in the same column and to ADC 1040, nanopore cell array 1000 includes M rows of nanopore cells 1002, where each of the M rows may be selected by a row select line, nanopore cells 1002 in nanopore cell array 1000 may be organized into N groups, where N may be any number between 2 and the total number of nanopore cells in the column, in embodiments, nanopore cell array 1000 may be organized such that each of the N groups may include nanopore cells in M/N rows, 633 in other embodiments, with each of nanopore cells 1002 may be organized in N groups, such that each of the N groups may include nanopore cells in M/N rows, and, in a further embodiment, nanohole cell arrays 1000 may be organized in pairs of different groups, such as pairs of nanopore cells 633, 9.

The counter electrode of each nanopore cell 1002 in the nanopore cell array 1000 may be connected to a common signal V_LIQ(not shown), the common signal V_LIQMay be a constant voltage level. The working electrodes of the nanopore cells 1002 in each of the N groups may be connected to a common signal V_PRE(1030) Wherein the signals V for the N groups_PRE(i.e., V)_PRE1、V _PRE2、……、V_PREN) may be applied independently to the N groups and may be in different phases from each other. For example, signals V for N groups_PREMay come from the same signal source but may be delayed differently by a delay line or gate. The delay allows sampling primarily for bright periods, as illustrated below in fig. 11. In this way, more groups of nanopore cells may be utilized to receive different V' s_PREThe signals enable a higher granularity of control. Because by applying the same V_PREThe signal being applied to The nanopore cells in a group are electrically grouped, so that the grouping may be expandable and dynamically configurable when the working electrodes of the nanopore cells are independently addressable at the subgroup level or the cell level.

In implementations, each nanopore cell may include a switch, the switches may be connected to the switch 401 of FIG. 4, e.g., upstream of the switch 401 or in parallel with the switch 401, but with coordinated control signals_PRE405) For example, a switch may be controlled by an AC control signal, such as a square or rectangular wave signal, such that during a portion of a cycle of the AC control signal, the working electrode may be connected to a high voltage level and may be connected to a low voltage level during another portion of the cycle_LIQAnd the low voltage level may be lower than the common signal V_LIQ. As such, AC V may be_PREA signal is effectively applied to the nanopore cell. By applying different digital AC control signals (e.g., with different phase delays) to the nanopore cell, different Vs may be applied_PRE some of the nanopore cells may receive digital AC control signals having the same phase to form groups of N groups of nanopore cells in this manner, groups may include or more nanopore cells, and the grouping of nanopore cells may be more flexible and dynamic, for example, the grouping may be dynamically changed by changing the digital AC control signals applied to the nanopore cells, and nanopore cells that are not near the same may form groups .

In implementations, the nanopore sensorFor example, nanopore cells in different fluidic channels may be assigned to different groups, in implementations, nanopore cells in two or more fluidic channels may be grouped at _LIQTo drive the counter electrodes of the nanopore cells in different groups. For example, the AC signals used to drive the counter electrodes of nanopore cells in different groups may have different phases or delays. Due to V_LIQDifferent phases of the signal, nanopore cells in different groups may be in bright periods at different times, and thus the output of nanopore cells in different groups may be sampled at different times by the shared sampling circuit.

B.AC signal of different cell groups with different phases

Fig. 11 illustrates an example AC signal (V) for a nanopore cell array, such as nanopore cell array 1000, in accordance with certain aspects of the present disclosure_PRE) Nanopore cells in nanopore cell array 1000 having M rows may be organized into N groups fig. 11 includes a plurality of graphs, each graph showing an AC signal applied to groups of cells in the N groups and corresponding sample points fig. 11 the horizontal axis represents time during the sequencing process.

As shown in fig. 11, the counter electrode of each nanopore cell in the nanopore cell array 1000 may be connected to a common signal V_LIQThe common signal V_LIQMay be a constant voltage level. The working electrodes of the nanopore cells in each of the N groups may be connected to a signal V_PREWherein the signals V for the N groups_PRE(i.e., V)_PRE1、V _PRE2、……、V_PREN) may be in different phases from each other.

In embodiments, signals V for N groups_PREMay each be incrementally delayed by about with respect to the other groupsA period equal to the bright period. In the example shown in FIG. 11, N numbers of V_PREThe signal is a rectangular AC signal that switches between a high voltage level and a low voltage level. V_PREThe signals are delayed from each other such that when the nanopore cells in the groups are in the bright period (e.g., when V is_LIQBelow V_PRETime), the nanopore cells in the other groups are in a dark period.

For example, for nanopore cells in group 1, during the bright period, signal V _PRE1 may be at or above constant V_LIQHigh voltage level of and is subjected to V_PREThe integrating capacitor in each nanopore cell in group 1 controlled by 1 may be precharged first to V _PRE1, high voltage level. The integrating capacitor may then be coupled to V _PRE1 is disconnected and is fed by a low level signal V through the nanopore_LIQAfter the integration capacitor has been discharged for a selected period of time, the voltage level of the integration capacitor may be measured by a sampling and conversion circuit (e.g., ADC). during the bright period, or more samples may be captured from the nanopore cell in this manner.

When receiving V _PRE1 control of the width T of the nanopore cells in group 1 in the bright period_BIn the middle, the signal V is received_PRE2 to V_PREÑ the nanopore cells in groups 2-N, respectively, may be in a dark period. During this time period T_BDuring this time, ADCs may serve M/N instead of M nanopore cells in column_BDuring which K samples are captured for each nanopore cell, a sampling rate of (M K/T) may be used_B) An ADC of/N. In other words, the sampling rate is M K/T_BMay be able to operate for a time period T_BDuring which K × N (instead of K) samples are captured for each nanopore cell. Thus, each nanopore cell may be sampled at a faster rate (e.g., N times it), and may therefore be detected with a shorter holdA time-lapse event.

At signal V_PREAfter switching 1 to a low voltage level, the nanopore cells in group 1 may enter a dark period. During the dark period, the signal V _PRE1 may be at a voltage that may be below constant V_LIQIn various embodiments, for example, no data samples may be captured during the dark period, or or more data samples at the end (or beginning) of the dark period may be captured in each AC cycle for normalization purposesnOne dark period, such as every 8 dark periods. To capture data samples in the dark period, exposure to V may first be performed_PRE1-controlled integrating capacitor in each nanopore cell in group 1 precharged to V _PRE1, low voltage level. The integrating capacitor may then be coupled to V _PRE1 is disconnected and is signaled by a signal V via a nanopore_LIQAnd (6) charging. As mentioned above, the rate of charge depends on the resistance of the nanopore.

At signal V_PRESignal V after 1 switches to a low voltage level _PRE2 can be switched from a low voltage level to a high voltage level such that the signal V is generated_PREThe nanopore cells in group 2 controlled by 2 may enter a light period, and data samples from the nanopore cells in group 2 may be captured by the shared sampling and conversion circuitry as described above, multiple data samples may be captured for each nanopore cell in group 2 during the light period, and or more data samples from each nanopore cell in group 2 may be captured at the end of the dark period during or more AC cycles for purposes.

The nanopore cells in each of groups 3 through N may enter the light period sequentially, and multiple data samples from each of the nanopore cells in each group may be captured by the shared sampling and conversion circuitry in the manner described above after the nanopore cells from group N enter the dark period from the light period, the nanopore cells from group 1 may again enter the light period in a new AC cycle for sequencing the data samples.

Fig. 12 illustrates an example data sample captured from a nanopore cell in a column of a nanopore cell array, such as nanopore cell array 1000, in accordance with certain aspects of the present disclosure. The horizontal axis in fig. 12 represents time during the sequencing process. The grey boxes in the figure indicate row selection events. A column of the nanopore cell array includes M nanopore cells, each on a different row. The M nanopore cells are organized in N distinct groups, with M/N nanopore cells in each group. When receiving V_PREThe nanopore cells in group 1 of 1 (nanopore cells in rows 0 to M/N-1) are in the bright period T_BTime, signal V _PRE2 to V_PREThe nanopore cells in the controlled groups 2-N of N may be in a dark period, respectively. Therefore, in the bright period T_BDuring this time, the output from only nanopore cells in rows 0 to M/N-1 can be captured by the ADC serving the column.

Each of the nanopore cells in group 1 may be precharged to signal V _PRE1, and is signaled by a nanopore V_LIQAfter a selected discharge time, the ADC may capture data samples for nanopore cells in row 0, data samples for nanopore cells in row 1, … …, and data samples for nanopore cells in row M/N-1 after samplings of each cell in a column belonging to group 1, each of the nanopore cells in group 1 may again be precharged to signal V _PRE1, and passing signal V through the nanopore_LIQAnd (4) discharging. After the selected discharge time, the ADC may capture a second data sample for the nanopore cell in row 0, a second data sample for the nanopore cell in row 1, … …, and a second data sample for the nanopore cell in row M/N-1. During the bright period, the sequencing process may be repeated to capture multiple samples from each of the M/N nanopore cells in group 1. Thus, for a sampling rate of M K/T_BAnd the ADC shown in FIG. 9In the bright period T_BDuring which K samples are captured from each of the nanopore cells, for a bright period T_BDuring this time, a total number N × K samples may be captured from each of the M/N cells in group 1. Thus, each nanopore cell in nanopore cell array 1000 may be measured N times faster than a nanopore cell in nanopore cell array 800, without using faster sampling and conversion circuitry, and thus events of shorter duration may be detected.

Similarly, when the nanopore cells in group 2 (or any of groups 3 through N) are in a bright period and the nanopore cells in the other groups are in a dark period, in a bright period T_BA total number N x K samples may be captured from each of the M/N cells in group 2 (or any of groups 3 through N) during.

In this manner, the data sampling and conversion circuitry can sample and convert the output signal from each nanopore cell in the section of columns at a higher sampling rate by servicing only the section of nanopore cells at a given time, even though the overall speed of the data sampling and conversion circuitry has not changed.

Additionally or alternatively, the AC control signal (e.g., V) may be increased with or without increasing the number of samples captured during the bright period_PRE) In addition, because the effective sampling rate is higher for each nanopore cell having a shorter AC cycle, events having shorter durations may be detected.

FIGS. 11 and 12 illustrate different V's in different groups for nanopore cells_PREEmbodiments in which there may be no overlap between the bright periods of the signals. Such an embodiment may occur when the bright period of each AC control signal is shorter than the period of the AC cycle divided by the number of groups N, i.e. the duty cycle of the AC control signal is not greater than 1/N. For example, whenThe nanopore cells in the nanopore cell array are organized into two groups, and the duty cycle of the AC control signal is no greater than 50%, i.e., the bright period is equal to or shorter than the dark period, when V for the two groups of nanopore cells_PRENo overlap between the bright periods of the signals may occur. Although in FIGS. 11 and 12V is used for different groups_PREThe signals are shown to have somewhat similar properties (e.g., the same voltage level, duty cycle, and cycle time) and may be derived from the same signal source with different delays, but because the working electrode of each nanopore cell may be independent of the working electrodes of other nanopore cells, V for different groups_PREThe signals may be independent of each other. Thus, V_PREThe signals may have different voltage levels, duty cycles, cycle times and phases.

As described above, in implementations, each nanopore cell may include a switch that may be controlled by an AC control signal to switch the working electrode (and V) of the nanopore cell_PRE) Alternately to a high voltage level and a low voltage level. The high voltage level may be higher than the common signal V_LIQAnd the low voltage level may be lower than the common signal V_LIQ. Therefore, by using digital AC control signals having different phases for the switches, V having different phases can be applied_PREA signal is effectively applied to the working electrodes of different groups of nanopore cells or different individual nanopore cells.

As described above, in implementations, nanopore cells in a nanopore sensor chip may be grouped based on the fluidic channel in which they are located, and different AC signals V may be used_LIQTo drive the counter electrodes of nanopore cells in different groups, rather than using different AC signals V for each group as described with respect to fig. 11_PRE. In such an implementation, V is configured similarly to in FIG. 11_PREBy way of signals, V for different groups may be delayed differently_LIQA signal. The outputs of nanopore cells in different groups at different times through a shared sampling circuit (e.g., ADC)Sampling is performed in a manner similar to the manner in which nanopore cells are sampled as described with respect to fig. 12.

C.Adaptive and selective sampling

In implementations, V may be configured_PRESignal such that different V's are applied to different groups of nanopore cells_PREThere may be overlapping periods between the bright periods of the signal. In such implementations, the data sampling and conversion circuit or control circuit may be configured to determine which group of nanopore cells to sample and convert an output voltage signal from during each overlapping period in a different AC cycle.

Fig. 13 illustrates an example control signal (V) for a nanopore cell array, such as nanopore cell array 1000, in accordance with certain aspects of the present disclosure_PRE). The horizontal axis in fig. 13 represents time during the sequencing process. As shown in fig. 13, the counter electrode of each nanopore cell in the nanopore cell array 1000 may be connected to a common signal V_LIQThe common signal V_LIQMay be a constant voltage level. The working electrodes of the nanopore cells in each of the N groups may be connected to a signal V_PREWith signals V for N groups_PRE(i.e., V)_PRE1、V _PRE2、……，V_PREN) are in different phases from each other. For example, in the signal V_PREIs greater than 1/N, for N groups of signals V_PRE may each be incrementally delayed relative to the others by a period of time shorter than the bright period_PREThere may be overlapping periods between the bright periods of the signal. The advantage of this implementation is that a duty cycle with a bright period higher than 50% can be used, or the cells can be divided into more than two groups and can be controlled by more than two signals with different phases.

During non-overlapping bright periods, each nanopore cell in a group of the array of nanopore cells 1000 in a bright period may be sampled at a higher rate (as described above with respect to fig. 11 and 12) than the nanopore cell shown in fig. 8 in some cases, during overlapping periods, the data sampling and conversion circuit or control circuit may dynamically determine the group(s) of nanopore cells to sample while ignoring output signal(s) from other group(s) of nanopore cells in overlapping bright periods, in cases, during overlapping intervals, the sampling rate of nanopore cells in different groups in a bright period may be reduced since nanopore cells from more than groups are in a bright period, for example, in cases, the sampling rate of nanopore cells in different groups in a bright period may be at an equal but reduced rate relative to the sampling rate when only groups are in a bright period.

D.Advantages of the invention

The techniques described in this disclosure enable nanopore cells to be controlled at higher granularity, such as at an individual cell level or at a group level, rather than applying a common control signal to all nanopore cells. As such, the number of cells in the bright period may be more constant over time, and the sampling and conversion rate for each cell in the bright period may be increased without changing the overall speed of the data sampling and conversion circuitry, as the available resources are more efficiently utilized at any given time.

In addition to increasing the effective sampling rate for each nanopore cell as described above to detect events with short durations and reduce sequencing time, other advantages may be achieved using the techniques disclosed herein. For example, the bandwidth of the data sampling and conversion circuitry may be leveraged at any given time to capture data samples useful for sequencing. Thus, nanopore sensor chips with higher densities or higher numbers of cells may become possible. Additionally or alternatively, with reduced sampling data captured from cells in the dark period, the amount of data to be transmitted from the nanopore sensor chip and processed by subsequent storage or processing circuitry may be reduced, which may reduce the cost of the sequencing system, as circuitry with lower performance (e.g., speed or bandwidth) or capacity (e.g., storage space or data channel) may be used.

Furthermore, because some nanopore cells are in the dark period during the same time, while other nanopore cells are in the light period, and because of a common V_LIQIs applied to (shared by) the counter electrode of all cells, so the total current on the counter electrode can be at least partially reduced at any given time. This is due to the opposite polarity of the current from the cell in the dark period to the current from the cell in the bright period, where the flow of electrons into and out of the counter electrode reduces the net current on the counter electrode. Furthermore, because of V_PREThe AC nature of the signal, the currents on the counter and working electrodes for each cell may also be balanced over time.

In implementations, V applied to the counter electrode_LIQThe signal may also be an AC signal, rather than a constant voltage level, but at a frequency below V applied to the working electrode_PREA signal. In this way, even if there is any imbalance between the current from the cell in the dark period and the current from the cell in the bright period at a given time (e.g., because the duty cycle is different than 50%, and the number of groups of nanopore cells in the dark period may be different than the number of groups of nanopore cells in the bright period), the total current on the counter electrode may be balanced over time. In this way, the voltage drop across the counter electrode and hence the offset of the output voltage can be reduced.

Input range control

In many cases, the integration capacitor to be measured (e.g., integration capacitor 408 (n)_cap) ) may be aggregated over some smaller range. Thus, if the input range of the ADC is fixed, the dynamic range of the ADC may not be fully utilized. To fully utilize the dynamic range of the ADC, the output of the ADC can be dynamically changed by adaptively changing the reference level of the ADC based on the estimated voltage level to be measuredAnd (4) entering the range.

In an ADC, the full-scale input range and common-mode voltage of the ADC may depend on a reference level of the ADC, such as a positive reference level (V)_REFP) And a negative reference level (V)_REFN). The common mode voltage may be V_REFPAnd V_REFNAverage value of (a). When the signal to be sampled by the ADC is at approximately V_REFNAt a voltage level of (d), the input of the ADC may be close to the zero scale, that is, the output of the ADC may represent a value close to the lowest possible value; the exact output code may depend on the coding scheme of the ADC, which may include, for example, direct binary, offset binary, two's complement, etc.

When the signal to be sampled by the ADC is at approximately V_REFPThe input of the ADC may be near full scale and the output of the ADC may represent a value near the maximum possible value. In order to fully utilize the dynamic range of the ADC (i.e., the range of signal amplitudes that the ADC can resolve), it is desirable that the input signal level be at V_REFPAnd V_REFNMore fully distributed than aggregated within some smaller range. For an 8-bit ADC, if the input signal level is between 0V and 1V, then V_REFPCan be set to 1V, and V_REFNCan be set to 0V; and the ADC may be capable of distinguishing between two signal levels that differ by more than about 4 mV. If the input signal level is between 0.25V and 0.5V, V can be set to be_REFPSet to 0.5V, and V may be set_REFNSet to 0.25V; and the ADC may be capable of distinguishing between two signal levels that differ by more than about 1 mV.

However, as shown in fig. 11 or 13, in some cases, the voltage level of the integration capacitor to be sampled by the ADC may not be distributed at V_PREThe full range between the low voltage level and the high voltage level of the signal. Thus, at a fixed reference level (V) of the ADC_REFPAnd V_REFN) Next, the dynamic range of the ADC may not be fully utilized, since the output code may only include portions of all possible codes (e.g., 256 different codes for an 8-bit ADC), or in other words, the signal level may not be resolved at the maximum possible resolution of the ADC.

Fig. 14A shows a fixed reference level for an ADC in a nanopore sensor chip. As shown in FIG. 14A, the voltage level 1430 of the integrating capacitor to be sampled by the ADC may be close to V_PRE Low voltage level 1410 or V _PRE1420. Thus, to include all of these voltage levels in the input range of the ADC, V for the ADC may be included_REFPIs set slightly higher than V_PRETo avoid saturation (e.g., caused by overshoot) and/or distortion near the full scale input. V of ADC_REFNCan be set slightly below V_PRE Low voltage level 1410. However, the voltage level of the integrating capacitor does not fall in the middle portion of the input range (shown as patterned region 1440). Thus, the mid-range of the ADC full-scale input range may not be used at all, and the dynamic range of the ADC may not be fully utilized. To fully utilize the dynamic range of the ADC, the reference level of the ADC may be dynamically changed so that the input range of the ADC does not include the patterned region 1440.

Fig. 14B illustrates a variable reference level of an ADC in a nanopore sensor chip. The reference level comprises a reference level V_REFPAnd V_REFNThey determine the full scale input range and the common mode voltage of the ADC. Reference level V_REFPAnd V_REFNCan follow V_PREThe signal changes which brings the nanopore cell into light and dark periods. Thus, the full scale input range and/or common mode of the ADC may be different between the light and dark periods. In this way, the output voltage signal of the nanopore cell may better fit within the full-scale input range of the ADC, rather than being distributed only within some narrow range of the full-scale input range of the ADC.

For example, as shown in FIG. 14B, to capture a data sample 1430 in the dark period, V can be set_REFPIs set below V_LIQLevel 1450 of. Can be combined with V_REFNIs set below V_PRESuch that the input range of the ADC (shown as the shaded area) includes only below V_LIQAnd a region between 1450 and 1460, but not including at level 1450 toThe region above, wherein none of the voltage levels of the integration capacitors fall within the region. To capture data samples in the bright period, V may be set_REFPIs set higher than V_PREAnd V may be V, and a level 1470 of the high voltage level 1475_REFNIs set higher than V_LIQLevel 1480. Thus, the input range of the ADC (shown as the shaded region) may only include above V_LIQAnd a region between levels 1470 and 1480, but not a region below 1480 into which none of the voltage levels of the integration capacitors fall. When no data sample is captured from a nanopore cell in a group and a data sample is captured from a nanopore cell in another group that is in a bright period, the same reference setting may be used during the dark period 1490 of the group.

In this way, the input range (and common mode input) of the ADC can be dynamically changed to include only the range within which the voltage level of the integrating capacitor may fall. Thus, the voltage level within the input range may be determined to have the maximum possible resolution of the ADC.

Example method to increase sampling rate per cell

FIG. 15 is a flow chart 1500 illustrating an example method of nucleic acid sequencing using a sensor comprising sets of cells in accordance with certain aspects of the present disclosure the method illustrated by flow chart 1500 may apply AC signals having different phases to different groups of nanopore cells in a nanopore sensor chip. As a result, when of the nanopore cells are in a dark period, of the other nanopore cells are in a light period and are sampled by a shared sampling and conversion circuit.

In block 1510, an th circuit, such as circuit 622 of FIG. 6, may apply th AC signals to the th group of cells of the sets of cells in the nanopore sensor chip As described above with respect to FIG. 10, the sets of cells in the nanopore sensor chip may be organized into a plurality of groups, where each group may be independent of the other groups and have corresponding circuitry for applying AC signals to the th group of nanopore cells the AC signals may be rectangular waves and may have a selected duty cycle.

In block 1520, the second circuit may provide a second AC signal to a second group of cells in the set of cells in embodiments, the AC signal and the second AC signal may have different phases and may be derived from the same signal source with different delays in embodiments, the AC signal and the second AC signal may also be different in at least of amplitude, duty cycle, or frequency.

In block 1530, during part of the th AC signal, the group of cells may be in a light period and the analog-to-digital converter may sample output signals from the group of cells during the same time period, the second group of cells may be in a dark period due to a phase difference between the th AC signal and the second AC signal and output signals from the second group of cells may not be sampled by the analog-to-digital converter signals, therefore, during part of the th AC signal, the analog-to-digital converter may serve only the group of cells.

In block 1540, during the second portion of the AC signal, the group of cells may be in a dark period and the analog-to-digital converter may not sample the output signals from the group of cells during the same time period, the second group of cells may be in a bright period and the output signals from the second group of cells may be sampled by the analog-to-digital converter.

Note that although FIG. 15 describes data processing as a sequential process, many of the operations can be performed in parallel or concurrently.

VII. computer System

Any of the computer systems mentioned herein may utilize any suitable number of subsystems an example of such subsystems is shown in FIG. 16 in computer system 10. in embodiments, a computer system includes a single computer device, where the subsystems may be components of the computer device.

The subsystems shown in fig. 16 are interconnected via a system bus 75. Additional subsystems, such as printer 74, keyboard 78, storage device(s) 79, monitor 76 coupled to display adapter 82, and sequencing are shown. Peripherals and input/output (I/O) devices, which are coupled to the I/O controller 71, can be connected to the computer system by any number of means known in the art, such as an input/output (I/O) port 77 (e.g., USB, FireWire)^®) For example, I/O port 77 or external interface 81 (e.g., Ethernet, Wi-Fi, etc.) may be used to connect computer system 10 to domain networks, such as the Internet, a mouse input device, or a scannerThe interconnection via system bus 75 allows a central processor 73 to communicate with each subsystem and control the execution of a plurality of instructions from system memory 72 or storage device(s) 79 (e.g., a fixed disk, such as a hard drive or optical disk), and the exchange of information between subsystems the system memory 72 and/or storage device(s) 79 may embody a computer readable medium.

A computer system may include multiple identical components or subsystems that are connected together, for example, through external interfaces 81, through internal interfaces, or via removable storage devices that may be connected and removed from components to components in some embodiments computers may communicate over a network in some instances computers may be considered clients and computers are servers, where each may be part of the same computer system.