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WO2020010589A1 - Appareil de séquençage de biopolymère - Google Patents

Appareil de séquençage de biopolymère Download PDF

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Publication number
WO2020010589A1
WO2020010589A1 PCT/CN2018/095522 CN2018095522W WO2020010589A1 WO 2020010589 A1 WO2020010589 A1 WO 2020010589A1 CN 2018095522 W CN2018095522 W CN 2018095522W WO 2020010589 A1 WO2020010589 A1 WO 2020010589A1
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WIPO (PCT)
Prior art keywords
sensors
nanopore
detector
electrical
chemical compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2018/095522
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English (en)
Inventor
Peiyan CAO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Genorivision Technology Co Ltd
Original Assignee
Shenzhen Genorivision Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Shenzhen Genorivision Technology Co Ltd filed Critical Shenzhen Genorivision Technology Co Ltd
Priority to PCT/CN2018/095522 priority Critical patent/WO2020010589A1/fr
Priority to TW108121517A priority patent/TWI804639B/zh
Publication of WO2020010589A1 publication Critical patent/WO2020010589A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8827—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis

Definitions

  • 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.
  • biopolymers e.g., DNAs, RNAs, proteins
  • chemical compounds e.g., units of the biopolymers
  • 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.
  • 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 ⁇ -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.
  • 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 sacrificial device comprises a fuse.
  • the chemical compound is a nucleotide.
  • the nanopore comprises a protein
  • the nanopore comprises an inorganic material.
  • the electrical signal is an electrical current through the nanopore.
  • the nanopores of the sensors are arranged in an array.
  • the apparatus further comprises a voltage source configured to apply a voltage across the nanopore.
  • the interaction is a partial blockage of the nanopore by the chemical compound.
  • 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 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 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.
  • irreversibly severing the electrical connections is by breaking a fuse.
  • the method determining the quality of performance of each of the sensors is based on the electrical signal from that sensor.
  • the subset consists of those among the plurality of sensors, the electrical signals output by which are above a threshold.
  • the subset consists of those among the plurality of sensors, the electrical signals output by which are within a range.
  • the chemical compound is a nucleotide.
  • the nanopore comprises a protein
  • the nanopore comprises an inorganic material.
  • the electrical signal is an electrical current through the nanopore.
  • the nanopores of the sensors are arranged in an array.
  • the apparatus further comprises a voltage source configured to apply a voltage across the nanopore.
  • the interaction is a partial blockage of the nanopore by the chemical compound.
  • 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.
  • 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.
  • the electrical signals are electrical currents through the nanopores 105.
  • 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) .
  • 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.
  • 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.
  • the chemical compound 102 may interact with that nanopore and causes a change in the electrical current through that nanopore.
  • 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.
  • 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.
  • the fuses 128 may be respectively connected to the electrodes 109 of the sensors 110.
  • 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.
  • 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.
  • 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.
  • 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.
  • the apparatus 100 is obtained.
  • 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.
  • electrical signals e.g., electrical currents
  • 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.
  • 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.
  • 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.

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  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Nanotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un appareil et un procédé d'utilisation associé, l'appareil comprenant : une pluralité de capteurs, dont chacun comprend un nanopore et est configuré pour émettre un signal électrique en fonction d'une interaction d'un composé chimique avec le nanopore ; un détecteur, configuré pour recevoir des signaux électriques provenant des capteurs par l'intermédiaire d'un dispositif sacrificiel, le dispositif sacrificiel étant configuré pour sectionner sélectivement et irréversiblement des connexions électriques entre le détecteur et l'un quelconque des capteurs ; et le procédé comprenant : l'obtention d'un appareil comprenant une pluralité de capteurs, dont chacun comprend un nanopore et est configuré pour émettre un signal électrique en fonction d'une interaction d'un composé chimique avec le nanopore, un détecteur étant configuré pour recevoir des signaux électriques provenant des capteurs ; la détermination d'une qualité de performance de chacun des capteurs ; la sélection d'un sous-ensemble à partir des capteurs en fonction des qualités de performance ; le sectionnement irréversible de connexions électriques entre le détecteur et le sous-ensemble.
PCT/CN2018/095522 2018-07-12 2018-07-12 Appareil de séquençage de biopolymère Ceased WO2020010589A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2018/095522 WO2020010589A1 (fr) 2018-07-12 2018-07-12 Appareil de séquençage de biopolymère
TW108121517A TWI804639B (zh) 2018-07-12 2019-06-20 用於生物聚合物定序的設備及定序方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/095522 WO2020010589A1 (fr) 2018-07-12 2018-07-12 Appareil de séquençage de biopolymère

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110192723A1 (en) * 2010-02-08 2011-08-11 Genia Technologies, Inc. Systems and methods for manipulating a molecule in a nanopore
CN105259229A (zh) * 2015-10-22 2016-01-20 清华大学 一种检测药物的单分子分析方法
CN106596645A (zh) * 2016-12-13 2017-04-26 中国科学院重庆绿色智能技术研究院 单分子操纵的石墨烯纳米孔dna测序仪
WO2017090087A1 (fr) * 2015-11-24 2017-06-01 株式会社日立ハイテクノロジーズ Analyseur d'échantillon biologique et procédé d'analyse d'échantillon biologique
WO2017098322A1 (fr) * 2015-12-08 2017-06-15 Katholieke Universiteit Leuven Ku Leuven Research & Development Nanopores modifiés, compositions les comprenant et leurs utilisations
WO2017102852A1 (fr) * 2015-12-14 2017-06-22 Dubois Valentin Structures de fissures, jonctions de tunnelisation utilisant des structures de fissures et leurs procédés de fabrication
WO2017184790A1 (fr) * 2016-04-19 2017-10-26 Takulapalli Bharath Capteur de nanopores, structure et dispositif comprenant le capteur, et procédés de formation et d'utilisation correspondants
CN107533045A (zh) * 2015-02-05 2018-01-02 哈佛大学校长及研究员协会 包括流体通道的纳米孔感测器
CN207318408U (zh) * 2017-10-25 2018-05-04 深圳宣泽生物医药有限公司 高通量纳米孔检测装置

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI223064B (en) * 1999-11-15 2004-11-01 Matsushita Electric Industrial Co Ltd Biological sensor, formation method of thin film electrode, quantity determination device and quantity determination method
EP3298389B1 (fr) * 2015-05-20 2021-09-29 Quantum-Si Incorporated Procédé de détermination de la séquence d'un acide nucléique au moyen de la luminescence à résolution temporelle

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110192723A1 (en) * 2010-02-08 2011-08-11 Genia Technologies, Inc. Systems and methods for manipulating a molecule in a nanopore
CN107533045A (zh) * 2015-02-05 2018-01-02 哈佛大学校长及研究员协会 包括流体通道的纳米孔感测器
CN105259229A (zh) * 2015-10-22 2016-01-20 清华大学 一种检测药物的单分子分析方法
WO2017090087A1 (fr) * 2015-11-24 2017-06-01 株式会社日立ハイテクノロジーズ Analyseur d'échantillon biologique et procédé d'analyse d'échantillon biologique
WO2017098322A1 (fr) * 2015-12-08 2017-06-15 Katholieke Universiteit Leuven Ku Leuven Research & Development Nanopores modifiés, compositions les comprenant et leurs utilisations
WO2017102852A1 (fr) * 2015-12-14 2017-06-22 Dubois Valentin Structures de fissures, jonctions de tunnelisation utilisant des structures de fissures et leurs procédés de fabrication
WO2017184790A1 (fr) * 2016-04-19 2017-10-26 Takulapalli Bharath Capteur de nanopores, structure et dispositif comprenant le capteur, et procédés de formation et d'utilisation correspondants
CN106596645A (zh) * 2016-12-13 2017-04-26 中国科学院重庆绿色智能技术研究院 单分子操纵的石墨烯纳米孔dna测序仪
CN207318408U (zh) * 2017-10-25 2018-05-04 深圳宣泽生物医药有限公司 高通量纳米孔检测装置

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