WO2020010589A1

WO2020010589A1 - An apparatus for biopolymer sequencing

Info

Publication number: WO2020010589A1
Application number: PCT/CN2018/095522
Authority: WO
Inventors: Peiyan CAO
Original assignee: Shenzhen Genorivision Technology Co Ltd
Current assignee: Shenzhen Genorivision Technology Co Ltd
Priority date: 2018-07-12
Filing date: 2018-07-12
Publication date: 2020-01-16
Anticipated expiration: 2021-01-12
Also published as: TW202006355A; TWI804639B

Abstract

An apparatus and a method use of it, wherein the 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; and the method including: 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

AN APPARATUS FOR BIOPOLYMER SEQUENCING

Technical Field

The disclosure herein relates to an apparatus suitable for sequencing biopolymers (e.g., DNAs, RNAs, proteins) by sensing electrical signals from interaction of chemical compounds (e.g., units of the biopolymers) with nanopores.

Background

DNA sequencing is the process of determining the sequence of nucleotides (e.g., Adenine (A) , Guanine (G) , Cytosine (C) , and Thymine (T) ) in a strand of DNA. The classical DNA sequencing method (e.g., The Sanger method is based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. The next-generation sequencing method is improved based on the Sanger method to conduct a large scale of sequencing in parallel, which makes it much quicker and cheaper than the Sanger method.

DNA sequencing using nanopores is a third-generation method. A nanopore is a structure with a small hole with an internal diameter of the order of 1 nanometer. One type of nanopore is membrane protein complexes such as α-Hemolysin, MspA (Mycobacterium Smegmatis Porin A) or CsgG. Another type of nanopore is solid-state nanopores such as a film of silicon nitride and aluminum oxide, with a small hole. When a nanopore is immersed in a conducting fluid and a voltage is applied across it, an electric current due to conduction of ions through the nanopore can be observed. The amount of current is very sensitive to the size and shape of the nanopore. The change in the current through the nanopore as a DNA molecule passes through the nanopore represents a direct reading of the DNA sequence. Other biopolymers such as RNAs and proteins may also be sequenced using nanopores.

Summary

Disclosed herein is an apparatus, comprising: a plurality of sensors, each of which comprising a nanopore and configured to output an electrical signal that is dependent on an interaction of a chemical compound with the nanopore; a detector configured to receive the 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.

According to an embodiment, the sacrificial device comprises a fuse.

According to an embodiment, the chemical compound is a nucleotide.

According to an embodiment, the nanopore comprises a protein.

According to an embodiment, the nanopore comprises an inorganic material.

According to an embodiment, the electrical signal is an electrical current through the nanopore.

According to an embodiment, the nanopores of the sensors are arranged in an array.

According to an embodiment, the apparatus further comprises a voltage source configured to apply a voltage across the nanopore.

According to an embodiment, the interaction is a partial blockage of the nanopore by the chemical compound.

According to an embodiment, the sacrificial device is configured to selectively and irreversibly sever electrical connections between the detector and any of the sensors when the electrical signal from that sensor is greater than a threshold.

According to an embodiment, the sacrificial device is configured to selectively and irreversibly sever electrical connections between the detector and any of the sensors when the electrical signal from that sensor is within a range.

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 that is dependent on an interaction of a chemical compound with the nanopore, and a detector configured to receive the 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.

According to an embodiment, irreversibly severing the electrical connections is by breaking a fuse.

According to an embodiment, the method determining the quality of performance of each of the sensors is based on the electrical signal from that sensor.

According to an embodiment, the subset consists of those among the plurality of sensors, the electrical signals output by which are above a threshold.

According to an embodiment, the subset consists of those among the plurality of sensors, the electrical signals output by which are within a range.

According to an embodiment, the chemical compound is a nucleotide.

According to an embodiment, the nanopore comprises a protein.

According to an embodiment, the nanopore comprises an inorganic material.

Brief Description of Figures

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

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

Fig. 2B schematically shows an example of a sacrificial device of the apparatus, according to an embodiment.

Fig. 3 schematically shows a component diagram of a detector of the apparatus, according to an embodiment.

Fig. 4 schematically shows a flow chart of a biopolymer sequencing method using the apparatus, according to an embodiment.

Fig. 5 schematically shows an example using the apparatus described herein.

Detailed Description

Fig. 1 schematically shows a cross-sectional view of a portion of an apparatus 100, according to an embodiment. The apparatus 100 comprises a plurality of sensors 110. Each of the sensors 110 comprises a nanopore 105, which may be disposed in a substrate 106. The nanopore 105 may be mostly organic materials (e.g., a transmembrane protein) , or inorganic materials such as silicon nitride, aluminum oxide or a combination thereof. The sensors 110 may produce electrical signals that reflect interactions of chemical compounds with the nanopores 105 in the sensors 110. For example, the electrical signals are electrical currents through the nanopores 105. Examples of the chemical compounds may include nucleotides, nucleosides and amino acids. An example of the interactions is partial blockage of the nanopores 105 by the chemical compounds as the chemical compounds pass through the nanopores 105. The partial blockage may be transient.

Fig. 1 also schematically shows the operation of the apparatus 100. The sensors 110 are immersed in a conductive fluid 103 (e.g., a salt solution) . When a voltage across the nanopores 105 is established (e.g., by a voltage source 104, which may be part of the apparatus 100) , electrical currents flow through the nanopores. The electrical currents 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 that are in contact with the conductive fluid 103 and separated by the nanopores 105. When a chemical compound 102 in the conductive fluid 103 passes through one of the nanopores 105, for example driven by the voltage, the chemical compound 102 may interact with that nanopore and causes a change in the electrical current through that nanopore. For example, the chemical compound 102 may partially block that nanopore and causes a decrease in the electrical current through that nanopore. Characteristics (e.g., magnitude, duration, waveform) of the change may depend on characteristics (e.g., size, shape, structure, chemical composition) of the chemical compound 102. Therefore, the chemical compound 102 may be identified based on the characteristics of the change.

The apparatus 100 has a detector 125 that receives the electrical signals from the sensors 110 through a sacrificial device 126. The detector 125 may have analog circuitry such as a filter network, amplifiers, integrators, and comparators, or digital circuitry such as a microprocessor and a memory. The detector 125 may include components shared by the sensors 110 or components dedicated to each of the sensors 110. For example, the detector 125 may include an amplifier dedicated to each of the sensors 110 and a microprocessor shared among all of the sensors 110.

The sacrificial device 126 is configured to selectively and irreversibly sever electrical connections 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 signals are defective (e.g., produce electrical signals orders of magnitudes larger than the electrical signals from the rest of the sensors 110) , the sacrificial device 126 may irreversibly sever the electrical connections between these defective sensors and the detector 125 but leave the electrical connections between the rest of the sensors 110 and the detector 125 intact.

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

Fig. 2B schematically shows an example of the sacrificial device 126, according to an embodiment. The sacrificial device 126 may include a multiplexer 127. The multiplexer 127 is connected to the detector 125. The multiplexer 127 is 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 breaking one of the fuses 128, the electrical connection between the detector 125 and the sensor 110 connected through that fuse is selectively and irreversibly severed.

Fig. 3 schematically shows a component diagram of the detector 125, according to an embodiment. The detector 125 comprises an amplifier 301 with a feedback loop. The detector 125 may comprise other circuits, such as an integrator, a noise filter, or a feedback control logic. The detector 125 may also comprise other functional components such as an ADC converter. The detector 125 may include a controller 300, a memory 320, and a communication module 330. The amplifier 301 is configured to receive the electrical current from the sensor 110 via its electrode 109. The amplifier 301 may be configured to monitor the electrical current directly, or calculate the average over a period of time. The amplifier 301 may be controllably activated or deactivated by the controller 300. The amplifier 301 may be configured to be activated continuously, and monitor electrical current continuously. The amplifier 301 may have a high speed to allow the detector 125 to operate for a large-scale sequencing in parallel.

The detector 125 is configured to receive the electrical signals from the sensors 110 through the sacrificial device 126. When the electrical signal from one of the sensors 110 is greater than a certain threshold or within a certain range, which may indicate that the nanopore 105 in this particular sensor 110 is defective, the sacrificial device 126 may selectively and irreversibly sever the electrical connection between the detector 125 and this particular sensor 110 by breaking the fuse 128 therebetween. For example, as shown in Fig. 3, the electrical connection between one instance 109A of the electrodes 109 and the detector 125 remains intact via one instance 128A of the fuses 128; and the electrical connection between one instance 109B of the electrodes 109 and the detector 125 is selectively and irreversibly severed by an instance 128B, which is open circuit, of the fuses 128.

The controller 300 may be configured to cause the sacrificial device 126 to selectively and irreversibly sever electrical connection between any of the sensors 110 and the detector 125, for example, by breaking any of the fuses 128, according to an embodiment.

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

The controller 300 may be configured to cause the voltage source to supply different voltages to the

electrodes

108 and 109 across the nanopores 105, and the supplied voltages may be subsequently maintained throughout the current measurement. In some examples, a waveform of suitable shapes is supplied to the

electrodes

108 and 109, comprising triangular waveforms, sine waveforms, sawtooth waveforms, or square waveforms.

The memory 320 may be a random-access memory, flash memory, hard disk, with high read/write speed that matches with the speed of sample sequencing.

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

Fig. 4 schematically shows a flow chart of a biopolymer sequencing method using the apparatus 100, according to an embodiment. In step 901, the apparatus 100 is obtained. In step 902, determining, a quality of performance of each of the sensors 110 of the apparatus 100 is determined, e.g., by using the detector 125 and the controller 300. In one example, electrical signals (e.g., electrical currents) output by the sensors 110 are measured and compared with a reference value. In step 903, a subset of the sensors 110 is selected based on the qualities of performance, e.g., by the controller 300. For example, the subset consists of those sensors 110 that output electrical signals that are greater than a certain threshold or fall into a certain range. In step 904, electrical connections between the detector 125 and the subset of the sensors 110 are selectively and irreversibly severed, e.g., by breaking the fuses 128 therebetween.

Fig. 5 schematically shows the apparatus 100 described herein used in a portable DNA sequencing application. The apparatus 100 may be used for inspecting and identifying goods in transportation systems such as shipping containers, vehicles, ships, luggage, etc. The apparatus 100 may comprise one or multiple detectors. The prepared DNA sample 502 from an object (e.g., shipping containers, vehicles, ships, etc. ) and be brought to a surface of the apparatus 100. The sample may be sieved, screened, and mixed with chemical reagents in the system. The extracted substance that contains useful DNA strands are sequenced by the sensors 501 and the sample is therefore identified.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be 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, with the true scope and spirit being indicated by the following claims.

Claims

An apparatus, comprising:

a plurality of sensors, each of which comprising a nanopore and configured to output an electrical signal that is dependent on an interaction of a chemical compound with the nanopore;

a detector configured to receive the 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.
The apparatus of claim 1, wherein the sacrificial device comprises a fuse.
The apparatus of claim 1, wherein the chemical compound is a nucleotide.
The apparatus of claim 1, wherein the nanopore comprises a protein.
The apparatus of claim 1, wherein the nanopore comprises an inorganic material.
The apparatus of claim 1, wherein the electrical signal is an electrical current through the nanopore.
The apparatus of claim 1, wherein the nanopores of the sensors are arranged in an array.
The apparatus of claim 1, further comprising a voltage source configured to apply a voltage across the nanopore.
The apparatus of claim 1, wherein the interaction is a partial blockage of the nanopore by the chemical compound.
The apparatus of claim 1, wherein the sacrificial device is configured to selectively and irreversibly sever electrical connections between the detector and any of the sensors when the electrical signal from that sensor is greater than a threshold.
The apparatus of claim 1, wherein the sacrificial device is configured to selectively and irreversibly sever electrical connections between the detector and any of the sensors when the electrical signal from that sensor is within a range.
A method comprising:

obtaining an apparatus comprising a plurality of sensors, each of which comprising a nanopore and configured to output an electrical signal that is dependent on an interaction of a chemical compound with the nanopore, and a detector configured to receive the 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.
The method of claim 12, wherein irreversibly severing the electrical connections is by breaking a fuse.
The method of claim 12, wherein determining the quality of performance of each of the sensors is based on the electrical signal from that sensor.
The method of claim 12, wherein the subset consists of those among the plurality of sensors, the electrical signals output by which are above a threshold.
The method of claim 12, wherein the subset consists of those among the plurality of sensors, the electrical signals output by which are within a range.
The method of claim 12, wherein the chemical compound is a nucleotide.
The method of claim 12, wherein the nanopore comprises a protein.
The method of claim 12, wherein the nanopore comprises an inorganic material.
The method of claim 12, wherein the electrical signal is an electrical current through the nanopore.
The method of claim 12, wherein the nanopores of the sensors are arranged in an array.
The method of claim 12, wherein the apparatus further comprises a voltage source configured to apply a voltage across the nanopore.
The method of claim 12, wherein the interaction is a partial blockage of the nanopore by the chemical compound.