WO2019015315A1 - 基因测序结构、芯片、系统和基因测序方法 - Google Patents

基因测序结构、芯片、系统和基因测序方法 Download PDF

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WO2019015315A1
WO2019015315A1 PCT/CN2018/076265 CN2018076265W WO2019015315A1 WO 2019015315 A1 WO2019015315 A1 WO 2019015315A1 CN 2018076265 W CN2018076265 W CN 2018076265W WO 2019015315 A1 WO2019015315 A1 WO 2019015315A1
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electrode
gene sequencing
semiconductor layer
layer
sequencing structure
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PCT/CN2018/076265
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English (en)
French (fr)
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蔡佩芝
庞凤春
刘华哲
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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 US16/084,670 priority Critical patent/US11155866B2/en
Priority to JP2018548384A priority patent/JP6797931B2/ja
Priority to EP18765347.2A priority patent/EP3656843B1/en
Publication of WO2019015315A1 publication Critical patent/WO2019015315A1/zh

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    • 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
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
    • 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
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • 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
    • H—ELECTRICITY
    • H10—SEMICONDUCTOR DEVICES; ELECTRIC SOLID-STATE DEVICES NOT OTHERWISE PROVIDED FOR
    • H10N—ELECTRIC SOLID-STATE DEVICES NOT OTHERWISE PROVIDED FOR
    • H10N30/00—Piezoelectric or electrostrictive devices
    • H10N30/30—Piezoelectric or electrostrictive devices with mechanical input and electrical output, e.g. functioning as generators or sensors
    • H10N30/302—Sensors

Definitions

  • the present disclosure belongs to the field of gene sequencing and biological detection, and specifically relates to a gene sequencing structure, a gene sequencing chip, a gene sequencing system and a gene sequencing method.
  • Gene sequencing technology is a commonly used technique in modern molecular biology research. Gene sequencing can analyze the complete sequence of genes from blood or saliva, and predict the possibility of multiple diseases, such as cancer or leukemia. So far, gene sequencing technology has made considerable progress.
  • the current gene sequencing technologies include the first generation of sanger sequencing technology, the second generation of high-throughput sequencing technology, the third generation of single-molecule sequencing technology, and the current mainstream sequencing technology is still The second generation of high-throughput sequencing is the mainstay, single-molecule sequencing technology is still in the research and development stage, and there is no substantial commercialization progress.
  • the second-generation high-throughput sequencing technology mainly includes edge synthesis sequencing technology, ion semiconductor (semiconductor) sequencing technology, ligation sequencing technology and pyrosequencing technology, especially the side synthesis side sequencing technology as the mainstream, occupying the leading position in the market.
  • both sequencing and sequencing methods require fluorescent labeling of bases, complex laser sources and optical systems, which complicates the sequencing system and makes labeling reagents particularly expensive, leading to sequencing costs.
  • High in sequencing time, and increased sequencing time; pyrosequencing technology does not require laser light source and optical system, but also needs fluorescent labeling;
  • ion semiconductor sequencing technology requires CMOS technology to produce an ion sensor and two field effect transistors. It is difficult to make.
  • Embodiments of the present disclosure provide a gene sequencing structure, a gene sequencing chip, a gene sequencing system, and a gene sequencing method to alleviate or alleviate the above problems existing in the prior art.
  • the gene sequencing structure proposed by the embodiments of the present disclosure includes first and second electrodes, a semiconductor layer, a sensing electrode, an insulating layer, and a sensitive film layer spaced apart from each other.
  • the first electrode is connected to the second electrode via the semiconductor layer, the sensing electrode is in contact with the sensitive film layer, and the insulating layer separates each of the sensitive film layer and the sensing electrode
  • Each of the first electrode, the second electrode, and the semiconductor layer are isolated, wherein the sensitive film layer generates a charge in response to receiving ions generated by base pairing during gene sequencing.
  • the gene sequencing structure may further include a microporous layer above the sensitive membrane layer, and micropores for containing raw materials used in the sequencing process of the gene are formed in the microporous layer, the micropores The bottom surface of the hole is the upper surface of the sensitive film layer.
  • the material of the sensitive film layer comprises a material that generates a charge in response to receiving hydrogen ions.
  • the material of the sensitive film layer comprises silicon nitride.
  • the first electrode and the second electrode are located in the same layer of the genetic sequencing structure, the semiconductor layer covering a portion of the first electrode and a portion of the second electrode.
  • the semiconductor layer covers a portion of an upper surface of the first electrode and the second electrode, respectively, the insulating layer is disposed on a side of the semiconductor layer away from the first electrode and the second electrode .
  • the sensing electrode is disposed on a side of the insulating layer away from the semiconductor layer, and an orthographic projection of the sensing electrode on a horizontal plane at least partially overlaps with an orthographic projection of the semiconductor layer on a horizontal plane.
  • the sensitive film layer is disposed on a side of the sensing electrode away from the insulating layer.
  • the first electrode and the second electrode are identical in shape and are symmetrically disposed in mirror images of each other.
  • the first electrode and the second electrode are L-shaped in a cross section parallel to a horizontal plane, and the L-shaped openings of the first electrode and the second electrode are oppositely disposed.
  • the ends of the first electrode and the second electrode are respectively provided with a connecting line to introduce a test signal.
  • a cross-sectional shape of the sensing electrode parallel to a horizontal plane is the same as a cross-sectional shape of the semiconductor layer parallel to a horizontal plane, and an orthographic projection of the semiconductor layer on a horizontal plane falls into the sensing electrode at a horizontal plane. Inside the orthographic projection.
  • the pores range in size from 1 to 100 [mu]m.
  • a further embodiment of the present disclosure provides a gene sequencing chip comprising a plurality of gene sequencing structures as described in any of the preceding embodiments.
  • the plurality of gene sequencing structures are arranged in an array on a glass substrate.
  • the ends of the first electrode and the second electrode in each of the gene sequencing structures are respectively provided with a connecting line for introducing a test signal, wherein the first electrode is located in a peer or the same sequence of gene sequencing structures
  • the connecting lines are respectively connected to a first test disc at the periphery of the array of the gene sequencing structure, and the connecting lines of the second electrodes in the peer or the same sequence of the gene sequencing structure are respectively connected to the periphery of the array of the gene sequencing structure Two test disks.
  • a further embodiment of the present disclosure provides a gene sequencing system comprising the gene sequencing chip as described in the above embodiments and a test instrument detachably coupled to the gene sequencing chip.
  • the test instrument is configured to load a test signal to the first electrode and the second electrode of the genetic sequencing chip via a flexible circuit board, or to the genetic sequencing chip with a probe of the test instrument The first electrode and the second electrode load a test signal.
  • a further embodiment of the present disclosure provides a gene sequencing method based on the gene sequencing structure of any one of the foregoing embodiments of the gene sequencing structure of the present disclosure, the method comprising: placing a single strand of DNA to be tested a bottom of the micropore; applying a voltage to the first electrode or the second electrode; sequentially adding four deoxyribonucleotides to the micropore; detecting including the first electrode, the semiconductor layer, and Whether a current is generated in the loop of the second electrode to determine the type of base on the single strand of the DNA to be tested is determined according to the deoxyribonucleotide added when the current is generated.
  • the deoxyribonucleotide when performing the pairing reaction, does not need fluorescent labeling, and does not need a laser light source and an optical system; moreover, the gene sequencing structure can be used for preparation of the existing thin film transistor. The process is fully produced and the cost of the chip is lower. Correspondingly, the gene sequencing system and corresponding gene sequencing methods greatly reduce sequencing time and cost, improve efficiency, and simplify the sequencer equipment.
  • FIG. 1 is a cross-sectional view of a gene sequencing structure provided in accordance with an embodiment of the present disclosure
  • Figure 2 is a partial plan view of the gene sequencing structure of Figure 1;
  • FIG. 3 is a top plan view of a gene sequencing chip provided by an embodiment of the present disclosure.
  • FIG. 4 is a flow chart of a gene sequencing method provided by an embodiment of the present disclosure.
  • 5A and 5B are diagrams illustrating the principle of a gene sequencing method provided in accordance with an embodiment of the present disclosure.
  • 1-glass substrate 2-first electrode; 3-second electrode; 4-semiconductor layer; 5-insulating layer; 6-sensing electrode; 7-sensitive film layer; 8-microporous layer; 9-microporous;
  • the current gene sequencing method generally requires an optical system and a base fluorescent label, which leads to a problem of high cost of gene sequencing.
  • Embodiments of the present disclosure provide a gene sequencing structure, which has a simple structure and is beneficial for reducing genes. The cost of sequencing.
  • the gene sequencing structure includes first and second electrodes, a semiconductor layer, an insulating layer, a sensing electrode, and a sensitive film layer spaced apart from each other.
  • the first electrode is connected to the second electrode via a semiconductor layer
  • the sensing electrode and the sensitive film layer are in contact with the insulating layer
  • the insulating layer separates each of the sensitive film layer and the sensing electrode from the first electrode and the second electrode
  • Each phase in the semiconductor layer is isolated.
  • the sensitive membrane layer is configured to generate a charge in response to receiving ions generated by base pairing during gene sequencing.
  • the gene sequencing structure provided by the embodiments of the present disclosure may be the smallest functional unit in the gene test chip or the genetic test element.
  • the corresponding ions are usually produced.
  • hydrogen ions can be released, which can induce a charge on the surface of the sensitive membrane to generate an electric potential on the sensing electrode.
  • the semiconductor layer (including the p-doped semiconductor and the n-doped semiconductor) in the genetic test structure may be substantially in a non-conducting state, and when the sensing electrode is responsive to receipt in the gene sequencing
  • the semiconductor layer can be electrically conducted by the influence of the potential.
  • the gene sequencing structure may include a first electrode 2 and a second electrode 3 which are disposed at intervals; a semiconductor layer 4 which is in contact with the first electrode 2 and the second electrode 3, respectively, to connect the first electrode and the second electrode 3 is connected to each other; the insulating layer 5 is disposed on a side of the semiconductor layer 4 away from the first electrode and the second electrode; the sensitive film layer 7 is disposed above the insulating layer 5, and at least comprises responsive to receiving the pair base pairing A sensitive material that generates ions to generate electricity.
  • the gene sequencing structure further includes the sensing electrode 6 in contact with the sensitive film layer 7 and is isolated from the first electrode, the second electrode, and the semiconductor layer via the insulating layer 5. The sensing electrode 6 may be disposed on a side of the insulating layer 5 away from the first electrode and the second electrode.
  • the gene sequencing structure can be arranged above the glass substrate 1 to sense base pairing through the sensitive membrane layer 7 in the gene sequencing structure, so that the gene sequencing structure is simple, which is beneficial to reducing the cost of gene sequencing.
  • the gene sequencing structure may further include a microporous layer 8 located above the sensitive membrane layer 7, and a microporous layer 9 for accommodating the raw materials used in the sequencing process of the gene, microporous 9 is formed in the microporous layer 8.
  • the bottom surface is the upper surface of the sensitive film layer 7.
  • the first electrode 2 and the second electrode 3 have the same shape and are mirror-symmetrical to each other.
  • the first electrode 2 and the second electrode 3 have the same structure and function, so that there is no need to distinguish between functions when performing gene sequencing, which is convenient for testing.
  • the cross-sectional shape of the first electrode 2 and the second electrode 3 parallel to the horizontal plane is L-shaped, and the opening of the L-shaped cross section of the first electrode 2 and the second electrode 3 Relative settings. In this way, it is convenient to extract test lines for loading test signals from the genetic test structure.
  • the semiconductor layer 4 may include an a-Si material, a polysilicon material, or the like, and the semiconductor layer 4 has a cross-sectional shape of any one of a square, a rectangle, and a circle to facilitate sensing the presence of ions on the sensitive film layer.
  • the cross-sectional shape of the semiconductor layer 4 in the gene sequencing structure may also be other shapes, and is not limited by the examples herein.
  • the first electrode 2, the second electrode 3, the sensing electrode 6, and the semiconductor layer 4 form a structure similar to a top-gate thin film transistor, and the semiconductor layer 4 is appropriate. In the case of a conductive channel can be formed.
  • the sensitive film layer when the sensitive film layer generates a charge in response to receiving ions generated during the sequencing of the gene, when the potential is generated on the sensing electrode, the semiconductor layer 4 is in a conductive state, the first electrode 2, the semiconductor layer 4, and the second electrode 3 can form a conductive loop.
  • the shape of the cross section of the sensing electrode 6 parallel to the horizontal plane is the same as the cross-sectional shape of the semiconductor layer 4 parallel to the horizontal plane, and the orthographic projection of the semiconductor layer 4 on the horizontal plane falls on the horizontal surface of the sensing electrode 6.
  • the orthographic projection area of the sensing electrode 6 on the horizontal plane may be greater than or equal to the orthographic projection area of the semiconductor layer 4 on the horizontal plane.
  • the material forming the sensitive film layer 7 includes a material sensitive to hydrogen ions (H+), that is, a material that generates a charge in response to receiving hydrogen ions, although the present invention does not exclude the use of a base capable of sensing Other sensitive materials that cause ions to change when ions are paired to form a sensitive film layer.
  • the sensitive film layer 7 comprises silicon nitride.
  • the micropores 9 are regularly etched on the top of the gene sequencing structure, and the micropores 9 in the microporous layer 8 are on the order of micrometers, and the pores 9 have a size ranging from 1 to 100 ⁇ m.
  • the shape of the micropores is not limited in any way herein, and the size of the micropores refers to the maximum distance between points on the edge of the micropores.
  • the ends of the first electrode 2 and the second electrode 3 are respectively provided with a connecting line to introduce a test signal. That is, the first electrode 2 and the second electrode 3 are connected to a test line, and are connected to an external test instrument through a test line. In an embodiment, the first electrode 2 and the second electrode 3 may be respectively connected to a test pad by using a metal lead, and the test electrical signal may be loaded by using a flexible printed circuit (FPC), or directly used. Device probe mode loading.
  • FPC flexible printed circuit
  • ions for example, H+
  • the sensitive membrane layer can sense the presence of H+ to generate a charge, thereby causing the potential of the sensing electrode.
  • a change occurs, at which time the conductivity of the semiconductor layer can change (from no conductivity to conduction).
  • whether or not base pairing occurs can be determined based on whether or not a current change occurs in the loop of the first electrode and the second electrode.
  • the deoxyribonucleotide does not need to be fluorescently labeled, nor does it require a laser light source and an optical system; moreover, the gene sequencing structure can be fabricated by means of a thin film transistor fabrication process. Therefore, the cost of gene sequencing and the cost of making genetic sequencing products are low.
  • Another embodiment of the present disclosure provides a gene sequencing chip comprising a plurality of gene sequencing structures as described in any of the preceding embodiments.
  • the gene sequencing chip utilizes a sensitive membrane layer to sense base pairing, so that the structure of the gene sequencing chip is simple, and the cost of gene sequencing is reduced.
  • a plurality of gene sequencing structures 101 are arranged in an array on a glass substrate.
  • the plurality of gene sequencing structures 101 can also be uniformly distributed on the glass substrate.
  • the multiple gene sequencing structures 101 can also be other distribution methods, and are not limited herein.
  • the first line 2 of the first electrode 2 located in the peer or inline gene sequencing structure 101 can be first tested to the periphery of the array of the gene sequencing structure, respectively.
  • a disk; a connecting line 102 of the second electrode 3 located in the peer or inline gene sequencing structure 101 can be coupled to a second test disk at the periphery of the array of gene sequencing structures, respectively.
  • An external measuring instrument can apply a test electrical signal to the first electrode or the second electrode via the first test disc and the second test disc. In this way, it is beneficial to reduce the space of the gene sequencing chip and obtain better test efficiency.
  • Yet another embodiment of the present disclosure provides a gene sequencing system that can greatly reduce the time and cost of gene sequencing and improve the efficiency of gene sequencing.
  • the gene sequencing system comprises a gene sequencing chip and a test instrument detachably connected to the gene sequencing chip, and the gene sequencing chip can be the gene sequencing chip described in the foregoing embodiment.
  • the test instrument can load a test signal to the first electrode and the second electrode of the genetic sequencing chip through the flexible circuit board, or load the test signal to the first electrode and the second electrode of the genetic sequencing chip with the probe of the test instrument.
  • the manner in which the signals are loaded is not limited by the embodiments of the present disclosure.
  • the embodiments of the present disclosure also provide a gene sequencing method based on a gene sequencing structure, which is based on the gene sequencing structure described in the foregoing embodiments of the present disclosure, so that the cost of gene sequencing is greatly reduced.
  • the gene sequencing method comprises the steps of:
  • Step S1) The single strand of DNA to be tested is placed at the bottom of the microwell. As shown in FIG. 5A, the DNA single strand 10 to be tested is immobilized at the bottom of the microwell 9.
  • a voltage of 0.5 to 20 V can be applied to the first electrode 2.
  • the semiconductor layer 4 is substantially in an insulating state, and the current between the first electrode 2 and the second electrode 3 is approximately zero.
  • the sensing electrode 6 has an electric potential, the conductivity of the semiconductor layer 4 is greatly enhanced, and at this time, a certain current can be detected between the first electrode 2 and the second electrode 3.
  • Step S3) four kinds of deoxyribonucleotides are sequentially introduced into the micropores.
  • A, T, C, and G are four nucleotides in the DNA strand, wherein A is adenine, T is thymine, C is cytosine, and G is guanine.
  • the gene sequencing system and the corresponding gene sequencing method proposed in the embodiments of the present disclosure do not need fluorescent labeling, and do not require complicated laser light source and optical system, thereby greatly reducing sequencing time and cost, improving efficiency, and simplifying the sequencer device.

Abstract

本公开涉及基因测序结构、芯片、系统和方法。该基因测序结构包括：彼此间隔的第一电极和第二电极、半导体层、感应电极、绝缘层以及敏感膜层。所述第一电极经由所述半导体层连接至所述第二电极，所述感应电极与所述敏感膜层接触，所述绝缘层将所述敏感膜层和感应电极中的每个与所述第一电极、第二电极和半导体层中的每个相隔离，其中所述敏感膜层响应于接收到基因测序过程中的碱基配对产生的离子而产生电荷。

Description

基因测序结构、芯片、系统和基因测序方法
相关申请的交叉引用
本申请要求于2017年7月17日向中国专利局提交的专利申请201710580687.4的优先权利益，并且在此通过引用的方式将该在先申请的内容并入本文。
技术领域
本公开属于基因测序及生物检测领域，具体涉及一种基因测序结构、基因测序芯片、基因测序系统和基因测序方法。
背景技术
基因测序(Gene sequencing)技术是现代分子生物学研究中常用的技术，基因测序能够从血液或唾液中分析测定基因全序列，预测罹患多种疾病的可能性，如癌症或白血病。至今，基因测序技术取得了相当大的发展，当前的基因测序技术包括第一代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，目前市场主流的测序技术仍以第二代高通量测序为主，单分子测序技术还处在研发阶段，并未有实质的商业化进展。
第二代高通量测序技术主要包括边合成边测序技术、离子半导体(semiconductor)测序技术、连接法测序技术和焦磷酸测序技术等，尤其以边合成边测序技术为主流，占据市场龙头地位。其中，边合成边测序技术和连接法测序技术都需要对碱基进行荧光标记，还需要有复杂的激光光源和光学系统，这样使得测序系统变得复杂，而且标记化学试剂特别昂贵，导致测序成本居高不下，还增加测序时间；焦磷酸测序技术虽然无需激光光源和光学系统，但同样也需要进行荧光标记；离子半导体测序技术需要采用CMOS工艺制作一个离子传感器和两个场效应晶体管，工艺复杂，制作困难。
发明内容
本公开的实施例提供一种基因测序结构、基因测序芯片、基因测序系统和基因测序方法，以缓解或减轻现有技术中存在的上述问题。
本公开实施例提出的基因测序结构包括彼此间隔的第一电极和第二电极、半导体层、感应电极、绝缘层以及敏感膜层。所述第一电极经由所述半导体层连接至所述第二电极，所述感应电极与所述敏感膜层接触，所述绝缘层将所述敏感膜层和感应电极中的每个与所述第一电极、第二电极和半导体层中的每个相隔离，其中所述敏感膜层响应于接收到基因测序过程中的碱基配对产生的离子而产生电荷。
在本公开的实施例中，基因测序结构还可包括位于所述敏感膜层上方的微孔层，所述微孔层中形成有用于容纳基因测序过程中使用的原料的微孔，所述微孔的底面为所述敏感膜层的上表面。
进一步地，在一些实施例中，敏感膜层的材料包括响应于接收到氢离子而产生电荷的材料。在一个示例中，敏感膜层的材料包括四氮化三硅。
在一些实施例中，所述第一电极和第二电极位于所述基因测序结构的同一层，所述半导体层覆盖一部分第一电极和一部分第二电极。
在一些实施例中，所述半导体层分别覆盖所述第一电极和第二电极的上表面的一部分，所述绝缘层设置在所述半导体层远离所述第一电极和第二电极的一侧。
在一些实施例中，所述感应电极设置于所述绝缘层远离所述半导体层的一侧，且所述感应电极在水平面上的正投影与所述半导体层在水平面上的正投影至少部分重叠。
在一些实施例中，所述敏感膜层设置在所述感应电极远离所述绝缘层的一侧。
在一些实施例中，所述第一电极和所述第二电极的形状相同，且互为镜像对称设置。
在一些实施例中，所述第一电极和所述第二电极沿平行于水平面的横截面的形状为L型，且所述第一电极和所述第二电极的L型的开口相对设置。
在一些实施例中，所述第一电极和所述第二电极的端部分别设置有一根连接线，以引入测试信号。
在一些实施例中，所述感应电极平行于水平面的横截面形状与所述半导体层平行于水平面的横截面形状相同，且所述半导体层在水平面上的正投影落入所述感应电极在水平面上的正投影内。
在一些实施例中，微孔的尺寸范围为1～100μm。
本公开的另外的实施例提供了一种基因测序芯片，包括多个如前述实施例中任一实施例所述的基因测序结构。
在一些实施例中，所述多个基因测序结构呈阵列排布在玻璃基板上。
在一些实施例中，每个基因测序结构中的第一电极和第二电极的端部分别设置有一根用于引入测试信号的连接线，其中位于同行或同列的基因测序结构中的第一电极的连接线分别连接至处于基因测序结构的阵列外围的第一测试盘，所述位于同行或同列的基因测序结构中的第二电极的连接线分别连接至处于基因测序结构的阵列的外围的第二测试盘。
本公开的另外的实施例提供了一种基因测序系统，包括如上述实施例所述的基因测序芯片以及与所述基因测序芯片可拆卸连接的测试仪器。
在一些实施例中，所述测试仪器被配置成通过柔性线路板向所述基因测序芯片的第一电极和第二电极加载测试信号，或者以所述测试仪器的探针向所述基因测序芯片的第一电极和第二电极加载测试信号。
本公开的又一实施例提供了一种基于本公开前述基因测序结构实施例中任一实施例所述的基因测序结构的基因测序方法，该方法包括：将待测的DNA单链置于所述微孔的底部；向所述第一电极或所述第二电极施加电压；向所述微孔依次加入四种脱氧核糖核苷酸；检测包括所述第一电极、所述半导体层和所述第二电极的回路中是否产生电流，以根据产生电流时加入的脱氧核糖核苷酸确定所述待测DNA单链上的碱基类型。
基于本公开实施例提供的基因测序结构，在进行配对反应时，脱氧核糖核苷酸无需荧光标记，也不需要激光光源和光学系统；而且，该基因测序结构可借鉴现有的薄膜晶体管的制备工艺进行完整制作，芯片成本更低。相应的，该基因测序系统和相应的基因测序方法，大大降低测序时间和成本，提升效率，同时简化测序仪设备。
附图说明
图1为根据本公开的实施例提供的基因测序结构的剖视图；
图2为图1所述的基因测序结构的局部俯视图；
图3为本公开的实施例提供的基因测序芯片的俯视图；
图4为本公开实施例提供的基因测序方法的流程图；
图5A和图5B用于图示根据本公开实施例提供的基因测序方法的原理。
具体实施方式
为使本领域技术人员更好地理解本公开实施例的技术方案，下面结合附图对本公开实施例提供的基因测序结构、基因测序芯片、基因测序系统和基因测序方法作进一步详细描述。
在下面的描述中，可能使用以下附图标记：
1-玻璃基板；2-第一电极；3-第二电极；4-半导体层；5-绝缘层；6-感应电极；7-敏感膜层；8-微孔层；9-微孔；
10-待测DNA单链；11-配对碱基；
101-基因测序结构；102-连接线。
针对目前的基因测序方式通常需要光学系统及碱基荧光标记，导致基因测序成本居高不下的问题，本公开的实施例提供一种基因测序结构，该基因测序结构的结构简单，有利于降低基因测序的成本。
根据本公开的一个实施例，该基因测序结构包括彼此间隔的第一电极和第二电极、半导体层、绝缘层、感应电极以及敏感膜层。第一电极经由半导体层连接至第二电极，感应电极与所述敏感膜层接触绝缘层，绝缘层将所述敏感膜层和感应电极中的每个与所述第一电极、第二电极和半导体层中的每个相隔离。敏感膜层被配置成响应于接收到基因测序过程中的碱基配对而产生的离子而产生电荷。
本公开实施例所提供的基因测序结构可以是基因测试芯片或基因测试元件中的最小功能单元。在基因测序过程中，通常产生会产生相应的离子。例如，当样本基因与所加入的核苷酸配对时，可释放出氢离子，这些氢离子可以在敏感膜层表面感应出电荷，从而在感应电极上产生电势。在感应电极没有电势的情况下，基因测试结构中的半导体层(包括p型掺杂半导体和n型掺杂半导体)可能基本上处于不导电的状态，而当感应电极响应于接收到在基因测序过程中产生的离子 而具有电势时，半导体层可受该电势的影响而导电。此时，通过向第一电极和第二电极施加测试信号，可以检测到半导体层受感应电极的电势的影响而导电这一事实的存在，从而判断样本基因与加入的核苷酸发生配对，进而实现基因测试。
下面通过具体的示例来详细说明本公开实施例提出的基因测序结构。
如图1所示，基因测序结构可包括间隔设置的第一电极2和第二电极3；半导体层4，其分别与第一电极2和第二电极3接触以将第一电极和第二电极3彼此连接；绝缘层5，设置于半导体层4远离第一电极和第二电极的一侧；敏感膜层7，设置于绝缘层5的上方，其至少包含有响应于接收到对碱基配对产生的离子而产生电荷的敏感材料。基因测序结构还包括感应电极6与敏感膜层7接触，并经由绝缘层5而与第一电极、第二电极和半导体层相隔离。感应电极6可设置于绝缘层5远离第一电极和第二电极的一侧。
基因测序结构整体可以设置在玻璃基板1的上方，通过基因测序结构中的敏感膜层7来感测碱基配对，使得基因测序结构简单，有利于降低基因测序成本。
在图1的示例中，基因测序结构还可包括位于敏感膜层7上方的微孔层8，微孔层8中形成有用于容纳基因测序过程中使用的原料的微孔9，微孔9的底面为敏感膜层7的上表面。由此，在进行基因测序时，可以向微孔9加入样本基因、各种核苷酸等基因测序用原料。
在一些实施例中，第一电极2和第二电极3的形状相同，且互为镜像对称设置。这里，第一电极2和第二电极3结构、功能相同，因此在进行基因测序时无需做功能区分，方便测试。
在一些实施例中，如图2所示，第一电极2和第二电极3的平行于水平面的横截面形状为L型，且第一电极2和第二电极3的L型横截面的开口相对设置。如此，可以方便从基因测试结构引出用于加载测试信号的测试线。
半导体层4可以包括a-Si材料、多晶硅材料等，半导体层4的横截面形状为正方形、矩形、圆形中的任一种，以利于感测敏感膜层上的离子的存在。当然，基因测序结构中半导体层4的横截面形状也可以为其他的形状，并不受这里的示例的限制。本图1和图2所示的基 因测序结构中，第一电极2、第二电极3、感应电极6和半导体层4形成了一种类似于顶栅式薄膜晶体管的结构，半导体层4在适当的情况下可形成导电沟道。例如，当敏感膜层响应于接收到在基因测序过程中产生的离子而产生电荷，在感应电极上产生电势时，半导体层4处于导电状态，第一电极2、半导体层4和和第二电极3可形成导电回路。
在一些实施例中，感应电极6的平行于水平面的横截面的形状与半导体层4平行于水平面的横截面形状相同，且半导体层4在水平面上的正投影落入感应电极6在水平面上的正投影内。也就是说，感应电极6在水平面上的正投影面积可以大于或等于半导体层4在水平面上的正投影面积。由此，可以使得即使在制作基因测序结构的过程中感应电极6和半导体层4的对位出现少许偏差的情况下，半导体层4也可以尽可能地被感应电极6覆盖，使得半导体层4的功能充分发挥出来，从而获得较佳的测试效果。
在一些本实施例中，形成敏感膜层7的材料包括对氢离子(H+)敏感的材料，即，响应于接收到氢离子而产生电荷的材料，当然本发明不排除采用能够在感知到碱基配对时产生的离子而引起电压变化的其他敏感材料来形成敏感膜层。在一个示例中，敏感膜层7包括四氮化三硅。
在图1的示例中，在基因测序结构的顶部规则刻蚀出微孔9，微孔层8中的微孔9为微米级，微孔9的尺寸范围为1～100μm。本文并不对微孔的形状作任何的限制，微孔的尺寸指的是微孔的边缘上的各个点之间的最大距离。
在实施例中，第一电极2和第二电极3的端部分别设置有一根连接线，以引入测试信号。即，第一电极2和第二电极3连接至测试线，通过测试线连接至外部的测试仪器。在实施例中，可以用金属引线分别将第一电极2和第二电极3连接到测试盘(pad)，测试电信号可以使用柔性线路板(Flexible Printed Circuit，简称FPC)方式加载，或直接用设备探针方式加载。
基于以上实施例所述的基因测序结构，当发生碱基配对时，可以释放出离子(例如，H+)至敏感膜层，敏感膜层感知H+的存在后可产生电荷，从而引起感应电极的电势发生变化，此时，半导体层的导电性可发生变化(从不导电变成导电)。由此，可根据第一电极和第二 电极的回路中是否发生电流变化，从而判断碱基配对是否发生。
利用该基因测序结构进行碱基配对的检测时，脱氧核糖核苷酸无需荧光标记，也不需要激光光源和光学系统；而且，该基因测序结构可借助于薄膜晶体管的制备工艺进行制作。因此基因测序的成本以及制作基因测序产品的成本都较低。
本公开的另一实施例提供一种基因测序芯片，该基因测序芯片包括多个如前述实施例中任一实施例所述的基因测序结构。该基因测序芯片利用敏感膜层来感测碱基配对，使得基因测序芯片的结构简单，基因测序成本得以降低。
如图3所示，在一些实施例中，多个基因测序结构101呈阵列排布在玻璃基板上。多个基因测序结构101也可以均匀地分布在玻璃基板上。当然，多个基因测序结构101也可以为其他的分布方式，这里并不做限制。
在具有多个基因测序结构101的基因测试芯片的实施例中，位于同行或同列的基因测序结构101中的第一电极2的连接线102可分别至处于基因测序结构的阵列外围的第一测试盘；位于同行或同列的基因测序结构101中的第二电极3的连接线102可分别连接至处于基因测序结构的阵列的外围的第二测试盘。外部的测量仪器可经由第一测试盘和第二测试盘向第一电极或第二电极施加测试电信号。这样，可有利于减小基因测序芯片的空间，获得较佳的测试效率。
本公开的又一实施例提供一种基因测序系统，可大大降低基因测序的时间和成本，提升基因测序的效率。
该基因测序系统包括基因测序芯片以及与基因测序芯片可拆卸连接的测试仪器，基因测序芯片可以为前述实施例所述的基因测序芯片。
测试仪器可通过柔性线路板向基因测序芯片的第一电极和第二电极加载测试信号，或者以测试仪器的探针向基因测序芯片的第一电极和第二电极加载测试信号。这里，信号的加载方式并不受本公开实施例的限制。
本公开实施例还提供一种基于基因测序结构的基因测序方法，该方法基于本公开中前述实施例所述的基因测序结构，使得基因测序的成本大大降低。
如图4所示，该基因测序方法包括步骤：
步骤S1)：将待测的DNA单链置于微孔的底部。如图5A所示：将待测DNA单链10固定在微孔9的底部。
步骤S2)：向第一电极或第二电极施加电压。例如，可以向第一电极2施加0.5～20V的电压。当敏感膜层没有产生电荷时，半导体层4基本上处于绝缘状态，第一电极2和第二电极3之间的电流近似于0。当感应电极6具有电势时，半导体层4的导电性大大增强，此时，可以检测到第一电极2和第二电极3之间有一定的电流。
步骤S3)：向微孔依次通入四种脱氧核糖核苷酸。
在进行DNA测序时，可以依次向微孔加入A、T、C、G四种配对碱基11。其中，A、T、C、G为DNA链中的四种核苷酸，其中A为腺嘌呤，T为胸腺嘧啶，C为胞嘧啶，G为鸟嘌呤。
步骤S4)：检测包括第一电极、半导体层和第二电极的回路中是否产生电流，以根据产生电流时加入的脱氧核糖核苷酸确定待测DNA单链上的碱基类型。
在一个示例中，如图5B所示，当配对碱基11与待测DNA链10发生配对反应时会释放氢离子(H+)，从而引起微孔9中PH值发生变化，而微孔9底部的敏感膜层7探测到这种变化后会产生电荷，从而引起感应电极6的电势升高，感应电极6的电势升高后，半导体层4的导电性发生变化(例如，由绝缘态变成导通态)，在第一电极2和第二电极3间加载外部电压的情况下，第一电极、半导体层和第二电极的回路中产生电流。因此，当检测到电流变化时，便可判断发生碱基配对反应，从而测出未知的DNA序列。
本公开实施例提出的基因测序系统和相应的基因测序方法，碱基无需荧光标记，也不需要复杂的激光光源和光学系统，大大降低测序时间和成本，提升效率，同时简化测序仪设备。
可以理解的是，以上实施例仅仅是为了说明本公开的原理而采用的示例性实施方式，然而本发明并不局限于此。对于本领域内的普通技术人员而言，在不脱离本公开的精神和实质的情况下，可以做出各种变型和改进，这些变型和改进也视为本发明的保护范围。
通过研究附图、公开内容和随附权利要求，本领域技术人员在实践所要求保护的发明时，可以理解和实现对所公开的实施例的其它变型。在权利要求中，词语“包括”不排除其它元件或步骤，并且不定 冠词“一”或“一个”不排除多个。即使某些特征被记载在不同的从属权利要求中，本发明也涉及共同包括这些特征的实施例。权利要求中的任何参考标记不应当解释为限制范围。

Claims (19)

  1. 一种基因测序结构，包括：彼此间隔的第一电极和第二电极、半导体层、感应电极、绝缘层以及敏感膜层，
    其中所述第一电极经由所述半导体层连接至所述第二电极，所述感应电极与所述敏感膜层接触，所述绝缘层将所述敏感膜层和感应电极中的每个与所述第一电极、第二电极和半导体层中的每个相隔离，其中所述敏感膜层响应于接收到基因测序过程中的碱基配对产生的离子而产生电荷。
  2. 根据权利要求1所述的基因测序结构，其中所述基因测序结构还包括位于所述敏感膜层上方的微孔层，所述微孔层中形成有用于容纳基因测序过程中使用的原料的微孔，所述微孔的底面为所述敏感膜层的上表面。
  3. 根据权利要求1-2中任一项所述的基因测序结构，其中所述敏感膜层的材料包括响应于接收到氢离子而产生电荷的材料。
  4. 根据权利要求3所述的基因测序结构，其中所述敏感膜层的材料包括四氮化三硅。
  5. 根据权利要求1-2中任一项所述的基因测序结构，其中所述第一电极和第二电极位于所述基因测序结构的同一层，所述半导体层覆盖一部分第一电极和一部分第二电极。
  6. 根据权利要求1-2中任一项所述的基因测序结构，其中所述半导体层分别覆盖所述第一电极和第二电极的上表面的一部分，所述绝缘层设置在所述半导体层远离所述第一电极和第二电极的一侧。
  7. 根据权利要求6所述的基因测序结构，其中所述感应电极设置于所述绝缘层远离所述半导体层的一侧，且所述感应电极在水平面上的正投影与所述半导体层在水平面上的正投影至少部分重叠。
  8. 根据权利要求7所述的基因测序结构，其中所述敏感膜层设置在所述感应电极远离所述绝缘层的一侧。
  9. 根据权利要求1-2中任一项所述的基因测序结构，其中所述第一电极和所述第二电极的形状相同，且互为镜像对称设置。
  10. 根据权利要求9所述的基因测序结构，其中所述第一电极和所述第二电极沿平行于水平面的横截面的形状为L型，且所述第一电极 和所述第二电极的L型的开口相对设置。
  11. 根据权利要求1-2中任一项所述的基因测序结构，其中所述第一电极和所述第二电极的端部分别设置有一根连接线，以引入测试信号。
  12. 根据权利要求1-2中任一项所述的基因测序结构，其中所述感应电极平行于水平面的横截面形状与所述半导体层平行于水平面的横截面形状相同，且所述半导体层在水平面上的正投影落入所述感应电极在水平面上的正投影内。
  13. 根据权利要求2所述的基因测序结构，其中所述微孔的尺寸范围为1～100μm。
  14. 一种基因测序芯片，包括多个如权利要求1-13中任一项所述的基因测序结构。
  15. 根据权利要求14所述的基因测序芯片，其中所述多个基因测序结构呈阵列排布在玻璃基板上。
  16. 根据权利要求15所述的基因测序芯片，其中每个基因测序结构中的第一电极和第二电极的端部分别设置有一根用于引入测试信号的连接线，其中位于同行或同列的基因测序结构中的第一电极的连接线分别连接至处于基因测序结构的阵列外围的第一测试盘，所述位于同行或同列的基因测序结构中的第二电极的连接线分别连接至处于基因测序结构的阵列的外围的第二测试盘。
  17. 一种基因测序系统，包括如权利要求14-16中任一项的所述基因测序芯片以及与所述基因测序芯片可拆卸连接的测试仪器。
  18. 根据权利要求17所述的基因测序系统，其中所述测试仪器被配置成通过柔性线路板向所述基因测序芯片的第一电极和第二电极加载测试信号，或者以所述测试仪器的探针向所述基因测序芯片的第一电极和第二电极加载测试信号。
  19. 一种基于权利要求2-13中任一项的所述基因测序结构的基因测序方法，包括：
    将待测的DNA单链置于所述微孔的底部；
    向所述第一电极或所述第二电极施加电压；
    向所述微孔依次加入四种脱氧核糖核苷酸；
    检测包括所述第一电极、所述半导体层和所述第二电极的回路中 是否产生电流，以根据产生电流时加入的脱氧核糖核苷酸确定所述待测DNA单链上的碱基类型。
PCT/CN2018/076265 2017-07-17 2018-02-11 基因测序结构、芯片、系统和基因测序方法 WO2019015315A1 (zh)

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