TWI804639B - Apparatus for biopolymer sequencing and sequencing method - Google Patents

Abstract

Disclosed herein is an apparatus, comprising: a plurality of sensors, each of which comprising a nanopore and configured to output an electrical signal depending on an interaction of a chemical compound with the nanopore; a detector configured to receive electrical signals from the sensors through a sacrificial device, wherein the sacrificial device is configured to selectively and irreversibly sever electrical connections between the detector and any of the sensors. Disclosed herein is a method obtaining an apparatus comprising a plurality of sensors, each of which comprising a nanopore and configured to output an electrical signal depending on an interaction of a chemical compound with the nanopore, a detector configured to receive electrical signals from the sensors; determining a quality of performance of each of the sensors; selecting a subset from the sensors based on the qualities of performance; irreversibly severing electrical connections between the detector and the subset.

Description

Apparatus and sequencing method for biopolymer sequencing

The present invention relates to a device suitable for sequencing biopolymers (eg, DNA, RNA, proteins) by sensing electrical signals from the interaction of chemical compounds (eg, units of the biopolymer) with nanopores.

DNA sequencing is the process of determining the sequence of nucleotides, such as adenine (A), guanine (G), cytosine (C), and thymine (T), in a DNA strand. Classical DNA sequencing methods (eg, the Sanger method) are based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerases during in vitro DNA replication. The next-generation sequencing method is an improvement on the basis of the Sanger method to perform massively parallel sequencing, making it faster and cheaper than the Sanger method.

DNA sequencing using nanopores is a third generation method. A nanopore is a structure of small pores with an inner diameter on the order of 1 nanometer. One type of nanopore is a membrane protein complex such as α-hemolysin, MspA (Mycobacterium tuberculosis A) or CsgG. Another type of nanopore is a solid-state nanopore, such as a membrane of silicon nitride and aluminum oxide, which has small pores. When the nanopore is immersed in a conductive fluid and a voltage is applied to it, it can be observed The current generated by the conduction of ions through the nanopore was observed. The magnitude of the current is very sensitive to the size and shape of the nanopore. As a DNA molecule passes through a nanopore, the change in electrical current passing through the nanopore represents a direct readout of the DNA sequence. Other biopolymers such as RNA and proteins can also be sequenced using nanopores.

The invention provides a device including a plurality of sensors and detectors. Each sensor includes a nanopore and is configured to output an electrical signal dependent on the interaction of the chemical compound with the nanopore. The detector is configured to receive an electrical signal from the sensor and through the sacrificial device. The sacrificial device is configured to selectively and irreversibly sever the electrical connection between the detector and the sensor.

In an embodiment of the present invention, the sacrificial device includes a fuse.

In one embodiment of the present invention, the above-mentioned chemical compound is a nucleotide.

In an embodiment of the present invention, the nanopore includes protein.

In an embodiment of the present invention, the aforementioned nanoholes include inorganic materials.

In an embodiment of the present invention, the electrical signal is a current passing through the nanopore.

In an embodiment of the present invention, the nanopores of the aforementioned sensors are arranged in an array.

In an embodiment of the present invention, the above-mentioned device further includes a voltage source configured to apply a voltage to the nanopore.

In an embodiment of the present invention, the above-mentioned interaction is that the nanopore is partially blocked by a chemical compound.

In an embodiment of the invention, when the electrical signal from the sensor is greater than a threshold, the sacrificial device is configured to selectively and irreversibly cut off the electrical connection between the detector and the sensor.

In an embodiment of the invention, said sacrificial means is configured to selectively and irreversibly sever the electrical connection between the detector and the sensor when the electrical signal from that sensor is within a range.

The invention provides a method comprising: obtaining a device comprising a plurality of sensors and detectors configured to receive electrical signals from the sensors, each sensor comprising a nanopore and configured to output a chemically dependent Electrical signal of compound interaction with nanopore; determination of performance qualities of each sensor; selection of subsets from sensors based on performance qualities; irreversible severing of electrical connection between detectors and subsets.

In an embodiment of the present invention, the electrical connection is irreversibly cut off by breaking the fuse.

In an embodiment of the invention, the determination of the performance quality of each sensor is based on electrical signals from the sensors.

In an embodiment of the invention, the subset is composed of sensors whose electrical signal output is higher than a threshold.

In an embodiment of the present invention, the above-mentioned subset consists of sensors whose electrical signal output is within a range.

In one embodiment of the present invention, the above-mentioned chemical compound is a nucleotide.

In an embodiment of the present invention, the nanopore includes protein.

In an embodiment of the present invention, the aforementioned nanoholes include inorganic materials.

In an embodiment of the present invention, the electrical signal is a current passing through the nanopore.

In an embodiment of the present invention, the nanopores of the aforementioned sensors are arranged in an array.

In an embodiment of the present invention, the above-mentioned device further includes a voltage source configured to apply a voltage to the nanopore.

In one embodiment of the present invention, the above-mentioned interaction is that the nanopore is partially blocked by a chemical compound.

100: equipment

110, 501: sensor

102: Chemical compounds

103: Conductive fluid

104: Voltage source

105: nanopore

106: Substrate

108, 109, 109A, 109B: electrodes

125: detector

126: Sacrificial device

127: multiplexer

128, 128A, 128B: Fuse

300: controller

301: Amplifier

320: memory

330: Communication module

502:DNA sample

901, 902, 903, 904: steps

Fig. 1 schematically shows a cross-sectional view of a part of an apparatus according to an embodiment.

Fig. 2A schematically shows that according to an embodiment, the nanopores of the sensors of the device may be arranged in an array.

Figure 2B schematically illustrates an example of a sacrificial means of an apparatus according to an embodiment.

Fig. 3 schematically shows a component diagram of a detector of a device according to an embodiment.

Fig. 4 schematically shows a flowchart of a method for sequencing biopolymers using a device according to an embodiment.

Figure 5 schematically shows an example of use of the device described herein.

FIG. 1 schematically shows a cross-section of part of an apparatus 100 according to an embodiment. face map. The device 100 includes a plurality of sensors 110 . Each of the sensors 110 includes a nanopore 105 which may be arranged in a substrate 106 . The nanopore 105 can be mainly made of organic material (eg, penetrating protein), or inorganic material such as silicon nitride, aluminum oxide or a combination thereof. The sensor 110 can generate an electrical signal that reflects the interaction of the chemical compound with the nanopore 105 in the sensor 110 . For example, the electrical signal is a current passing through the nanopore 105 . Examples of chemical compounds may include nucleotides, nucleosides and amino acids. An example of such an interaction is that when a chemical compound passes through the nanopore 105, the nanopore 105 is partially blocked by the chemical compound. The partial blockage may be transient.

FIG. 1 also schematically illustrates the operation of the device 100 . The sensor 110 is immersed in a conductive fluid 103, such as a saline solution. When a voltage to the nanopore 105 is established (eg, by the voltage source 104, which may be part of the device 100), current flows through the nanopore. The current may be carried by ions in the conductive fluid 103 . In the example shown in FIG. 1 , the voltage is applied between two electrodes 108 and 109 in contact with a conductive fluid 103 and separated by a nanopore 105 . When a chemical compound 102 in a conductive fluid 103 passes through one of the nanopores 105, for example driven by a voltage, the chemical compound 102 can interact with the nanopore and cause a change in the current flow through that nanopore. For example, the chemical compound 102 can partially block the nanopore and cause a decrease in current flow through the nanopore. The characteristics of the change (eg, magnitude, duration, waveform) may depend on characteristics of the chemical compound 102 (eg, size, shape, structure, chemical composition). Accordingly, the chemical compound 102 can be identified based on the changing characteristics.

Device 100 has a detector 125 that receives an electrical signal from sensor 110 through sacrificial device 126 . Detector 125 may have analog circuits, such as filter networks, amplifiers, integrators, and comparators, or digital circuits, such as microprocessors and memory. Detector 125 may include components common to sensors 110 or components specific to each of sensors 110 . For example, detector 125 may include amplifiers dedicated to each of sensors 110 and a microprocessor shared between all sensors 110 .

The sacrificial device 126 is configured to selectively and irreversibly sever the electrical connection between the detector 125 and any of the sensors 110 . If one or more of the sensors 110 from which the detector 125 receives the electrical signal is defective (e.g., generates an electrical signal that is orders of magnitude larger than the electrical signal from the rest of the sensors 110), the sacrificial device 126 may be irreversible The electrical connection between these defective sensors and detector 125 is severed, but the integrity of the electrical connection between the remainder of sensors 110 and detector 125 is preserved.

FIG. 2A schematically shows that nanopores 105 of sensors 110 may be arranged in an array. The device 100 may have at least 100, 10,000, or 1,000,000 sensors 110 .

Figure 2B schematically illustrates an example of a sacrificial device 126 according to an embodiment. The sacrificial device 126 may include a multiplexer 127 . A multiplexer 127 is connected to the detector 125 . The multiplexers 127 are also respectively connected to the sensors 110 through fuses 128 . For example, the fuses 128 may be respectively connected to the electrodes 109 of the sensors 110 . By opening one of the fuses 128, the electrical connection between the detector 125 and the sensor 110 connected by that fuse The connection is selectively and irreversibly severed.

Fig. 3 schematically shows a component diagram of a detector 125 according to an embodiment. The detector 125 includes an amplifier 301 with a feedback loop. Detector 125 may include other circuits such as integrators, noise filters, or feedback control logic. The detector 125 may also include other functional components, such as ADC converters. The detector 125 may include a controller 300 , a memory 320 , and a communication module 330 . Amplifier 301 is configured to receive current from sensor 110 (via its electrodes 109). Amplifier 301 may be configured to monitor the current directly, or to calculate an average value over a period of time. Amplifier 301 can be controllably activated or deactivated by controller 300 . Amplifier 301 may be configured to be continuously active and to continuously monitor current. Amplifier 301 may be of high speed to allow detectors 125 to operate in parallel for large scale sequencing.

Detector 125 is configured to receive an electrical signal from sensor 110 through sacrificial device 126 . When the electrical signal from one of the sensors 110 is greater than a certain threshold or within a certain range, it may indicate that the nanopore 105 in that particular sensor 110 is defective, and the sacrificial device 126 may be selected The electrical connection between the detector 125 and this particular sensor 110 is permanently and irreversibly severed (by opening the fuse 128 therebetween). For example, as shown in FIG. 3 , the electrical connection between one instance 109A of electrode 109 and detector 125 remains intact through one instance 128A of fuse 128; and the electrical connection between one instance 109B of electrode 109 and detector 125 Selectively and irreversibly cut by one instance 128B (open circuit) of fuse 128 .

According to an embodiment, controller 300 may be configured to cause sacrificial device 126 to selectively and irreversibly switch off any of sensors 110 and detectors 125 electrical connection between the fuses 128, for example by disconnecting any of the fuses 128.

The controller 300 can be configured to cause the memory 320 to store the DNA sequencing results

The controller 300 can be configured such that the voltage source provides different voltages to the electrodes 108 and 109 across the nanopore 105, and the provided voltage can then be maintained throughout the current measurement. In some examples, a suitably shaped waveform is provided to electrodes 108 and 109, including a triangular, sinusoidal, sawtooth, or square waveform.

The memory 320 can be random access memory, flash memory, hard disk, with high read/write speed matching the speed of sample sequencing.

The communication module 330 can send and receive signals or data to internal components or external devices.

Fig. 4 schematically shows a flowchart of a method for sequencing biopolymers using the device 100 according to an embodiment. In step 901, the device 100 is obtained. In step 902 , the performance quality of each of the sensors 110 of the device 100 is determined, for example by using the detector 125 and the controller 300 . In one example, an electrical signal (eg, current) output by the sensor 110 is measured and compared with a reference value. In step 903, for example by the controller 300, a subset of sensors 110 is selected based on performance qualities. For example, the subset consists of those sensors 110 whose output electrical signal is greater than a certain threshold or falls within a certain range. In step 904, the electrical connection between detectors 125 and a subset of sensors 110 is selectively and irreversibly severed (eg, by opening fuses 128 therebetween).

Figure 5 schematically shows the device 100 described herein being used in a portable DNA sequencing application. The device 100 can be used to inspect and identify goods in a transportation system, Examples include shipping containers, vehicles, ships, luggage, etc. Device 100 may include one or more detectors. The prepared DNA sample 502 is brought to the surface of the device 100 from an object (eg, shipping container, vehicle, ship, etc.). The sample can be sieved, screened and mixed with chemical reagents in the system. The sample is identified by sequencing the extract containing the useful DNA strands by the sensor 501 .

While the present disclosure discloses various aspects and embodiments, other aspects and embodiments will become apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, the true scope and spirit of which is indicated by the following claims.

100: Equipment

501: sensor

502:DNA sample

Claims (23)

An apparatus for sequencing biopolymers, comprising: a plurality of sensors, each of the plurality of sensors comprising a nanopore and configured to output a an electrical signal; and a detector configured to receive the electrical signal from the plurality of sensors through a sacrificial device, wherein the sacrificial device is configured to selectively and irreversibly switch off the detector and an electrical connection between any of the plurality of sensors. The apparatus of claim 1, wherein the sacrificial device comprises a fuse. The device as described in claim 1 of the patent application, wherein the chemical compound is a nucleotide. The device according to claim 1, wherein the nanopore comprises protein. The device as described in claim 1, wherein the nanopores comprise inorganic materials. The device according to claim 1, wherein the electrical signal is an electric current passing through the nanopore. The device according to claim 1, wherein the nanopores of the sensors are arranged in an array. The device according to claim 1, further comprising a voltage source configured to apply a voltage to the nanopore. The device of claim 1, wherein the interaction is that the nanopore is partially blocked by the chemical compound. The device according to claim 1, wherein when the electrical signal from any one of the plurality of sensors is greater than a threshold, the sacrificial device is configured to selectively and irreversibly cut off all An electrical connection between the detector and any one of the plurality of sensors. The apparatus of claim 1, wherein said sacrificial means is configured to selectively and irreversibly switch off when said electrical signal from any one of said plurality of sensors is within a range An electrical connection between the detector and any one of the plurality of sensors. A method of sequencing comprising: obtaining a device comprising a plurality of sensors and a detector configured to receive electrical signals from the plurality of sensors, each of the plurality of sensors comprising nanopore and configured to output said electrical signal dependent on the interaction of a chemical compound with said nanopore; determining a performance quality of each of said plurality of sensors; based on said performance quality from said plurality of selecting a subset of sensors; and irreversibly breaking electrical connections between said detectors and said subset. The method of claim 12, wherein the electrical connection is irreversibly severed by breaking a fuse. The method of claim 12, wherein determining the performance quality of each of the plurality of sensors is based on the electrical signal from each of the plurality of sensors. 12. The method of claim 12, wherein said subset consists of those said plurality of sensors for which said electrical signal output is above a threshold. The method of claim 12, wherein said subset consists of those said plurality of sensors for which said electrical signal output is within a range. The method as described in claim 12, wherein the chemical compound is a nucleotide. The method according to claim 12, wherein the nanopore comprises protein. The method according to claim 12, wherein the nanopores comprise inorganic materials. The method according to claim 12, wherein the electrical signal is a current passing through the nanopore. The method of claim 12, wherein the nanopores of the plurality of sensors are arranged in an array. The method of claim 12, wherein the apparatus further comprises a voltage source configured to apply a voltage to the nanopore. The method of claim 12, wherein the interaction is that the nanopore is partially blocked by the chemical compound.

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