Abstract: Techniques for replacing nanopores within a nanopore based sequencing chip are provided. A first electrolyte solution is added to the external reservoir of the sequencing chip, introducing an osmotic imbalance between the reservoir and the well chamber located on the opposite side of a lipid bilayer membrane. The osmotic imbalance causes the membrane to change shape, and a nanopore within the membrane to be ejected. A second electrolyte solution is then added to the external reservoir to provide replacement nanopores and to restore the membrane shape. The replacement nanopores can be inserted into the membrane, effectively replacing the initial pore without causing the destruction of the membrane.

Abstract: A method of analyzing a molecule is disclosed. A voltage source is selectively connected to or disconnected from a capacitor using a switch controlled by a reset signal. A charge is stored in a capacitor when the voltage source is connected to the capacitor. The capacitor is discharged through a nanopore in a membrane when the voltage source is disconnected from the capacitor. A duty cycle of the reset signal is determined such that the voltage source and the capacitor is connected for at least a one tenth portion of a reset signal period and disconnected for a remaining portion of the reset signal period, such that a voltage across the nanopore is maintained at a higher level during the portion of the reset signal period in which the connection is maintained than during the remaining portion of the reset signal period in which the connection is not maintained.

Abstract: A device for controlling, detecting, and measuring a molecular complex is disclosed. The device comprises a common electrode. The device further comprises a plurality of measurement cells. Each measurement cell includes a cell electrode and an integrator electronically coupled to the cell electrode. The integrator measures the current flowing between the common electrode and the cell electrode. The device further comprises a plurality of analog-to-digital converters, wherein an integrator from the plurality of measurement cells is electrically coupled to one analog-to-digital converter of the plurality of analog-to-digital converters.

Abstract: Described herein are variants of alpha-hemolysin having at least one amino acid substitution at H35G, E111N, M113A, and/or K147N in the mature, wild-type alpha-hemolysin amino acid sequence. In certain examples, the variant may have a substitution at E111S, M113S, T145S, K147S, or L135I in the mature alpha-hemolysin amino acid sequence. The ?-hemolysin variants may also include a substitution at H144A and/or a series of glycine residues spanning residues 127 to 131 of the mature, wild-type alpha hemolysin. Also provided are nanopore assemblies including the alpha-hemolysin variants, the assembly having an increased nanopore lifetime. Further, provided are variants that, in addition to providing increased lifetime, provide a decreased time-to-thread. Hence, the variants provided herein both increase nanopore lifetime and improve efficiency and accuracy of DNA sequencing reactions using nanopores comprising the variants.

Abstract: A nanopore measurement circuit includes a first analog memory configured to store a first electrical value corresponding to a first measurement sample of a nanopore and a second analog memory configured to store a second electrical value corresponding to a second measurement sample of the nanopore. The nanopore measurement circuit also includes a measurement circuitry configured to provide an output indicating a difference between the first electrical value of the first analog memory and the second electrical value of the second analog memory.

Abstract: A method of using a sequencing cell includes applying voltage across the sequencing cell, acquiring one or more signal values from the sequencing cell, and acquiring one or more correlated signal values that are correlated with respective values of the plurality of acquired signal values thereby forming a plurality of two-dimensional data points. The plurality of two-dimensional data points comprise values in a first dimension that equal the plurality of acquired signal value and values in a second dimension that equal the plurality of correlated signal values. The method can further include computing a plurality of transformed signal values by applying a two-dimensional transformation to the plurality of two-dimensional data points.

Abstract: This disclosure provides systems and methods for molecular identification and polymer (e.g., nucleic acid) sequencing using nanopores. The polymer may be passed through or in proximity to the nanopore and various subunits of the polymer may affect the current flowing through the nanopore. The various subunits may be identified by measuring the current at a plurality of voltages applied across the nanopore and/or membrane. In some cases, the polymerization of tagged nucleotides presents tag molecules to the nanopore that can be identified by measuring the current at a plurality of voltages applied across the nanopore and/or membrane. Also provided herein are systems and methods for sequencing both the sense and anti-sense strand of a double stranded nucleic acid molecule with a nanopore and methods for using ribonucleic acid (RNA) speed bump molecules to slow the passage of a nucleic acid molecule through or in proximity to a nanopore.

Abstract: The present disclosure provides biochips and methods for making biochips. A biochip can comprise a nanopore in a membrane (e.g., lipid bilayer) adjacent or in proximity to an electrode. Methods are described for forming the membrane and insert-ing the nanopore into the membrane. The biochips and methods can be used for nucleic acid (e.g., DNA) sequencing. The present disclosure also describes methods for detecting, sorting, and binning molecules (e.g., proteins) using biochips.

Abstract: A method of detecting a state of a lipid membrane in a cell of a nanopore based sequencing chip is disclosed. A lipid membrane is coupled with an integrating capacitor, wherein the lipid membrane is between a working electrode and a counter electrode. An alternating current (AC) voltage is applied to the counter electrode. A voltage across the integrating capacitor is periodically sampled by an analog-to-digital converter (ADC). A change in the sampled voltage across the integrating capacitor in response to an intermediate change in the AC voltage is determined. A state of the lipid membrane is determined based on the determined change in the sampled voltage across the integrating capacitor in response to the intermediate change in the AC voltage.

Abstract: Methods of detecting molecules using an apparatus comprising a plurality of magnetic sensors are disclosed. A method may include binding a first molecule to a proximal wall of a fluid chamber of the apparatus, and adding, to the fluid chamber, a magnetically-labeled molecule comprising a cleavable magnetic label, wherein the magnetically-labeled molecule is configured to bind to or be incorporated by the first molecule. The method may use at least one address line and at least one selector element of the apparatus to detect a characteristic of at least a portion of the plurality of magnetic sensors, wherein the characteristic indicates whether the magnetically-labeled molecule has bound to or been incorporated by the first molecule.

Abstract: Systems and methods for inserting a single pore into a membrane are described herein. A stepped or ramped voltage waveform can be applied across the membranes of the cells of an array, where the voltage waveform starts at first voltage and increases in magnitude over a period of time to a second voltage. The first voltage is selected to be low enough to reduce the risk of damaging the membrane, while the rate of voltage increase is selected to provide sufficient time for the pores to insert into the membranes. Once a pore is inserted into the membrane, the voltage across the membrane rapidly drops, thereby reducing the risk of damaging the membrane even if the applied voltage between the electrodes is further increased.

Abstract: The invention is a novel method of making and using a template for nucleic acid sequencing. The templates include circular and linear templates with symmetric and asymmetric adaptors. The methods include utilizing the templates in an asymmetric fashion.

Abstract: A method of forming a plurality of lipid bilayers over an array of cells in a nanopore based sequencing chip is disclosed. Each of the cells comprises a well. A salt buffer solution is flowed over the array of cells in the nanopore based sequencing chip to substantially fill the wells in the cells with the salt buffer solution. A lipid and solvent mixture is flowed over the array of cells to deposit the lipid and solvent mixture over at least some of the wells in the cells. A first portion of the cells, each having a lipid bilayer over its well, is detected. A second portion of the cells, each having a lipid membrane but not a lipid bilayer over its well, is detected. An electrical lipid-thinning stimulus is selectively applied to the second portion of the cells but not to the first portion of the cells.

Abstract: The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.

Abstract: Techniques described herein can apply AC signals with different phases to different groups of nanopore cells in a nanopore sensor chip. When a first group of nanopore cells is in a dark period and is not sampled or minimally sampled by an analog-to-digital converter (ADC) to capture useful data, a second group of nanopore cells is in a bright period during which output signals from the second group of nanopore cells are sampled by the analog-to-digital converter. The reference level setting of the ADC is dynamically changed based on the applied AC signals to fully utilize the dynamic range of the ADC.

Abstract: Devices for sequencing linear biomolecules (e.g., DNA, RNA, polypeptides, proteins, and the like) using quantum tunneling effects, and methods of making and using such devices, are provided. A nanofabricated device can include a small gap formed by depositing a thin film between two electrodes, and subsequently removing the film using an etching process. The width of the resulting gap can correspond with the size of a linear biomolecule such that when a part of the biomolecule (e.g., a nucleobase or amino acid) is present in the gap, a change in tunneling current, voltage, or impedance can be measured and the part of the biomolecule identified. The gap dimensions can be precisely controlled at the atomic-scale by, for example, atomic layer deposition (ALD) of the sacrificial film. The device can be made using existing integrated circuit fabrication equipment and facilities, and multiple devices can be formed on a single chip.

Abstract: Embodiments of the present technology may allow for the analysis of molecules by tunneling recognition at a tunneling junction. A tunneling junction of the present technology can include an insulating layer between two electrodes. A voltage may be applied to the electrodes. When a molecule makes contact with both electrodes, the molecule allows current to tunnel through the molecule. The characteristics of the current may aid in identifying a portion of the molecule, for example, a particular nucleotide or base present in a nucleic acid molecule. Methods and systems for analysis of molecules are described.

Abstract: Described herein are variants of alpha-hemolysin having at least one mutation selected from T12R, T12K, N17R, N17K or combinations of T12 and N17 mutations. The variants in some embodiments may further comprise H144A. The ?-hemolysin variants have a decreased time to thread.

Abstract: The invention comprises circular single-stranded templates for nucleic acid sequencing, methods of making and using the same.

Abstract: A method of forming a nanopore in a lipid bilayer is disclosed. A nanopore forming solution is deposited over a lipid bilayer. The nanopore forming solution has a concentration level and a corresponding activity level of pore molecules such that nanopores are substantially not formed un-stimulated in the lipid bilayer. Formation of a nanopore in the lipid bilayer is initiated by applying an agitation stimulus level to the lipid bilayer. In some embodiments, the concentration level and the corresponding activity level of pore molecules are at levels such that less than 30 percent of a plurality of available lipid bilayers have nanopores formed un-stimulated therein.