WO2018196307A1 - 基因测序芯片、装置以及方法 - Google Patents

基因测序芯片、装置以及方法 Download PDF

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WO2018196307A1
WO2018196307A1 PCT/CN2017/107437 CN2017107437W WO2018196307A1 WO 2018196307 A1 WO2018196307 A1 WO 2018196307A1 CN 2017107437 W CN2017107437 W CN 2017107437W WO 2018196307 A1 WO2018196307 A1 WO 2018196307A1
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gene sequencing
substrate
groove
liquid crystal
sequencing chip
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PCT/CN2017/107437
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English (en)
French (fr)
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蔡佩芝
庞凤春
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京东方科技集团股份有限公司
北京京东方光电科技有限公司
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Priority to US15/776,224 priority Critical patent/US20190256902A1/en
Publication of WO2018196307A1 publication Critical patent/WO2018196307A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J4/00—Measuring polarisation of light
    • G01J4/04—Polarimeters using electric detection means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • 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
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/333—Ion-selective electrodes or membranes
    • G—PHYSICS
    • G02—OPTICS
    • G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
    • G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
    • G02F1/01—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the intensity, phase, polarisation or colour 
    • G02F1/13—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the intensity, phase, polarisation or colour  based on liquid crystals, e.g. single liquid crystal display cells
    • G02F1/1313—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the intensity, phase, polarisation or colour  based on liquid crystals, e.g. single liquid crystal display cells specially adapted for a particular application
    • G—PHYSICS
    • G02—OPTICS
    • G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
    • G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
    • G02F1/01—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the intensity, phase, polarisation or colour 
    • G02F1/13—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the intensity, phase, polarisation or colour  based on liquid crystals, e.g. single liquid crystal display cells
    • G02F1/133—Constructional arrangements; Operation of liquid crystal cells; Circuit arrangements
    • G02F1/1333—Constructional arrangements; Manufacturing methods
    • G02F1/1343—Electrodes
    • G02F1/13439—Electrodes characterised by their electrical, optical, physical properties; materials therefor; method of making
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • G—PHYSICS
    • G02—OPTICS
    • G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
    • G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
    • G02F1/01—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the intensity, phase, polarisation or colour 
    • G02F1/13—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the intensity, phase, polarisation or colour  based on liquid crystals, e.g. single liquid crystal display cells
    • G02F1/133—Constructional arrangements; Operation of liquid crystal cells; Circuit arrangements
    • G02F1/1333—Constructional arrangements; Manufacturing methods
    • G02F1/1335—Structural association of cells with optical devices, e.g. polarisers or reflectors
    • G02F1/133528—Polarisers
    • G—PHYSICS
    • G02—OPTICS
    • G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
    • G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
    • G02F1/01—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the intensity, phase, polarisation or colour 
    • G02F1/13—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the intensity, phase, polarisation or colour  based on liquid crystals, e.g. single liquid crystal display cells
    • G02F1/133—Constructional arrangements; Operation of liquid crystal cells; Circuit arrangements
    • G02F1/1333—Constructional arrangements; Manufacturing methods
    • G02F1/1339—Gaskets; Spacers; Sealing of cells

Definitions

  • Embodiments of the present disclosure relate to a gene sequencing chip, a gene sequencing device, and a gene sequencing method.
  • the second generation of high-throughput sequencing technologies include Illumina's edge-synthesis sequencing technology, Thermo Fisher's ion semiconductor sequencing technology, ligation sequencing technology and Roche's pyrosequencing technology, among which Illumina relies on its ultra-high throughput and relative The advantage of long read lengths accounts for more than 70% of the market.
  • the usual gene sequencing technology performs different fluorophore modification on various bases.
  • these bases are paired with the gene fragment to be tested, the fluorophore is released; at this time, the base color can be determined by detecting the fluorescence color by the optical system. The species, thus obtaining the sequence of the gene fragment to be tested.
  • At least one embodiment of the present disclosure provides a gene sequencing chip, a gene sequencing device, and a gene sequencing method.
  • the gene sequencing chip includes a first substrate, a common electrode, a second substrate, and a liquid crystal layer.
  • the first substrate is disposed opposite to the second substrate, and the liquid crystal layer is disposed between the first substrate and the second substrate.
  • the side of the second substrate away from the first substrate includes at least one groove recessed into the second substrate, and the groove is used for placing the test In the sample, each groove is disposed near the bottom of the first substrate with an ion-sensitive membrane configured to sense ions generated by a gene sequencing reaction occurring in the groove to generate a voltage and generate an electric field with the common electrode, thereby driving the liquid crystal
  • the gene sequencing chip can provide simpler, lower cost gene sequencing.
  • At least one embodiment of the present disclosure provides a gene sequencing chip, including: a first substrate; a common electrode; a second substrate disposed opposite to the first substrate; a liquid crystal layer disposed on the first substrate and the first Between the two substrates, a side of the second substrate away from the first substrate includes at least one groove recessed into the second substrate, the groove is configured to place a sample to be tested, each of the grooves An ion sensitive membrane is disposed adjacent the bottom of the first substrate, the ion sensitive membrane being configured to sense ions generated by a gene sequencing reaction occurring within the recess to generate a voltage and generate an electric field with the common electrode.
  • the ion sensitive membrane comprises a hydrogen ion sensitive membrane.
  • the at least one groove includes a plurality of the grooves, and the plurality of grooves are arranged in an array on the second substrate.
  • the common electrode is disposed on a side of the first substrate adjacent to the liquid crystal layer.
  • the common electrode includes a plurality of strip-shaped common electrodes, and the common electrode and the ion-sensitive film are disposed in the same layer, and each of the strip-shaped common electrodes is disposed at Between adjacent grooves.
  • the common electrode includes a plurality of strip-shaped common electrodes
  • the ion-sensitive film includes a plurality of strip-shaped sensitive films, the common electrode and the ion-sensitive film In the same layer, the strip-shaped common electrode and the strip-shaped sensitive film are alternately spaced apart at the bottom of the groove.
  • the gene sequencing chip further includes: a first polarizer; and a second polarizer, wherein the first polarizer and the second polarizer are disposed on both sides of the liquid crystal layer.
  • the gene sequencing chip according to an embodiment of the present disclosure further includes: a backlight disposed on a side of the first polarizer away from the second polarizer, or disposed on the second polarizer away from the One side of the first polarizer.
  • the shape of the groove parallel to the cross section of the first substrate includes at least one of a circular shape and a regular polygon.
  • the groove is parallel to the
  • the maximum dimension of the cross section of the first substrate ranges from 10 to 100 ⁇ m.
  • a gene sequencing chip further includes: a third substrate, an inlet, and a sample outlet, wherein the third substrate is disposed on a side of the second substrate away from the first substrate, the first The three substrate includes at least one flow channel, the flow channel is in communication with the groove, and the injection port and the sample outlet are disposed on the third substrate and communicate with the flow channel.
  • At least one embodiment of the present disclosure provides a gene sequencing device comprising: a gene sequencing chip; and a photosensitive device comprising the gene sequencing chip according to any one of the above, the photosensitive device configured to sense Light exiting at the location of the at least one groove.
  • the photosensitive device includes a CCD image sensor.
  • At least one embodiment of the present disclosure provides a gene sequencing method for a gene sequencing chip, comprising the gene sequencing chip according to any one of the above, the gene sequencing method comprising: placing a sample to be tested in a groove And sequentially adding four different deoxyribonucleoside triphosphates to the groove and respectively sensing ions released by the base pairing reaction through the ion sensitive membrane and generating an induced voltage, the voltage generating an electric field with the common electrode; and detecting the liquid crystal In the case of deflection, the deoxyribonucleoside triphosphate in which the pairing reaction occurs is judged by the state of the liquid crystal deflection.
  • the case where the liquid crystal is deflected is detected, and the deoxyribonucleoside triphosphate in which the pairing reaction occurs is determined by the liquid crystal deflection: including the photosensitive device and the polarizer
  • the case where the polarized light is reflected by the liquid crystal is sensed to detect the deflection of the liquid crystal.
  • FIG. 1 is a schematic structural diagram of a gene sequencing chip according to an embodiment of the present disclosure
  • FIG. 2a is a schematic structural diagram of another gene sequencing chip according to an embodiment of the present disclosure.
  • 2b is a schematic structural diagram of another gene sequencing chip according to an embodiment of the present disclosure.
  • FIG. 3 is a schematic plan view of a groove according to an embodiment of the present disclosure.
  • FIG. 4 is a schematic structural diagram of another gene sequencing chip according to an embodiment of the present disclosure.
  • FIG. 5 is a schematic plan view of a gene sequencing chip according to an embodiment of the present disclosure.
  • FIG. 6 is a schematic diagram of a working principle of a gene sequencing chip according to an embodiment of the present disclosure
  • FIG. 7 is a schematic structural diagram of a gene sequencing apparatus according to an embodiment of the present disclosure.
  • FIG. 8 is a flowchart of a gene sequencing method according to an embodiment of the present disclosure.
  • Embodiments of the present disclosure provide a gene sequencing chip, a gene sequencing device, and a gene sequencing method.
  • the gene sequencing chip includes a first substrate, a common electrode, a second substrate, and a liquid crystal layer.
  • the first substrate is disposed opposite to the second substrate, and the liquid crystal layer is disposed between the first substrate and the second substrate.
  • the side of the second substrate away from the first substrate comprises at least one groove recessed into the second substrate, the groove is used for placing the sample to be tested, and the groove is disposed near the bottom of the first substrate with an ion sensitive film, and the ion sensitive film can be
  • the ions generated by the gene sequencing reaction occurring in the sensing groove generate a voltage to generate an electric field with the common electrode to drive the liquid crystal molecules in the liquid crystal layer to deflect.
  • the gene sequencing chip can utilize an ion-sensitive membrane to sense ions (eg, hydrogen ions) generated in a base pairing reaction and generate a voltage, and generate a liquid crystal layer in the liquid crystal layer.
  • ions eg, hydrogen ions
  • the sub-deflected electric field is used to determine whether a base pairing reaction occurs by using a liquid crystal optical switching technique, thereby realizing gene sequencing.
  • the gene sequencing technology using the gene sequencing chip does not require fluorescent labeling of various bases, and does not require a laser light source and an optical system, the system using the gene sequencing technology of the gene sequencing chip is simpler and lower in cost. .
  • An embodiment of the present disclosure provides a gene sequencing chip.
  • 1 is a gene sequencing chip according to the present embodiment; as shown in FIG. 1, the gene sequencing chip includes a first substrate 110, a common electrode 120, a second substrate 130, and a liquid crystal layer 140.
  • the first substrate 110 and the second substrate 130 are oppositely disposed, and the liquid crystal layer 140 is disposed between the first substrate 110 and the second substrate 130.
  • the side of the second substrate 130 away from the first substrate 110 includes at least one groove 136 recessed into the second substrate 130, the groove 136 can be placed on the sample to be tested and used for gene sequencing of the sample to be tested; the groove 136 is close to the first substrate
  • the bottom of the 110 is provided with an ion-sensitive membrane 132 that senses a gene sequencing reaction occurring in the groove 136, for example, a base pairing reaction generates ions and generates a voltage to form an electric field with the common electrode 120 to control the second substrate.
  • the liquid crystal molecules in the liquid crystal layer 140 on the side close to the first substrate 110 are rotated by 130.
  • the ion sensitive membrane can be used to induce a gene sequencing reaction occurring in the groove, such as a base pairing reaction, generating ions (for example, hydrogen ions) and generating a voltage, such as a Nernst voltage. And generating an electric field that controls the deflection of the liquid crystal molecules in the liquid crystal layer (for example, generating an electric field with the common electrode), thereby using liquid crystal optical switching technology to determine whether a base pairing reaction occurs, thereby realizing gene sequencing.
  • a gene sequencing reaction occurring in the groove such as a base pairing reaction
  • generating ions for example, hydrogen ions
  • a voltage such as a Nernst voltage
  • generating an electric field that controls the deflection of the liquid crystal molecules in the liquid crystal layer for example, generating an electric field with the common electrode
  • the ion sensitive membrane induces ions (eg, hydrogen ions) released by the base pairing reaction and Generating a voltage to form an electric field with the common electrode and controlling deflection of the liquid crystal molecules in the liquid crystal layer at the position where the groove is located; as shown by the groove on the right side in FIG. 1, if the sample to be tested and the currently added deoxyribonucleoside
  • the base pairing reaction does not occur in the phosphoric acid
  • the ion-sensitive film does not generate a voltage
  • the liquid crystal molecules in the liquid crystal layer at the position where the groove is located are not deflected.
  • the analyzer and the light sensing device are used to detect whether there is light passing through to determine whether there is a voltage on the ion sensitive membrane, thereby judging whether the sample to be tested and the currently added deoxyribonucleoside triphosphate have a base pairing reaction, thereby realizing the gene Sequencing.
  • the gene sequencing technology of the chip does not require fluorescent labeling of various bases, nor does it require a laser light source and an optical system. Therefore, the system using the gene sequencing technology of the gene sequencing chip is simpler and lower in cost. It should be noted that since the base pairing reaction of the single sample to be tested with the deoxyribonucleoside triphosphate releases less ions, the sample to be tested can be amplified, and multiple base pairing reactions occur simultaneously, thereby making the ions Sensitive membranes are capable of sensing and generating voltage.
  • the first substrate may include a glass substrate, a plastic substrate, or other transparent substrate to facilitate light transmission.
  • the common electrode may be a transparent metal electrode such as an indium tin oxide (ITO) electrode.
  • ITO indium tin oxide
  • embodiments of the present disclosure include, but are not limited to, the common electrode may also employ an opaque electrode, and a plurality of openings are provided on the common electrode to achieve light transmission.
  • the recess can be formed by etching the second substrate.
  • embodiments of the present disclosure include, but are not limited to, the grooves may be formed by other methods.
  • the shape of the cross section of the groove parallel to the first substrate includes at least one of a circular shape and a regular polygon.
  • embodiments of the present disclosure include but are not limited thereto.
  • the maximum dimension of the cross section of the groove parallel to the first substrate ranges from 10 to 100 ⁇ m. It should be noted that when the groove is parallel to the cross section of the first substrate, the largest dimension is a circular diameter. When the cross section of the groove is a regular polygon, the maximum dimension is a diagonal of a regular polygon.
  • the ion sensitive membrane may include a hydrogen ion sensitive membrane.
  • the potential of the hydrogen ion sensitive membrane may vary in response to hydrogen ions.
  • a hydrogen ion sensitive membrane may utilize a hydrogen ion recognition material immobilized on a hydrogen ion sensitive membrane, for example, silicon trinitride (Si 3 N 4 ) selectively binds hydrogen ions, thereby causing a change in membrane potential or membrane current.
  • Si 3 N 4 silicon trinitride
  • the embodiments of the present disclosure include, but are not limited to, the ion sensitive membrane may also adopt other ion sensitive membranes according to actual conditions.
  • the hydrogen ion sensitive membrane is transparent to facilitate observation of the transmission of polarized light.
  • the material of the hydrogen ion sensitive film includes an organic material or an electrodeless material.
  • the material of the hydrogen ion sensitive film may be selected from one or more of silicon nitride (SiN x ), lithium glass, silicon dioxide (SiO 2 ), and aluminum oxide (Al 2 O 3 ).
  • the common electrode 120 is disposed on a side of the first substrate 110 close to the liquid crystal layer 140.
  • the common electrode 120 shown in FIG. 1 is disposed on the entire surface of the first substrate 110, so that the process of patterning the common electrode 120 can be reduced.
  • embodiments of the present disclosure include, but are not limited to, the common electrode may also be disposed corresponding to the ion sensitive film. That is, the common electrode is disposed only on the first substrate at the position where the groove is located.
  • the gene sequencing chip further includes a sealant 190 disposed between the first substrate 110 and the second substrate 130 and located on the first substrate 110.
  • the peripheral region is to seal the liquid crystal layer 140 between the first substrate 110 and the second substrate 130.
  • the at least one groove includes a plurality of grooves, and the plurality of grooves are arranged in an array on the second substrate.
  • a plurality of grooves are arranged in an array on the second substrate to facilitate numbering of the plurality of grooves.
  • FIG. 2a shows a gene sequencing chip according to the present embodiment.
  • the common electrode 120 includes a plurality of strip-shaped common electrodes 1200, and the common electrode 120 and the ion-sensitive film 132 are disposed in the same layer, and the strip-shaped common electrodes 1200 are disposed adjacent to each other. Between slots 136.
  • the strip-shaped common electrode disposed between the adjacent grooves can generate a transverse electric field with the ion-sensitive film to drive the liquid crystal molecules of the liquid crystal layer to deflect.
  • the analyzer and the light sensing device are used to detect whether there is light passing through to determine whether there is a voltage on the ion sensitive membrane, thereby judging whether the sample to be tested and the currently added deoxyribonucleoside triphosphate have a base pairing reaction, thereby realizing the gene Sequencing.
  • Figure 2b shows a gene sequencing chip in accordance with the present embodiment.
  • the common electrode 120 includes a plurality of strip-shaped common electrodes 1200
  • the ion-sensitive film 132 includes a plurality of strip-shaped sensitive films 1320.
  • the common electrode 120 and the ion-sensitive film 132 are disposed in the same layer, and the strip-shaped common electrodes 1200 and strips are provided.
  • the sensitive films 1320 are alternately spaced apart at the bottom of the recess 136.
  • the strip-shaped common electrode and the strip-shaped sensitive film can generate a transverse electric field, and the liquid crystal molecules that drive the liquid crystal layer are deflected.
  • the analyzer and the light sensing device are used to detect whether there is light passing through to determine whether there is a voltage on the ion sensitive membrane, thereby determining whether the sample to be tested and the currently added deoxyribonucleoside triphosphate are base paired. In this way, gene sequencing can be achieved.
  • FIG. 3 is a schematic plan view of a groove in a gene sequencing chip according to the present embodiment.
  • the common electrode 120 includes a plurality of strip-shaped common electrodes 1200, and the plurality of strip-shaped common electrodes 1200 are connected through the common electrode connecting portion 1201.
  • the ion sensitive film 132 includes a plurality of strip-shaped sensitive films 1320, and the plurality of strip-shaped common electrodes 1320 are connected by the ion-sensitive film connecting portion 1321.
  • the common electrode 120 and the ion sensitive film 132 are disposed in the same layer, and the strip common electrode 1200 and the strip sensitive film 1320 are alternately spaced apart at the bottom of the groove 136.
  • the embodiments of the present disclosure include, but are not limited to, the common electrode and the ion sensitive film may be disposed in the same layer, as long as the strip-shaped common electrode and the strip-shaped sensitive film can generate a transverse electric field, and the liquid crystal molecules of the liquid crystal layer are deflected. Just fine.
  • FIG. 4 shows a gene sequencing chip according to the present embodiment.
  • the gene sequencing chip further includes a first polarizer 181 and a second polarizer 182.
  • the first polarizer 181 and the second polarizer 182 are disposed on both sides of the liquid crystal layer 140, and the transmission axis of the first polarizer 181 and the transmission axis of the second polarizer 182 are perpendicular or opposite to each other.
  • first polarizer and the second polarizer are linear polarizers
  • the transmission axis of the first polarizer and the transmission axis of the second polarizer are perpendicular to each other, and the first polarizer and the second polarizer
  • the transmission axis of the first polarizer is opposite to the transmission axis of the second polarizer.
  • the first polarizer and the second polarizer are disposed on both sides of the liquid crystal layer, and the transmission axis of the first polarizer and the transmission axis of the second polarizer are perpendicular to each other. Or turn to the opposite.
  • the transmission axis of the first polarizer and the transmission axis of the second polarizer are perpendicular to each other. Or turn to the opposite.
  • the ion sensitive membrane induces ions (eg, hydrogen ions) released by the base pairing reaction and can generate a voltage, thereby
  • the common electrode forms an electric field and controls the deflection of the liquid crystal molecules in the liquid crystal layer at the position where the groove is located, at a position where the groove is located, light incident from one side of the liquid crystal layer can be emitted from the other side of the liquid crystal layer. .
  • the gene sequencing technology using the gene sequencing chip is simple in operation and low in cost.
  • the embodiments of the present disclosure include, but are not limited to, in the gene sequencing chip provided in this embodiment, the transmission axis of the first polarizer and the transmission axis of the second polarizer may be the same, when the liquid crystal layer When the liquid crystal molecules in the liquid crystal molecules do not deflect, light incident from one side of the liquid crystal layer may be from the liquid crystal layer One side emerges.
  • the ion sensitive membrane induces ions (eg, hydrogen ions) released by the base pairing reaction and can generate a voltage, thereby
  • the common electrode forms an electric field and controls the deflection of the liquid crystal molecules in the liquid crystal layer at the position where the groove is located, at a position where the groove is located, light incident from one side of the liquid crystal layer cannot be emitted from the other side of the liquid crystal layer. .
  • the gene sequencing chip may further include a backlight 170, and the backlight 170 may be on a side of the first polarizer 181 away from the second polarizer 182; or, the backlight 170 may also be disposed on a side of the second polarizer 182 away from the first polarizer 181.
  • the backlight 170 is disposed on a side of the first polarizer 181 away from the second polarizer 182.
  • the backlight can be integrated in the gene sequencing chip, thereby expanding the use range of the gene detection substrate.
  • the gene sequencing chip may further include a third substrate 150 disposed on a side of the second substrate 130 away from the first substrate 110.
  • the third substrate 150 includes at least one flow path 163 that communicates with the groove 136.
  • the groove can be protected by the third substrate to provide a relatively stable reaction environment.
  • four different deoxyribonucleoside triphosphates can be simultaneously added to a plurality of grooves through the flow channel.
  • the gene sequencing chip further includes an inlet 161 and an outlet 162, and the inlet 161 and the outlet 162 are set in the third.
  • the substrate 150 is in communication with the flow path 163.
  • four different deoxyribonucleoside triphosphates or detergents can be added through the inlet, and four different deoxyribonucleoside triphosphates or detergents can be discharged through the sample port.
  • Fig. 5 shows a schematic plan view of a gene sequencing chip according to the present embodiment. As shown in FIG. 5, at least one of the grooves 136 includes a plurality of grooves 136, and the plurality of grooves 136 are arranged in an array.
  • the third substrate 150 includes a plurality of flow paths 163 corresponding to the respective rows of the plurality of grooves 136 provided in the array, and each of the flow paths 163 is at least one inlet 161 and one outlet. 162 is connected.
  • Fig. 6 shows a working principle diagram of a gene sequencing chip according to the present embodiment.
  • the sample to be tested is placed in a groove, and four different deoxyribonucleoside triphosphates are sequentially added to the groove.
  • the ion sensitive membrane senses the ions (eg, hydrogen ions) released by the base pairing reaction and A voltage can be generated to form an electric field with the common electrode and control the deflection of the liquid crystal molecules in the liquid crystal layer at the position where the groove is located; as shown by the groove on the right side of FIG.
  • FIG. 7 shows a gene sequencing device according to the present embodiment.
  • the gene sequencing device includes the gene sequencing chip according to any of the above embodiments.
  • the gene sequencing device further includes a photosensitive device for sensing light emission at a position of at least one groove in the genetic sequencing chip.
  • the light at the position of the above-mentioned groove refers to the light emitted by the ambient light or the backlight from the side of the gene sequencing chip and transmitted through the position of the groove on the gene sequencing chip. Thereby, whether or not the light incident from the side of the gene sequencing chip is emitted from the position of the groove can be judged by the photosensitive device.
  • the photosensitive device may include a CCD image sensor. Due to the high sensitivity of the CCD image sensor and the ability to convert optical signals into analog current signals, it is easy to analyze using a computer.
  • An embodiment of the present disclosure provides a gene sequencing method for a gene sequencing chip.
  • the gene sequencing chip employs the gene sequencing chip described in any of the above embodiments.
  • Fig. 8 shows a gene sequencing method according to the present embodiment. As shown in FIG. 8, the gene sequencing method includes steps S501-S503.
  • Step S501 placing a sample to be tested in the groove.
  • Step S502 sequentially adding four different deoxyribonucleoside triphosphates to the groove and respectively sensing ions released by the base pairing reaction through the ion sensitive membrane.
  • the ion sensitive membrane can control liquid crystal deflection by forming at least one of a transverse electric field, a longitudinal electric field, and a multi-dimensional electric field with the common electrode.
  • Step S503 detecting the state of liquid crystal deflection, and judging the occurrence of the paired reaction of deoxyribonucleoside triphosphate by the state of liquid crystal deflection.
  • the ion sensitive membrane can sense the ions released by the base pairing reaction and generate an induced voltage, and the voltage generates an electric field with the common electrode, so that the currently added deoxyribonucleoside can be judged by the deflection of the liquid crystal.
  • the triphosphate reacts with the sample to be tested.
  • the ion sensitive membrane does not generate a voltage, and the liquid crystal is not deflected, so that the currently added deoxyribonucleoside triphosphate can be judged by the liquid crystal without deflection. No base pairing reaction occurred in the sample to be tested.
  • the current base type of the sample to be tested in the groove can be known.
  • the base sequence of the sample to be tested can be obtained by the above process.
  • deoxyribonucleoside triphosphates including different bases for example, four kinds of deoxyribonucleoside triphosphates including different bases, wherein the four bases may be cytosine, Guanine, adenine and thymine or cytosine, guanine, adenine and uracil
  • four deoxyribonucleoside triphosphates comprising different bases in turn and the sample to be tested in the groove, such as a DNA fragment Contact
  • ions such as hydrogen ions are released.
  • the ion sensitive membrane can sense the ions released by the base pairing reaction and generate an induced voltage, and the voltage generates an electric field with the common electrode, so that the liquid crystal is deflected to judge the currently added deoxyribonucleoside triphosphate and the sample to be tested.
  • a base pairing reaction occurs; after a plurality of rounds of the above steps, the gene sequence of the sample to be tested can be determined.
  • the gene sequencing method can realize gene sequencing without performing fluorescent labeling of four bases in different colors, which can simplify the process of gene sequencing; and the system using the gene sequencing method is simpler and lower in cost, and is beneficial to gene sequencing technology. Promotion and use.
  • deoxyribonucleoside triphosphate is a reversible termination of deoxyribonucleoside triphosphate
  • the gene sequencing method further comprises: washing the reversible stop deoxyribonucleoside triphosphate added in the groove, and adding a radical-based reagent. After the base type detection at a position on the sample to be tested (for example, a DNA fragment) is completed, it is necessary to wash away the reversible termination of the deoxyribonucleoside triphosphate added in the groove and add the sulfhydryl reagent.
  • deoxyribonucleoside triphosphates unlike ordinary deoxyribonucleoside triphosphates, reversible termination of the 3' end of deoxyribonucleoside triphosphates is linked to an azide group, which does not form a phosphodiester bond during DNA synthesis, and thus is interrupted.
  • the azide group will be broken and a hydroxyl group will be formed at the original position.
  • the base type detection in the subsequent position can be continued, and the detection method is the same as the above method, and details are not described herein again.
  • the above-mentioned reversible termination of deoxyribonucleoside triphosphate may include reversible termination of adenine triphosphate deoxyribonucleotide, reversible termination of thymidine deoxygenation Ribonucleotides, reversible termination of cytosine deoxyribonucleotides and reversible termination of guanine deoxyribonucleotide triphosphate.
  • the base on the sample to be tested (for example, a DNA fragment) is thymine; if the groove The deoxyribonucleoside triphosphate added and reacted is thymidine triphosphate deoxyribonucleotide, and the base on the sample to be tested (for example, a DNA fragment) is adenine; if the groove is added And the reacted deoxyribonucleoside triphosphate is a cytosine deoxyribonucleotide triphosphate, and then the base on the sample to be tested (for example, a DNA fragment) is guanine; if the groove is added and reacts The deoxyribonucleoside triphosphate is a guanine deoxyribonucleotide triphosphate, and then the base on the sample to be tested (for example, a DNA fragment) is
  • the case where the liquid crystal is deflected can be detected by the photosensitive device and the polarizer sensing the case where the polarized light passes through the liquid crystal.
  • the polarization direction of the polarizer and the polarization direction of the polarized light are perpendicular or opposite to each other, if the liquid crystal does not deflect, the photosensitive device cannot sense the polarized light passing through the liquid crystal, and if the liquid crystal is deflected, the polarized light is polarized.
  • the direction changes due to the deflection of the liquid crystal, and the photosensitive device can sense the deflected light passing through the liquid crystal.
  • the above-mentioned polarized light can be generated by additionally providing a polarizer.
  • placing the sample to be tested in the groove may include: amplifying the sample to be tested to form a plurality of the same sample to be tested; and placing the plurality of identical samples The sample is placed in the groove. Since the base pairing reaction of the single sample to be tested with the deoxyribonucleoside triphosphate releases less ions, the sample to be tested can be amplified, and multiple identical samples to be tested simultaneously undergo multiple base pairing reactions simultaneously, thereby The ion sensitive membrane is capable of sensing and generating a voltage.

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Abstract

本发明提供了一种基因测序芯片、基因测序装置以及基因测序方法。该基因测序芯片包括第一基板(110)、公共电极(120)、第二基板(130)以及液晶层(140)。第一基板(110)与第二基板(130)相对设置，液晶层(140)设置在第一基板(110)与第二基板(130)之间。第二基板(130)远离第一基板(110)的一侧包括至少一个凹入第二基板(130)的凹槽(136)，凹槽(136)用于放置待测样本(210)，凹槽(136)靠近第一基板(110)的底部设置有离子敏感膜(132)，离子敏感膜(132)可感应凹槽(136)中基因测序反应产生的离子以产生电压并与公共电极产生电场。

Description

基因测序芯片、装置以及方法
交叉引用
本申请要求于2017年04月28日递交的中国专利申请第201710293304.5号的优先权，在此全文引用上述中国专利申请公开的内容以作为本申请的一部分。
技术领域
本公开的实施例涉及一种基因测序芯片、基因测序装置以及基因测序方法。
背景技术
随着基因测序技术的不断发展，基因测序技术逐渐成为现代分子生物学研究中最常用的技术，具有广泛的应用场景。因此，用于基因测序的装置具有较大的市场空间。
从1977年第一代基因测序发展至今，基因测序技术取得了相当大的发展，第一代sanger测序技术，第二代高通量测序技术，第三代单分子测序技术，第四代纳米孔测序技术，目前市场主流的测序技术仍以第二代高通量测序为主。
第二代高通量测序技术主要包括Illumina的边合成边测序技术，Thermo Fisher的离子半导体测序技术、连接法测序技术和Roche的焦磷酸测序技术等，其中Illumina凭借其超高通量和相对较长的读长的优势，占有超过70％的市场份额。
通常的基因测序技术会对各种碱基进行不同的荧光基团修饰，当这些碱基与待测基因片段配对时，荧光基团释放；此时，通过光学系统检测荧光颜色便可确定碱基的种类，从而得到待测基因片段序列。
发明内容
本公开至少一个实施例提供一种基因测序芯片、基因测序装置以及基因测序方法。该基因测序芯片包括第一基板、公共电极、第二基板以及液晶层。第一基板与第二基板相对设置，液晶层设置在第一基板与第二基板之间。第二基板远离第一基板的一侧包括至少一个凹入第二基板的凹槽，凹槽用于放置待测 样本，各凹槽靠近第一基板的底部设置有离子敏感膜，离子敏感膜被配置为感应凹槽内发生的基因测序反应产生的离子以产生电压并与公共电极产生电场，从而可驱动该液晶层中的液晶分子偏转。由此，该基因测序芯片可提供更简单，成本更低的基因测序。
本公开至少一个实施例提供一种基因测序芯片，其包括：第一基板；公共电极；第二基板，与所述第一基板相对设置；液晶层，设置在所述第一基板与所述第二基板之间，所述第二基板远离所述第一基板的一侧包括至少一个凹入所述第二基板的凹槽，所述凹槽被配置为放置待测样本，各所述凹槽靠近所述第一基板的底部设置有离子敏感膜，所述离子敏感膜被配置为感应所述凹槽内发生的基因测序反应产生的离子以产生电压并与所述公共电极产生电场。
例如，在本公开一实施例提供的基因测序芯片中，所述离子敏感膜包括氢离子敏感膜。
例如，在本公开一实施例提供的基因测序芯片中，所述至少一个凹槽包括多个所述凹槽，所述多个凹槽在所述第二基板上呈阵列设置。
例如，在本公开一实施例提供的基因测序芯片中，所述公共电极设置在所述第一基板靠近所述液晶层的一侧。
例如，在本公开一实施例提供的基因测序芯片中，所述公共电极包括多个条状公共电极，所述公共电极和所述离子敏感膜同层设置，各所述条状公共电极设置在相邻的所述凹槽之间。
例如，在本公开一实施例提供的基因测序芯片中，所述公共电极包括多个条状公共电极，所述离子敏感膜包括多个条状敏感膜，所述公共电极和所述离子敏感膜同层设置，所述条状公共电极和所述条状敏感膜交替间隔设置在所述凹槽的底部。
例如，本公开一实施例提供的基因测序芯片中还包括：第一偏光片；以及第二偏光片，所述第一偏光片和所述第二偏光片设置在所述液晶层的两侧。
例如，本公开一实施例提供的基因测序芯片中还包括：背光源，设置在所述第一偏光片远离所述第二偏光片的一侧，或者，设置在所述第二偏光片远离所述第一偏光片的一侧。
例如，在本公开一实施例提供的基因测序芯片中，所述凹槽平行于所述第一基板的横截面的形状包括圆形和正多边形至少之一。
例如，在本公开一实施例提供的基因测序芯片中，所述凹槽的平行于所述 第一基板的横截面的最大尺寸的范围为10-100μm。
例如，本公开一实施例提供的基因测序芯片还包括：第三基板、进样口和出样口，第三基板设置在所述第二基板远离所述第一基板的一侧，所述第三基板包括至少一个流道，所述流道与所述凹槽连通，所述进样口和所述出样口设置在所述第三基板上并与所述流道连通。
本公开至少一个实施例提供一种基因测序装置，其包括：基因测序芯片；以及感光装置，所述基因测序芯片包括上述任一项所述的基因测序芯片，所述感光装置被配置为感测所述至少一个凹槽所在位置处的出光。
例如，在本公开一实施例提供的基因测序装置中，所述感光装置包括CCD图像传感器。
本公开至少一个实施例提供一种基因测序芯片的基因测序方法，所述基因测序芯片包括上述任一项所述的基因测序芯片，所述基因测序方法包括：在凹槽中放入待测样本；依次向所述凹槽加入四种不同的脱氧核糖核苷三磷酸并分别通过离子敏感膜感应碱基配对反应释放的离子并产生感应电压，此电压与公共电极产生电场；以及检测所述液晶偏转的情况，并通过所述液晶偏转的情况判断发生配对反应的脱氧核糖核苷三磷酸。
例如，在本公开一实施例提供的基因测序装置中，检测所述液晶偏转的情况，并通过所述液晶偏转的情况判断发生配对反应的脱氧核糖核苷三磷酸包括：通过感光装置和偏光片感测偏振光通过所述液晶的情况来检测所述液晶偏转的情况。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例的附图作简单地介绍，显而易见地，下面描述中的附图仅仅涉及本公开的一些实施例，而非对本公开的限制。
图1为本公开一实施例提供的一种基因测序芯片的结构示意图；
图2a为本公开一实施例提供的另一种基因测序芯片的结构示意图；
图2b为本公开一实施例提供的另一种基因测序芯片的结构示意图；
图3为本公开一实施例提供的一种凹槽的平面示意图；
图4为本公开一实施例提供的另一种基因测序芯片的结构示意图；
图5为本公开一实施例提供的一种基因测序芯片的平面示意图；
图6为本公开一实施例提供的一种基因测序芯片的工作原理示意图；
图7为本公开一实施例提供的一种基因测序装置的结构示意图；以及
图8为本公开一实施例提供的一种基因测序方法的流程图。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将结合本公开实施例的附图，对本公开实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本公开的一部分实施例，而不是全部的实施例。基于所描述的本公开的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本公开保护的范围。
除非另外定义，本公开使用的技术术语或者科学术语应当为本公开所属领域内具有一般技能的人士所理解的通常意义。本公开中使用的“第一”、“第二”以及类似的词语并不表示任何顺序、数量或者重要性，而只是用来区分不同的组成部分。“包括”或者“包含”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同，而不排除其他元件或者物件。“连接”或者“相连”等类似的词语并非限定于物理的或者机械的连接，而是可以包括电性的连接，不管是直接的还是间接的。
在通常的基因测序技术中，需要对各种碱基进行不同的荧光基团修饰，当这些碱基与待测基因片段配对时，荧光基团释放；此时，通过光学系统检测荧光颜色便可确定碱基的种类，从而得到待测基因片段序列。也就是说，通常的基因测序技术不仅需要对碱基进行荧光标记，还需要有激光光源和光学系统。因此，通常的基因测序技术的基因测序系统比较复杂，并且对碱基进行荧光基团修饰标记的化学试剂比较昂贵，从而增加基因测序的时间和成本。
本公开实施例提供一种基因测序芯片、基因测序装置以及基因测序方法。该基因测序芯片包括第一基板、公共电极、第二基板以及液晶层。第一基板与第二基板相对设置，液晶层设置在第一基板与第二基板之间。第二基板远离第一基板的一侧包括至少一个凹入第二基板的凹槽，凹槽用于放置待测样本，各凹槽靠近第一基板的底部设置有离子敏感膜，离子敏感膜可感应凹槽中发生的基因测序反应产生的离子并产生电压，从而与公共电极产生电场以驱动该液晶层中的液晶分子偏转。由此，该基因测序芯片可利用离子敏感膜感应碱基配对反应中产生的离子(例如，氢离子)并产生电压，并产生控制液晶层中液晶分 子偏转的电场，从而利用液晶光学开关技术来判断是否发生碱基配对反应，进而实现基因测序。而且，由于采用该基因测序芯片的基因测序技术无需对各种碱基进行荧光标记，也不需要激光光源和光学系统，因此，采用该基因测序芯片的基因测序技术的系统更简单，成本更低。
下面结合附图对本公开实施例提供的基因测序芯片、基因测序装置以及基因测序方法进行说明。
本公开一本实施例提供一种基因测序芯片。图1为根据本实施例的一种基因测序芯片；如图1所示，该基因测序芯片包括第一基板110、公共电极120、第二基板130以及液晶层140。第一基板110和第二基板130相对设置，液晶层140设置在第一基板110和第二基板130之间。第二基板130远离第一基板110的一侧包括至少一个凹入第二基板130的凹槽136，凹槽136可放置待测样本并用于对待测样本进行基因测序；凹槽136靠近第一基板110的底部设置有离子敏感膜132，离子敏感膜132可感应凹槽136中发生的基因测序反应，例如碱基配对反应产生离子并产生电压以与公共电极120形成电场，以控制位于第二基板130靠近第一基板110的一侧的液晶层140中的液晶分子发生旋转。
在本实施例提供的基因测序芯片中，可利用离子敏感膜感应凹槽中发生的基因测序反应，例如碱基配对反应，产生的离子(例如，氢离子)并产生电压，例如能斯特电压，并产生控制液晶层中液晶分子偏转的电场(例如，与公共电极产生电场)，从而利用液晶光学开关技术来判断是否发生碱基配对反应，进而实现基因测序。例如，如图1所示，将待测样本210放入凹槽136中，并依次向凹槽136加入四种不同的脱氧核糖核苷三磷酸220。如图1中左边的凹槽所示，若待测样本与当前加入的脱氧核糖核苷三磷酸发生碱基配对反应，离子敏感膜感应碱基配对反应释放的离子(例如，氢离子)并可产生电压，从而与公共电极形成电场并控制该凹槽所在位置处的液晶层中的液晶分子偏转；如图1中右边的凹槽所示，若待测样本与当前加入的脱氧核糖核苷三磷酸不发生碱基配对反应，离子敏感膜不产生电压，该凹槽所在位置处的液晶层中的液晶分子不偏转。此时，可通过判断通过该凹槽所在位置处的液晶层的光线的偏振方向或者旋向是否发生改变(例如，向该液晶层的一侧照射偏振光，并在该液晶层的另一侧通过检偏器和光感应装置来检测是否有光通过)来判断离子敏感膜上是否有电压，从而判断待测样本与当前加入的脱氧核糖核苷三磷酸是否发生碱基配对反应，进而可实现基因测序。值得注意的是，由于采用该基因测序芯 片的基因测序技术无需对各种碱基进行荧光标记，也不需要激光光源和光学系统，因此，采用该基因测序芯片的基因测序技术的系统更简单，成本更低。需要说明的是，由于单个待测样本与脱氧核糖核苷三磷酸发生的碱基配对反应释放的离子较少，可通过对待测样本进行扩增，同时发生多次碱基配对反应，从而使得离子敏感膜能够感应并产生电压。
例如，第一基板可包括玻璃基板、塑料基板或其他透明基板，从而便于透光。
例如，公共电极可选用透明金属电极，例如，氧化铟锡(ITO)电极。当然，本公开实施例包括但不限于此，公共电极也可采用不透明电极，通过在公共电极上设置多个开口以实现透光。
例如，凹槽可通过刻蚀第二基板而形成。当然，本公开实施例包括但不限于此，凹槽也可采用其他方法形成。
例如，在本实施例一示例提供基因测序芯片中，凹槽平行于第一基板的横截面的形状包括圆形和正多边形至少之一。当然，本公开实施例包括但不限于此。
例如，在本实施例一示例提供基因测序芯片中，凹槽平行于第一基板的横截面的最大尺寸的范围为10-100μm。需要说明的是，当凹槽平行于第一基板的横截面为圆形时，最大尺寸为圆形的直径，当凹槽的横截面为正多边形时，最大尺寸为正多边形的对角线。
例如，在本实施例一示例提供的基因测序芯片中，离子敏感膜可包括氢离子敏感膜。需要说明的是，氢离子敏感膜的电位可响应于氢离子而变化。例如，氢离子敏感膜可利用固定在氢离子敏感膜上的氢离子识别材料，例如四氮化三硅(Si3N4)有选择性地结合氢离子，从而发生膜电位或膜电流的改变。当然，本公开实施例包括但不限于此，离子敏感膜也可根据实际情况采用其他离子敏感膜。
例如，氢离子敏感膜为透明的，从而便于观察偏振光的透过情况。
例如，氢离子敏感膜的材料包括有机材料或无极材料。例如，氢离子敏感膜的材料可选自氮化硅(SiNx)、锂玻璃、二氧化硅(SiO2)以及氧化铝(Al2O3)中的一种或多种。
例如，在本实施例一示例提供的基因测序芯片中，如图1所示，公共电极120设置在第一基板110靠近液晶层140的一侧。由此，当离子敏感膜132产 生电压时，可与公共电极120产生垂直于液晶层140的电场，从而驱动液晶层140中的液晶分子偏转。需要说明的是，图1示出的公共电极120整面设置在第一基板110上，从而可减少对公共电极120进行图案化的工艺。然而，本公开实施例包括但不限于此，公共电极也可与离子敏感膜对应设置。也就是说，公共电极仅设置在凹槽所在位置处的第一基板上。
例如，在本实施例一示例提供基因测序芯片中，如图1所示，该基因测序芯片还包括封框胶190，设置在第一基板110和第二基板130之间并位于第一基板110的周边区域以将液晶层140密封在第一基板110和第二基板130之间。
例如，在本实施例一示例提供的基因测序芯片中，至少一个凹槽包括多个凹槽，多个凹槽在第二基板上呈阵列设置。由此，通过设置多个凹槽可同时对多个待测样本进行检测，从而大大提高基因测序的效率。另外，多个凹槽在第二基板上呈阵列设置便于对多个凹槽进行编号。
本公开一实施例还提供一种基因测序芯片。图2a示出了根据本实施例的一种基因测序芯片。如图2a所示，与实施例一不同的是，公共电极120包括多个条状公共电极1200，公共电极120和离子敏感膜132同层设置，各条状公共电极1200设置在相邻的凹槽136之间。由此，当离子敏感膜产生电压时，设置在相邻的凹槽之间条状公共电极可与离子敏感膜产生横向电场，驱动液晶层的液晶分子偏转。此时，可通过判断通过该凹槽所在位置处的液晶层的光线的偏振方向或者旋向是否发生改变(例如，向该液晶层的一侧照射偏振光，并在该液晶层的另一侧通过检偏器和光感应装置来检测是否有光通过)来判断离子敏感膜上是否有电压，从而判断待测样本与当前加入的脱氧核糖核苷三磷酸是否发生碱基配对反应，进而可实现基因测序。
例如，图2b示出了根据本实施例的一种基因测序芯片。如图2b所示，公共电极120包括多个条状公共电极1200，离子敏感膜132包括多个条状敏感膜1320，公共电极120和离子敏感膜132同层设置，条状公共电极1200和条状敏感膜1320交替间隔设置在凹槽136的底部。由此，当离子敏感膜产生电压时，条状公共电极和条状敏感膜可产生横向电场，驱动液晶层的液晶分子偏转。此时，可通过判断通过该凹槽所在位置处的液晶层的光线的偏振方向或者旋向是否发生改变(例如，向该液晶层的一侧照射偏振光，并在该液晶层的另一侧通过检偏器和光感应装置来检测是否有光通过)来判断离子敏感膜上是否有电压，从而判断待测样本与当前加入的脱氧核糖核苷三磷酸是否发生碱基配对反 应，进而可实现基因测序。
例如，图3为根据本实施例的一种基因测序芯片中凹槽的平面示意图。如图3所示，在本实施例一实施例提供的基因测序芯片中，公共电极120包括多个条状公共电极1200，多个条状公共电极1200通过公共电极连接部1201相连。离子敏感膜132包括多个条状敏感膜1320，多个条状公共电极1320通过离子感应膜连接部1321相连。公共电极120和离子敏感膜132同层设置，条状公共电极1200和条状敏感膜1320交替间隔设置在凹槽136的底部。当然，本公开实施例包括但不限于此，公共电极和离子敏感膜同层设置时还可采用其他形状，只要条状公共电极和条状敏感膜可产生横向电场，驱动液晶层的液晶分子偏转即可。
本公开一实施例还提供一种基因测序芯片。图4示出了根据本实施例的一种基因测序芯片。如图4所示，该基因测序芯片还包括第一偏光片181和第二偏光片182。第一偏光片181和第二偏光片182设置在液晶层140的两侧，第一偏光片181的透过轴与第二偏光片182的透过轴相互垂直或旋向相反。需要说明的是，当第一偏光片和第二偏光片为线偏光片时，第一偏光片的透过轴与第二偏光片的透过轴相互垂直，当第一偏光片和第二偏光片为圆偏转片或椭圆偏振片时，第一偏光片的透过轴与第二偏光片的透过轴旋向相反。
在本实施例提供的基因测序芯片中，由于在液晶层的两侧设置了第一偏光片和第二偏光片，并且第一偏光片的透过轴与第二偏光片的透过轴相互垂直或旋向相反。当液晶层中的液晶分子不发生偏转时，从液晶层的一侧入射的光无法从该液晶层的另一侧出射。然而，当凹槽中的待测样本与当前加入的脱氧核糖核苷三磷酸发生碱基配对反应，离子敏感膜感应碱基配对反应释放的离子(例如，氢离子)并可产生电压，从而与公共电极形成电场并控制该凹槽所在位置处的液晶层中的液晶分子偏转时，在该凹槽所在的位置处，从液晶层的一侧入射的光可以从该液晶层的另一侧出射。由此，可通过观察或检测是否有光出射便可判断凹槽中的待测样本与当前加入的脱氧核糖核苷三磷酸是否发生碱基配对反应。因此，采用该基因测序芯片的基因测序技术操作简单，成本较低。
值得注意的是，本公开实施例包括但不限于此，在本实施例提供的基因测序芯片中，第一偏光片的透过轴与第二偏光片的透过轴方向可以相同，当液晶层中的液晶分子不发生偏转时，从液晶层的一侧入射的光可以从该液晶层的另 一侧出射。然而，当凹槽中的待测样本与当前加入的脱氧核糖核苷三磷酸发生碱基配对反应，离子敏感膜感应碱基配对反应释放的离子(例如，氢离子)并可产生电压，从而与公共电极形成电场并控制该凹槽所在位置处的液晶层中的液晶分子偏转时，在该凹槽所在的位置处，从液晶层的一侧入射的光无法从该液晶层的另一侧出射。由此，可通过观察或检测是否有光出射便可判断凹槽中的待测样本与当前加入的脱氧核糖核苷三磷酸是否发生碱基配对反应。
例如，在本实施例一示例提供的基因测序芯片中，该基因测序芯片还可包括背光源170，背光源170可在第一偏光片181远离第二偏光片182的一侧；或者，背光源170也可设置在第二偏光片182远离第一偏光片181的一侧。例如，如图4所示，背光源170设置在第一偏光片181远离第二偏光片182的一侧。由此，可将背光源集成在该基因测序芯片中，从而扩大该基因检测基板的使用范围。
例如，在本实施例一示例提供的基因测序芯片中，如图4所示，该基因测序芯片还可包括第三基板150，设置在第二基板130远离第一基板110的一侧。第三基板150包括至少一个流道163，流道163与凹槽136连通。由此，可通过第三基板对凹槽进行一定的保护，提供一个相对稳定的反应环境。另外，还可通过流道同时向多个凹槽加入四种不同的脱氧核糖核苷三磷酸。
例如，在本实施例一示例提供的基因测序芯片中，如图4所示，该基因测序芯片还包括进样口161和出样口162，进样口161和出样口162设置在第三基板150上并与流道163连通。由此，可通过进样口加入四种不同的脱氧核糖核苷三磷酸或洗涤剂，可通过出样口排出四种不同的脱氧核糖核苷三磷酸或洗涤剂。
图5示出了一种根据本实施例的基因测序芯片的平面示意图。如图5所示，至少一个凹槽136包括多个凹槽136，多个凹槽136呈阵列设置。
例如，如图5所示，第三基板150包括多个流道163，分别与阵列设置的多个凹槽136的各行对应，并且各流道163至少与一个进样口161和一个出样口162相连通。
图6示出了一种根据本实施例的基因测序芯片的工作原理图。如图6所示，将待测样本放入凹槽中，并依次向凹槽加入四种不同的脱氧核糖核苷三磷酸。如图6中左边的凹槽所示，若待测样本与当前加入的脱氧核糖核苷三磷酸发生碱基配对反应，离子敏感膜感应碱基配对反应释放的离子(例如，氢离子)并 可产生电压，从而与公共电极形成电场并控制该凹槽所在位置处的液晶层中的液晶分子偏转；如图6中右边的凹槽所示，若待测样本与当前加入的脱氧核糖核苷三磷酸不发生碱基配对反应，离子敏感膜不产生电压，该凹槽所在位置处的液晶层中的液晶分子不偏转。此时，从该基因测序芯片一侧入射的光在图6中左边的凹槽所在位置处射出，而从该基因测序芯片一侧入射的光在图6中其他位置无法从该基因测序芯片射出。由此，可在该基因测序芯片的另一侧观察或检测各凹槽所在位置处是否有光出射便可判断该凹槽中的待测样本与当前加入的脱氧核糖核苷三磷酸是否发生碱基配对反应。记录当前加入的脱氧核糖核苷三磷酸的种类，便可获知该凹槽中待测样本当前的碱基类型。通过多次上述的过程便可获取待测样本的碱基序列。
本公开一实施例提供一种基因测序装置。图7示出了根据本实施例的一种基因测序装置。如图7所示，该基因测序装置包括根据上述实施例中任一项的基因测序芯片。该基因测序装置还包括感光装置，感光装置用于感测该基因测序芯片中至少一个凹槽所在位置处的出光。需要说明的是，上述的凹槽所在位置处的出光指环境光或背光源发出的光从该基因测序芯片一侧入射并透过该基因测序芯片上凹槽所在位置处的出光。由此，可通过感光装置判断从该基因测序芯片一侧入射的光是否从凹槽所在位置处射出。
例如，在本实施例一示例提供的基因测序装置中，感光装置可包括CCD图像传感器。由于CCD图像传感器的灵敏度较高，并且可将光学信号转换为模拟电流信号，便于使用计算机进行分析。
本公开一实施例提供一种基因测序芯片的基因测序方法。该基因测序芯片采用上述实施例中任一项所描述的基因测序芯片。图8示出了根据本实施例的一种基因测序方法。如图8所示，该基因测序方法包括步骤S501-S503。
步骤S501：在凹槽中放入待测样本。
步骤S502：依次向凹槽加入四种不同的脱氧核糖核苷三磷酸并分别通过离子敏感膜感应碱基配对反应释放的离子。
例如，离子敏感膜可通过与公共电极形成横向电场、纵向电场以及多维电场中的至少之一来控制液晶偏转。
步骤S503：检测液晶偏转的情况，并通过液晶偏转的情况判断发生配对反应的脱氧核糖核苷三磷酸。
在本实施例提供的基因测序方法中，若当前加入的脱氧核糖核苷三磷酸与 待测样本发生碱基配对反应，离子敏感膜可感应该碱基配对反应释放的离子并产生感应电压，此电压与公共电极产生电场，从而可通过液晶发生偏转来判断当前加入的脱氧核糖核苷三磷酸与待测样本发生碱基配对反应。若当前加入的脱氧核糖核苷三磷酸与待测样本没有发生碱基配对反应，离子敏感膜不会产生电压，液晶不偏转，从而可通过液晶不偏转判断当前加入的脱氧核糖核苷三磷酸与待测样本没有发生碱基配对反应。记录当前加入的脱氧核糖核苷三磷酸的种类，便可获知该凹槽中待测样本当前的碱基类型。通过多次上述的过程便可获取待测样本的碱基序列。
例如，通过依次向凹槽加入四种包括不同的碱基的脱氧核糖核苷三磷酸(例如，四种包括不同的碱基的脱氧核糖核苷三磷酸，其中四种碱基可为胞嘧啶、鸟嘌呤、腺嘌呤和胸腺嘧啶或者胞嘧啶、鸟嘌呤、腺嘌呤和尿嘧啶)，四种包括不同的碱基的脱氧核糖核苷三磷酸依次与凹槽中的待测样本，例如DNA片段相接触，当待测样本上的碱基与当前加入的脱氧核糖核苷三磷酸发生碱基配对时，释放离子，例如氢离子。此时，离子敏感膜可感应该碱基配对反应释放的离子并产生感应电压，此电压与公共电极产生电场，从而可通过液晶发生偏转来判断当前加入的脱氧核糖核苷三磷酸与待测样本发生碱基配对反应；经过多轮上述的步骤便可确定待测样本的基因序列。该基因测序方法不需要对四种碱基进行不同颜色的荧光标记便可实现基因测序，可简化基因测序的流程；并且采用该基因测序方法的系统更简单，成本更低，利于基因测序技术的推广和利用。
例如，脱氧核糖核苷三磷酸为可逆终止脱氧核糖核苷三磷酸，该基因测序方法还包括：清洗凹槽中加入的可逆终止脱氧核糖核苷三磷酸，并加入疏基试剂。在完成待测样本(例如，DNA片段)上一个位置的碱基类型检测后，需要清洗掉在凹槽中加入的可逆终止脱氧核糖核苷三磷酸，并加入疏基试剂。需要说明的是，与普通的脱氧核糖核苷三磷酸不同，可逆终止脱氧核糖核苷三磷酸的3′端连接一个叠氮基团，在DNA合成过程中不能形成磷酸二酯键，因而会中断DNA的合成，如果加入疏基试剂，叠氮基团就会断裂，并在原来位置形成一个羟基。在加入疏基试剂后可继续进行后续位置的碱基类型检测，检测方法与上述方法相同，在此不再赘述。
例如，当待测样本为DNA片段时，上述的可逆终止脱氧核糖核苷三磷酸可包括可逆终止三磷酸腺嘌呤脱氧核糖核苷酸、可逆终止三磷酸胸腺嘧啶脱氧 核糖核苷酸、可逆终止三磷酸胞嘧啶脱氧核糖核苷酸和可逆终止三磷酸鸟嘌呤脱氧核糖核苷酸。若凹槽中加入的且发生反应的脱氧核糖核苷三磷酸为三磷酸腺嘌呤脱氧核糖核苷酸，则此时待测样本(例如，DNA片段)上的碱基为胸腺嘧啶；如果凹槽中加入的且发生反应的脱氧核糖核苷三磷酸为三磷酸胸腺嘧啶脱氧核糖核苷酸，则此时待测样本(例如，DNA片段)上的碱基为腺嘌呤；如果凹槽中加入的且发生反应的脱氧核糖核苷三磷酸为三磷酸胞嘧啶脱氧核糖核苷酸，则此时待测样本(例如，DNA片段)上的碱基为鸟嘌呤；如果凹槽中加入的且发生反应的脱氧核糖核苷三磷酸为三磷酸鸟嘌呤脱氧核糖核苷酸，则此时待测样本(例如，DNA片段)上的碱基为胞嘧啶。
例如，在本实施例一示例提供的基因测序方法中，可通过感光装置和偏光片感测偏振光通过液晶的情况来检测液晶偏转的情况。例如，当偏光片的偏振方向与偏振光的偏振方向相互垂直或旋向相反时，若液晶不发生偏转，则感光装置无法感测到通过液晶的偏振光，若液晶发生偏转，偏振光的偏振方向因液晶的偏转而改变，则感光装置可感测到通过液晶的偏转光。需要说明的是，上述的偏振光可通过另外设置偏光片来产生。
例如，在本实施例一示例提供的基因测序方法中，在凹槽内放入待测样本可包括：对待测样本进行扩增以形成多个相同的待测样本；以及将多个相同的待测样本放入凹槽。由于单个待测样本与脱氧核糖核苷三磷酸发生的碱基配对反应释放的离子较少，可通过对待测样本进行扩增，多个相同的待测样本同时发生多次碱基配对反应，从而使得离子敏感膜能够感应并产生电压。
有以下几点需要说明：
(1)本公开实施例附图中，只涉及到与本公开实施例涉及到的结构，其他结构可参考通常设计。
(2)在不冲突的情况下，本公开同一实施例及不同实施例中的特征可以相互组合。
以上所述，仅为本公开的具体实施方式，但本公开的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本公开揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本公开的保护范围之内。因此，本公开的保护范围应以所述权利要求的保护范围为准。

Claims (18)

  1. 一种基因测序芯片，包括：
    第一基板；
    公共电极；
    第二基板，与所述第一基板相对设置；
    液晶层，设置在所述第一基板与所述第二基板之间，
    其中，所述第二基板远离所述第一基板的一侧包括至少一个凹入所述第二基板的凹槽，所述凹槽被配置为放置待测样本，各所述凹槽靠近所述第一基板的底部设置有离子敏感膜，所述离子敏感膜被配置为感应所述凹槽内发生的基因测序反应产生的离子以产生电压并与所述公共电极产生电场。
  2. 根据权利要求1所述的基因测序芯片，其中，所述离子敏感膜包括氢离子敏感膜。
  3. 根据权利要求1所述的基因测序芯片，其中，所述至少一个凹槽包括多个所述凹槽，所述多个凹槽在所述第二基板上呈阵列设置。
  4. 根据权利要求1-3中任一项所述的基因测序芯片，其中，所述公共电极设置在所述第一基板靠近所述液晶层的一侧。
  5. 根据权利要求1-3中任一项所述的基因测序芯片，其中，所述公共电极包括多个条状公共电极，所述公共电极和所述离子敏感膜同层设置，各所述条状公共电极设置在相邻的所述凹槽之间。
  6. 根据权利要求1-3中任一项所述的基因测序芯片，其中，所述公共电极包括多个条状公共电极，所述离子敏感膜包括多个条状敏感膜，所述公共电极和所述离子敏感膜同层设置，所述条状公共电极和所述条状敏感膜交替间隔设置在所述凹槽的底部。
  7. 根据权利要求1-6中任一项所述的基因测序芯片，还包括：
    第一偏光片；以及
    第二偏光片，
    其中，所述第一偏光片和所述第二偏光片设置在所述液晶层的两侧。
  8. 根据权利要求7所述的基因测序芯片，还包括：
    背光源，设置在所述第一偏光片远离所述第二偏光片的一侧，或者，设置在所述第二偏光片远离所述第一偏光片的一侧。
  9. 根据权利要求1-8中任一项所述的基因测序芯片，其中，所述离子敏感膜为透明的。
  10. 根据权利要求1-8中任一项所述的基因测序芯片，还包括：封框胶，设置在所述第一基板和所述第二基板之间并位于所述第一基板的周边区域以将所述液晶层密封在所述第一基板和所述第二基板之间。
  11. 根据权利要求1-8中任一项所述的基因测序芯片，其中，所述凹槽平行于所述第一基板的横截面的形状包括圆形和正多边形至少之一。
  12. 根据权利要求1-8中任一项所述的基因测序芯片，其中，所述凹槽的平行于所述第一基板的横截面的最大尺寸的范围为10-100μm。
  13. 根据权利要求1-8中任一项所述的基因测序芯片，还包括：
    第三基板，设置在所述第二基板远离所述第一基板的一侧；
    进样口；以及
    出样口，
    其中，所述第三基板包括至少一个流道，所述流道与所述凹槽连通，所述进样口和所述出样口设置在所述第三基板上并与所述流道连通。
  14. 一种基因测序装置，包括：
    基因测序芯片；以及
    感光装置，
    其中，所述基因测序芯片包括根据权利要求1-13中任一项所述的基因测序芯片，所述感光装置被配置为感测所述至少一个凹槽所在位置处的出光。
  15. 根据权利要求14所述的基因测序装置，其中，所述感光装置包括CCD图像传感器。
  16. 一种基因测序芯片的基因测序方法，所述基因测序芯片包括根据权利要求1-13中任一项所述的基因测序芯片，所述基因测序方法包括：
    在凹槽中放入待测样本；
    依次向所述凹槽加入四种不同的脱氧核糖核苷三磷酸并分别通过离子敏感膜感应碱基配对反应释放的离子；以及
    检测所述液晶偏转的情况，并通过所述液晶偏转的情况判断发生配对反应的脱氧核糖核苷三磷酸。
  17. 根据权利要求16所述的基因测序方法，其中，检测所述液晶偏转的情况，并通过所述液晶偏转的情况判断发生配对反应的脱氧核糖核苷三磷酸包 括：
    通过感光装置和偏光片感测偏振光通过所述液晶的情况来检测所述液晶偏转的情况。
  18. 根据权利要求16或17所述的基因测序方法，其中，所述脱氧核糖核苷三磷酸包括可逆终止脱氧核糖核苷三磷酸，所述基因测序方法还包括：清洗所述凹槽中加入的所述可逆终止脱氧核糖核苷三磷酸，并加入疏基试剂。
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