WO2016206593A1 - 微孔电极及分析化学物质的方法 - Google Patents

微孔电极及分析化学物质的方法 Download PDF

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WO2016206593A1
WO2016206593A1 PCT/CN2016/086846 CN2016086846W WO2016206593A1 WO 2016206593 A1 WO2016206593 A1 WO 2016206593A1 CN 2016086846 W CN2016086846 W CN 2016086846W WO 2016206593 A1 WO2016206593 A1 WO 2016206593A1
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Prior art keywords
electrode
support member
material layer
molecule
microporous
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PCT/CN2016/086846
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English (en)
French (fr)
Inventor
李汉东
林建勋
云全新
向少华
德马纳克•拉多杰
德马纳克•斯内扎娜
张永卫
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深圳华大基因研究院
深圳华大基因科技有限公司
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Priority to EP16813709.9A priority Critical patent/EP3315461B1/en
Priority to JP2017566337A priority patent/JP6818995B2/ja
Priority to ES16813709T priority patent/ES2882583T3/es
Priority to CN201680021734.5A priority patent/CN107683337B/zh
Priority to DK16813709.9T priority patent/DK3315461T3/da
Priority to US15/738,114 priority patent/US20180180567A1/en
Publication of WO2016206593A1 publication Critical patent/WO2016206593A1/zh
Priority to HK18106691.9A priority patent/HK1247253A1/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
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81B—MICROSTRUCTURAL DEVICES OR SYSTEMS, e.g. MICROMECHANICAL DEVICES
    • B81B7/00—Microstructural systems; Auxiliary parts of microstructural devices or systems
    • B81B7/02—Microstructural systems; Auxiliary parts of microstructural devices or systems containing distinct electrical or optical devices of particular relevance for their function, e.g. microelectro-mechanical systems [MEMS]
    • 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
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
    • 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
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
    • 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

Definitions

  • the invention relates to the field of semiconductor technology and chemical substance analysis, in particular to a micropore electrode and a manufacturing method thereof, a micropore electrode array, a sensor chip and a sequencing system, and a micropore electrode, a micropore electrode array, a sensor chip or A method of analyzing chemical and nucleic acid molecules in a sequencing system.
  • a new sequencing technology is based on nanopore-based sequencing technology.
  • the basic idea of this technique is that when a single DNA molecule passes through a nanopore, the nanopore structure acts as both a restriction site and an incorporation site.
  • Oxford Nanopore Technology (ONT) a leading company in nanopore sequencing, recently released its first protein nanopore sequencer, which has a read length of 10,000 bases and a read speed. Up to 100 bases per second.
  • ONT's technology has reduced instrument cost and footprint by eliminating the need for optical equipment.
  • solid-state nanopores are more advantageous in terms of stability and scalability, stability and scalability are critical to the durability and low cost of a sequencing device, but currently based on protein nanopore sequencing technology.
  • the development is faster.
  • Solid-state nanopores still lack atomic-level accuracy and chemical specificity compared to protein nanopores. Retouched by different surfaces Technology can basically achieve chemical specificity, but repeated production of large nanopore arrays is still difficult in the manufacturing process.
  • Most current nanopore-based sequencing methods rely on 3-D nanoscale structures, not only are the pore diameters small enough, but the pores or electrodes are also as small as the distance between adjacent bases.
  • nanopores the most common method is to use ion beam etching or electron beam drilling on thin insulating materials such as silicon nitride or graphene, but this method is not a conventional method and is not compatible with standard semiconductor manufacturing processes. Compatible. This makes the nanopore preparation process very expensive and non-repeatable.
  • nanopore-based technology can provide a long read length, is superior to other existing technologies in terms of read length, and can greatly reduce the cost of sequencing.
  • Nabsys is developing a positioning sequencing platform to generate long-range sequencing maps of short probe sequences by detecting sequence-specific tags that bind to DNA templates when the DNA template is passed through a nanochannel ( ⁇ 100 nm).
  • nanopore-based DNA sequencing still faces many technical challenges. Therefore, how to overcome the above problems and propose a practical low-cost, high-throughput nanopore DNA sequencing solution has become the focus of science and technology worldwide, and develop a new generation of single-molecule DNA detection technology with independent intellectual property rights. China's future high-tech industry The layout will play an important role. Moreover, the integration and portability of its solutions will have a positive impact on many areas of China, such as disease diagnosis, food testing, and environmental monitoring.
  • a microporous electrode comprising: one or more first electrodes; a second electrode disposed opposite each of the first electrodes, each of the first electrodes and a second electrode opposite thereto There is a channel therebetween, at least one end of the channel is connected to a cavity; one or more guiding electrodes are located in the cavity.
  • the guide electrode is capable of directing charged species into the channel and/or controlling movement of the charged species in the channel.
  • the microporous electrode further comprises: a first support element for supporting the first electrode.
  • each first electrode is located on a side wall of the first support element.
  • the microporous electrode comprises a plurality of first electrodes, the first support element comprising a plurality of first support elements, each first electrode being respectively supported by a corresponding first support element.
  • the microporous electrode includes a plurality of second electrodes, the microporous electrode further comprising: a plurality of second support members, each second electrode being supported by a respective second support member.
  • each second electrode is located on a side wall of the second support element.
  • the first electrode comprises a plurality of spaced apart segments.
  • the second electrode comprises a plurality of spaced apart segments.
  • the first electrode includes a plurality of spaced apart segments
  • the microporous electrode further includes a plurality of first support members, each of the first electrodes being supported by a respective first support member.
  • the second electrode includes a plurality of spaced apart segments
  • the microporous electrode further includes a plurality of second support members, each of the second electrodes being supported by a respective second support member.
  • the first support element is a conductive element.
  • a voltage can be applied to the first electrode by the first support element.
  • the first support element is a non-conductive element.
  • the first supporting member functions mainly to support the first electrode, and preferably, a voltage may be applied to the first electrode through a wire embedded in the first supporting member.
  • the second support element is a conductive element.
  • a voltage can be applied to the second electrode by the second support element.
  • the second support element is a non-conductive element.
  • the second support member functions primarily to support the second electrode.
  • a voltage can be applied to the second electrode by a wire embedded in the second support member.
  • the microporous electrode further comprises a nanostructure capable of immobilizing an enzyme or a chemical to be detected.
  • the nanostructures are located at the bottom or sidewall of the cavity, or at the bottom or sidewall of the channel, or on the lead electrode.
  • the nanostructured material comprises a metal, a metal oxide, an inorganic polymer, an organic polymer, or a combination of the foregoing.
  • the channel has a width of 0.5-100 nm, such as 1 nm, 2 nm, 10 nm, 50 nm, 80 nm, etc.; and/or the channel has a length of 50 nm-100 ⁇ m, such as 100 nm, 500 nm, 5 ⁇ m, 10 ⁇ m, 30 ⁇ m or the like; and/or the depth of the channel is 0-10 ⁇ m, such as 100 nm, 300 nm, 1 ⁇ m, 2 ⁇ m, 8 ⁇ m, and the like.
  • the first electrode has a thickness of 1-1000 nm, such as 30 nm, 50 nm, 200 nm, 600 nm, 800 nm, and the like.
  • the second electrode has a thickness of 1-1000 nm, such as 30 nm, 50 nm, 200 nm, 600 nm, 800 nm, and the like.
  • the material of the first electrode is the same as the material of the second electrode
  • the material of the first electrode is different from the material of the second electrode.
  • the material of the first supporting member may be the same as or different from the material of the first electrode and the material of the second electrode.
  • the material of the first electrode comprises: platinum, gold, indium tin oxide, carbon-based material, silicon or other conductive material; and/or the material of the second electrode comprises: platinum, gold, oxidation Indium tin, a carbon based material, silicon or other electrically conductive material; and/or the material of the guiding electrode comprises: silicon, platinum, gold, indium tin oxide or a carbon based material.
  • the material of the conductive element comprises: silicon, platinum, gold, silver, oxygen Indium tin, carbon based materials or other conductive materials.
  • the material of the non-conductive element comprises: silicon oxide, silicon nitride, silicon oxynitride, borophosphosilicate glass, or the like.
  • the first support member has an elliptical, circular, polygonal or gear shape in a section parallel to the surface of the substrate.
  • the bottom surface of the cavity is in the same plane or a different plane than the bottom surface of the channel.
  • the microvia electrode further includes: a substrate and an insulating layer on the substrate; the first electrode, the second electrode, and the guiding electrode are located on the insulating layer.
  • the guiding electrode is substantially perpendicular to the first electrode or the second electrode. That is, after a voltage is applied to each of the leading electrode, the first electrode, and the second electrode, the direction of the electric field between the guiding electrodes and the direction of the electric field between the first electrode and the second electrode may be substantially perpendicular.
  • the microporous electrode further comprises: a passivation layer on a surface of the first electrode and/or the second electrode.
  • a microporous electrode array comprising: one or more microporous electrodes according to any one of the above embodiments.
  • the microwell electrode array includes a plurality of the microwell electrodes.
  • the number of microporous electrodes may be 100, 10,000, 10 6 or 10 8 or the like.
  • a plurality of the microporous electrodes are arranged in an array of microporous electrodes in an elliptical shape, a circular shape, a circular shape, a sector shape, a rectangular shape, a square shape, a zigzag shape, a gear shape, a matrix structure, or a top and bottom laminate shape. and many more.
  • the plurality of microporous electrodes are independent of one another or in series or in parallel.
  • the plurality of microporous electrodes in the array of microwell electrodes share a single lead electrode.
  • a sensor chip comprising: the micropore electrode array described in the above embodiments.
  • the present invention can be fabricated using a process that is compatible with a CMOS process to fabricate sensor chips and corresponding integrated circuits.
  • the number of microporous electrodes included in the micropore electrode array on the sensor chip can be determined according to factors such as the size of the micropore electrode, the nature of the molecule to be detected, and the cost.
  • the micropore electrode array can be exemplified. It is a 10 x 10 array, a 100 x 100 array, a 1000 x 1000 array or a 10 4 x 10 4 array, and the like.
  • a sequencing system comprising: the sensor chip described in the above embodiments.
  • a method of manufacturing a microporous electrode includes providing a substrate structure including a substrate having an insulating layer on a surface, and a first support member material layer on the insulating layer
  • the first support member material layer has a first electrode material layer, a sacrificial material layer, a second electrode material layer and a second support member material layer in sequence on the sidewall of the first support member material layer; the first support member material layer is patterned Determining a first electrode material layer, the sacrificial material layer, the second electrode material layer, and the second support member material layer, thereby forming one or more cavities, and forming a first support member, and sequentially located a first electrode, a sacrificial layer, a second electrode, and a second support member on a sidewall of the first support member; forming one or more guide electrodes in the cavity; removing a sidewall on the sidewall of the first support member a sacrificial layer to form a channel between the first electrode and the
  • the step of providing a substrate structure includes: providing a substrate having an insulating layer on a surface; forming a first support member material layer on a portion of the insulating layer; depositing a first electrode material layer to cover An upper surface and a sidewall of the first support member material layer; a first electrode material layer on the upper surface of the first support member material layer; a sacrificial material layer deposited to cover the first support member material layer Upper surface, upper surface and sidewalls of the remaining first electrode material layer; removal of the upper surface of the first support member material layer and the sacrificial material layer on the upper surface of the remaining first electrode material layer; deposition of the second a layer of electrode material to cover an upper surface of the first support member material layer, an upper surface of the remaining first electrode material layer, and an upper surface and a sidewall of the remaining sacrificial material layer; removing the first support member material An upper surface of the layer, an upper surface of the remaining first electrode material layer, and a second electrode material layer on the upper surface of the
  • the guiding electrode is substantially perpendicular to the first electrode; and/or the guiding electrode is substantially perpendicular to the second electrode.
  • the first support member before removing the sacrificial layer on the sidewall of the first support member, further comprising: at the first support member, the second support member, the first electrode, and the first A passivation layer is formed on a surface of at least one of the two electrodes.
  • removing the sacrificial layer on the sidewall of the first support member further comprising: removing a portion of the top of the first support member and the second support member to expose the first a portion of the electrode, the sacrificial layer and the second electrode; on the remaining first and second support members, and on the exposed first electrode, the sacrificial layer and the second electrode A passivation layer is deposited; the deposited passivation layer is planarized to form a passivation layer on the remaining first support member and the second support member and expose the sacrificial layer.
  • the patterning the first support member material layer, the first electrode material layer, the sacrificial material layer, the second electrode material layer, and the second support member material layer includes dividing the first electrode material layer and/or the second electrode material layer into spaced apart segments such that the formed first electrode and/or second electrode comprise spaced apart segments.
  • the method further comprises: forming a nanostructure capable of immobilizing an enzyme or a chemical to be detected on a bottom or sidewall of the cavity, or a bottom or sidewall of the channel, or the guiding electrode .
  • the nanostructured material comprises a metal, a metal oxide, an inorganic polymer, an organic polymer, or a combination of the foregoing.
  • the channel has a width of 0.5-100 nm, such as 1 nm, 2 nm, 10 nm, 50 nm, 80 nm, etc.; and/or the channel has a length of 50 nm-100 ⁇ m, such as 100 nm, 500 nm, 5 ⁇ m, 10 ⁇ m, 30 ⁇ m or the like; and/or the depth of the channel is 0-10 ⁇ m, such as 100 nm, 300 nm, 1 ⁇ m, 2 ⁇ m, 8 ⁇ m, and the like.
  • the first electrode has a thickness of 1-1000 nm, such as 30 nm, 50 nm, 200 nm, 600 nm, 800 nm, and the like.
  • the material of the first electrode and the material of the second electrode are the same.
  • the material of the first electrode and the material of the second electrode are different.
  • the material of the first electrode comprises: silicon, platinum, gold, silver, indium tin oxide or carbon-based material; and/or the material of the sacrificial layer comprises: chromium, tungsten, aluminum, aluminum An oxide of oxide, silicon, silicon or silicon; and/or a material of the second electrode comprising: silicon, platinum, gold, silver, indium tin oxide or a carbon-based material; and/or the guiding electrode Materials include: silicon, platinum, gold, silver, indium tin oxide or carbon-based materials.
  • the first support element and the second support element comprise electrically conductive elements.
  • the material of the conductive element comprises: silicon, platinum, gold, silver, indium tin oxide or a carbon-based material.
  • the first support member and/or the second support member are elliptical, circular, rectangular, square or gear-shaped in cross section in a direction parallel to the surface of the substrate.
  • the invention also provides a method of analyzing a chemical, the method comprising the steps of:
  • the first electrode, the second electrode, and/or the guiding electrode are used to determine the type of the charged molecule, thereby obtaining information of the chemical substance to be tested.
  • the first electrode, the second electrode, and/or the guiding electrode are used to determine the type of charged molecule by one or more methods selected from the group consisting of: redox effect, electrical resistance Effects, capacitive effects, field effects, and tunneling effects.
  • the chemical to be tested is selected from the group consisting of biomolecules (eg, nucleic acids, proteins, lipids, polysaccharides, etc.), compounds, and organic polymers, and the like.
  • the chemical substance to be tested is a nucleic acid such as DNA or RNA.
  • the chemical to be tested comprises or consists of one or more basic units (eg, nucleotides, amino acids, polymer monomers, etc.).
  • the basic unit of the chemical to be tested is unmodified.
  • the basic unit of the chemical to be tested is modified with a tag molecule.
  • the reaction solution contains a free, tagged molecule modified base unit which, upon the reaction, produces a free tag molecule.
  • the free tag molecule is charged (i.e., charged molecule) and is capable of entering the channel under the action of a lead electrode or accumulating in the channel under the action of a lead electrode.
  • the first electrode, the second electrode and/or the guiding electrode can be utilized to determine the type of the label molecule and thereby obtain information of the chemical substance to be tested.
  • the reaction solution contains one or more free, basic units modified with a tag molecule. In one embodiment, the reaction solution contains at least two (eg, three, four, or more) free, base units modified with a tag molecule. In one embodiment, different basic units are modified by the same tag molecule. In another embodiment, different basic units are modified by different tag molecules.
  • the free tag molecule is a redox active material and the first electrode and the second electrode are used as detection electrodes to determine the type of tag molecule by redox effect.
  • the first electrode and the second electrode are also utilized by one or more of a resistance effect, a capacitance effect, a field effect, and a tunneling effect. Electrodes and/or guide electrodes are used to determine the type of charged molecule.
  • one or more detection principles can be utilized to detect charged molecules to improve the accuracy of the detection results. For example, when an electric field is generated between the guiding electrodes, any molecules that appear in the channel will physically block the ion current, thereby producing a detectable drop in ion current. By selecting a suitably sized tag molecule and a channel of suitable length, the signal characteristics produced by the resistance effect will further improve the accuracy of detection of charged molecules.
  • field effects and redox effects can be The type of charged molecule is determined by combining or combining a capacitor with a redox effect, or combining a resistance with a redox effect, or combining a redox effect, a resistance effect, and a field effect.
  • the surface of the insulating layer at the bottom of the channel may also have a modified film.
  • the first electrode and the second electrode are used as a source electrode and a drain electrode, respectively, and the modified film is used as a conductive path between the two electrodes.
  • an identification point of the label molecule can be formed on the conductive path as a gate electrode to form a field effect transistor.
  • the first electrode and/or the second electrode may each be separated into a plurality of segments, that is, two or more segments (eg, 3-4 segments).
  • each segment of the electrode after segmentation, each segment of the electrode has a different voltage.
  • the voltage of each segment of the electrode corresponds to the response voltage (oxidation-reduction potential window) of one of the tag molecules, respectively.
  • the presence or absence of a signal of a segment of the electrode can determine the presence or absence of a tag molecule corresponding to the segment electrode.
  • This type of detection facilitates the separation of signals from noise.
  • This electrode design that provides dynamic information is also significantly different from traditional nanopore solutions.
  • the first electrode and/or the second electrode are separated into a plurality of segments, such as 2-4 segments of lateral electrodes, in the same channel, and each individual electrode can be individually controlled to produce a different voltage.
  • each microporous electrode can have multiple channels. Providing multiple channels in a microporous electrode can greatly increase the detection surface area, which not only increases signal strength, but also reduces potential contamination effects. The combination of the microwell electrode array and the cavity also provides more controllable sample injection modes.
  • the channel can be open. Open channel manufacturing process Easier and easier to sample and liquid, it is better to find a balance between speed and accuracy.
  • the channel can be closed. Closed channels help to control and reduce interference from external impurity signals. In practical applications, the open, closed or a combination of the two can be selected according to the specific situation.
  • the microporous electrode has an open channel, or a closed channel, or an open channel and a closed channel.
  • the chemical substance to be tested is a nucleic acid molecule, and, in the step (2), the reaction liquid is subjected to polymerization of nucleotides.
  • the method is used to analyze the composition, sequence, charge, size or concentration of a chemical, and the like.
  • the invention also provides a method of analyzing a nucleic acid molecule, the method comprising the steps of:
  • a polymerase such as DNA polymerase or RNA polymerase
  • reaction solution comprising the nucleic acid molecule to be tested, a primer, and at least one (for example, one, two, three, four) deoxygenation a ribonucleoside triphosphate (dNTP) molecule, a ribonucleoside triphosphate (NTP) molecule, or an analog thereof, wherein the primer is capable of hybridizing or annealing to a partial sequence of the nucleic acid molecule to be tested, and the at least one Each of the dNTPs, NTP molecules or their analogs are each modified with a tag molecule; subsequently, under appropriate conditions, the nucleic acid molecule to be tested is hybridized with the primer to form a complex;
  • dNTP ribonucleoside triphosphate
  • NTP ribonucleoside triphosphate
  • one of the dNTP, NTP molecule or an analog thereof modified with a tag molecule is incorporated (or incorporated) into the primer to form a nucleic acid complementary to the molecule to be tested.
  • An extension product, and the tag molecule carried by the dNTP, NTP molecule or their analog incorporated into the primer is removed to become a free tag molecule, the free tag molecule having a charge;
  • Steps (4), (5), and (6) are repeated until the extension reaction of the complex is completed.
  • the species of the tag molecule is determined by one or more of a redox effect, a resistance effect, a capacitive effect, a field effect, and a tunneling effect.
  • the species of the tag molecule is determined by a redox effect.
  • the free tag molecule is a redox active that can undergo a redox cycle reaction, or can be converted to a redox active that can undergo a redox cycle reaction.
  • the free tag molecule is converted, by physical or chemical action, to a redox active that can undergo a redox cycle reaction.
  • the redox active material undergoes a redox cycle reaction between the first electrode and the second electrode and generates a detectable current. Thereby, the type of the tag molecule can be determined based on the detectable current.
  • the species of the tag molecule is determined by one or more of a resistive effect, a capacitive effect, a field effect, and a tunneling effect.
  • a free label molecule (eg, a tag molecule having redox activity) is introduced and/or controlled using a lead electrode or other means to enter the channel and a potential is applied to the first and second electrodes of the channel, respectively.
  • the tag molecule is capable of undergoing a redox cycle reaction between the first electrode and the second electrode and generating a detectable redox current Pulse signal.
  • the specific recognition and detection of the tag molecule can be achieved by using the detectable redox current pulse signal.
  • a matching potential is set between the first electrode and the second electrode but the pulse signal is not detected, it indicates that no tag molecule exists.
  • the at least one dNTP, NTP molecule or the like each of the species each carries a tag molecule having a different window of redox potential.
  • by changing the potential of the first electrode and/or the second electrode, and measuring whether a redox current pulse signal is generated under various potential conditions, and optionally measuring the signal amplitude of the pulse signal, etc. can identify the type of label molecules in the channel.
  • each of the at least one dNTP, NTP molecule or analog thereof each carries a tag molecule having a different redox potential window.
  • the first electrode and/or the second electrode in the channel are divided into segments, and each segment is applied with a potential that matches the redox potential window of the different tag molecules.
  • the reaction solution further contains a phosphatase.
  • step (4) of the method the free tag molecule is further dephosphorylated by the action of a phosphatase.
  • the net charge of the tag molecule modified dNTP, NTP or analog thereof is neutral or has a negative charge.
  • the free tag molecule has a positive or negative charge.
  • the net charge of the dNTP, NTP or analog thereof modified with a tag molecule is neutral or has a negative charge, and the free tag molecule has a positive charge.
  • the positively charged free tag molecule can move along the channel under the action of an electric field, while the tag molecule modified dNTP, NTP molecule is not neutral or negatively charged in the cavity or Move in the channel.
  • the dNTP, NTP or analog thereof modified with a tag molecule has a negative charge and the free tag molecule has a negative charge.
  • the negatively charged free tag molecule, the dNTP modified with the tag molecule, the NTP molecule, and the unmodified dNTP, NTP molecule can collectively move along the channel under the action of an electric field.
  • the redox current signal can only be derived from free tag molecules.
  • the tag molecule is attached to a phosphate group, base or sugar group of a dNTP, NTP molecule or analog thereof.
  • the label molecule is selected from one or more of the following: amino acids, peptides, carbohydrates, metal compounds, dyes, chemiluminescent compounds, nucleotides, aliphatic acids, aromatic acids, alcohols, aminobenzenes A hydroxyphenyl, naphthyl, thiol, cyano, nitro, alkyl, alkenyl, alkynyl, azide or a derivative of the above.
  • the label molecule is selected from one or more of the following: an aminophenyl group, a hydroxyphenyl group, a naphthyl group, a metal compound of a valence state (such as ferrocene, hexacyanoferrate, hexacyanoferrite) Molecules such as hydrazine, methine blue, and derivatives of the above groups.
  • a metal compound of a valence state such as ferrocene, hexacyanoferrate, hexacyanoferrite
  • Molecules such as hydrazine, methine blue, and derivatives of the above groups.
  • the tag molecule is attached to a gamma-phosphate group of a dNTP, NTP molecule or the like, and, preferably, the tag molecule is selected from the group consisting of an aminophenyl group, a hydroxyphenyl group, a naphthyl group, and the above a derivative of a group.
  • the tag molecule is attached to a base or a sugar group of a dNTP, NTP molecule or the like, and, preferably, the tag molecule is selected from a metal compound of a variable state (eg, ferrocene, hexacyano) Molecules such as iron, hexacyanoferrate, hydrazine, and methine blue, and derivatives of the above groups.
  • a metal compound of a variable state eg, ferrocene, hexacyano
  • Molecules such as iron, hexacyanoferrate, hydrazine, and methine blue, and derivatives of the above groups.
  • each type of dNTP eg, dATP, dTTP, dCTP, dGTP, dUTP
  • NTP eg, ATP, TTP, CTP, GTP, UTP
  • all four types of dNTPs eg, selected from dATP, dTTP/dUTP, dCTP, dGTP
  • NTP eg, ATP, TTP, CTP, GTP, UTP
  • the amount of charge carried by the free tag molecule is adjusted by selecting a tag molecule to adjust the rate of movement of the free tag molecule under the action of the lead electrode.
  • the polymerase described in step (1) is immobilized on a bottom insulating layer within a cavity or channel, or is attached to a lead electrode.
  • the polymerase is immobilized at a portion of the bottom of the chamber near the entrance of the channel; preferably, the inlet of the channel is Designed in a variety of shapes (eg funnel shape) to accommodate the polymerase.
  • one polymerase is immobilized within each chamber or channel.
  • the nucleic acid molecule to be tested in the reaction solution is a single-stranded nucleic acid molecule.
  • each polymerase can capture a single stranded nucleic acid molecule or a complex formed by hybridization of a nucleic acid molecule and a primer.
  • the material of the insulating layer is selected from the group consisting of silicon dioxide, silicon oxynitride, silicon nitride, or other insulating materials (eg, carbon doped oxide (CDO), silicon carbide, such as polyimide, eight An organic polymer of fluorocyclobutane or polytetrafluoroethylene, fluorosilicate glass (FSG) and an organosilicate such as silsesquioxane, siloxane or organosilicate glass).
  • CDO carbon doped oxide
  • silicon carbide such as polyimide
  • FSG fluorosilicate glass
  • organosilicate such as silsesquioxane, siloxane or organosilicate glass
  • a functionalizable region and/or a molecular binding region is further included between the insulating layer and the polymerase.
  • the functionalizable zone comprises a functionalizable material such as silica, yttria, alumina, yttria, and/or zirconia.
  • the functionalizable material can be functionalized using a linker molecule selected from the group consisting of silane (eg, aminopropyltriethoxysilane), thiol (-SH), disulfide (-SS-), and Thiocyanates, alkenes and alkynes.
  • the molecular binding region comprises a probe molecule.
  • the probe molecule is for example selected from the group consisting of biotin, avidin, antibodies, antigens, receptors, ligands, DNA sequences, RNA sequences, proteins and ligands thereof.
  • the polymerase can be immobilized in a suitable orientation by selection of binding molecules.
  • the method is for analyzing the sequence, composition, charge, size or concentration of a nucleic acid molecule, and the like.
  • an electrolyte and a reaction liquid may be applied to the surface of the microporous electrode to fill all the cavities and channels with the electrolyte and the reaction liquid.
  • the voltages of different electrodes or different segments of the same electrode can be independently set to independently control or detect the reactive molecules.
  • the guiding electrode controls electrolyte and redox activity
  • the substance enters the channel, and the first electrode and the second electrode serve as redox reaction detecting means for detecting the redox active substance.
  • the electric field formed by the guiding electrode facilitates the entry of charged molecules (e.g., positively charged molecules) into the channel and accumulates in the channel to reduce the likelihood of these molecules diffusing out of the channel, thereby enhancing the detected signal.
  • charged molecules e.g., positively charged molecules
  • the voltage of the lead electrode can be adjusted to control the rate of transport of charged molecules.
  • the optimal rate of synthesis can be matched to the rate of molecular transport by engineering the polymerase.
  • the movement of molecules in the channel can be controlled by micro- or nano-scale fluid mechanics.
  • the nucleic acid molecule includes a polymer of deoxyribonucleotides (DNA) and ribonucleotides (RNA) and other analogs linked together by phosphodiester bonds.
  • a polynucleotide can be a fragment of a genome, a gene or a portion thereof, a cDNA or a synthetic polydeoxyribonucleic acid sequence.
  • Nucleotides comprising a polynucleotide are naturally occurring deoxyribonucleotides such as adenine, cytosine, guanine or thymine linked to 2'-deoxyribose, or glands such as linked to ribose A ribonucleotide such as purine, cytosine, guanine or uracil.
  • polynucleotides or oligonucleotides may also comprise nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides.
  • the redox cycle refers to an electrochemical method in which a molecule capable of reversibly oxidizing and/or reducing (ie, a redox active molecule) moves between at least two electrodes that are independently biased.
  • a molecule capable of reversibly oxidizing and/or reducing ie, a redox active molecule
  • One of the at least two electrodes is lower than the reduction potential of the detected redox active molecule and the other is higher than the oxidation potential, allowing electrons to shuttle back and forth between the independent bias electrodes (ie, the molecule is at the first electrode It is oxidized and diffuses to the second electrode where it is reduced, or vice versa, which is first reduced and then oxidized, depending on the potential at which the molecule and the electrode are biased).
  • the same molecule can thus contribute multiple electrons to the recorded current, resulting in a net amplification of the signal.
  • the signal from the redox active species can potentially be amplified more than 100 times, depending on factors such as the stability of the redox active species and the ability of the redox active species to diffuse into the sensing region.
  • the provision of the guiding electrode prevents the redox active substance from diffusing out of the channel, thereby Increase the effective concentration of the redox active substance.
  • the redox active material (or redox active molecule) has a general meaning in the art, and is a molecule which can be reversibly reversibly oxidized and/or reduced.
  • the phosphatase is, for example, selected from the group consisting of alkaline phosphatase, acid phosphatase, protein phosphatase, polyphosphate phosphatase, sugar phosphatase, and pyrophosphatase.
  • the incorporation of a tag molecule-modified dNTP, NTP or the like during the synthesis of the extension product releases the tag molecule pyrophosphate (PPi) into the solution.
  • the role of phosphatase is to remove pyrophosphate from the tag molecule.
  • the removal of the phosphate group further activates the redox active material, which in turn can be electrochemically detected to detect the presence of the redox active species.
  • the silane molecule may have a chemical equation X 3 -Si-YR", X2-Si-(N)YR” and X-Si-(N 2 )YR", wherein X is a leaving group, For example, -Cl, -OCH 3 or -OCH 2 CH 3 , R" is a reactive coupling group such as -NH 2 , -COOH, -COH, -CHCH 2 or -SH, and R is a non-reactive group, for example, Alkyl group.
  • the organic groups present for the surface-attached silane molecules for coupling may be, for example, carboxyl groups, aldehydes, esters, alkenes, alkynes, mercaptans, isothiocyanates, isocyanates, substituted amines, epoxides , such as small molecules such as biotin or ethanol.
  • Y is a non-reactive group such as a hydrocarbon having 1 to 16 carbon atoms. Examples of -YR" include -(CH 2 ) 3 NH 2 , -(CH 2 ) 2 COOH, and -(CH 2 ) 2 SH.
  • Some exemplary silanes include 3-aminopropyltriethoxysilane (APTS), Mercaptosilane and glycidoxytrimethoxysilane (having an epoxide reactive coupling group).
  • APTS 3-aminopropyltriethoxysilane
  • Mercaptosilane and glycidoxytrimethoxysilane (having an epoxide reactive coupling group).
  • the surface to be silylated can be reacted with, for example, a silane molecule in solution or as a silane gas.
  • the base is, for example, selected from the group consisting of adenine, guanine, cytosine, thymine, uracil, 7-deazaguanine, 7-deaza adenine, and 5-methylcytosine.
  • the primer (primer sequence) is a short, usually chemically synthesized oligonucleotide of appropriate length (eg, about 18-24 bases) sufficient to bind to a target nucleic acid (eg, single stranded) DNA) hybridizes and allows the addition or synthesis of oligonucleotide residues or polynucleotides therefrom under suitable conditions well known in the art.
  • the primer is A DNA primer, a primer consisting of a deoxyribonucleotide residue or consisting essentially of a deoxyribonucleotide residue.
  • the primer is designed to have a reverse complement of the region of the template/target nucleic acid (eg, single-stranded DNA) that hybridizes to the primer. Nucleotide residues are added to the 3' end of the primer by the formation of a phosphodiester bond to produce an extension product; the nucleotide residue is added to the 3' end of the extension product by the formation of a phosphodiester bond to create another extension. product.
  • the template/target nucleic acid eg, single-stranded DNA
  • the dNTP, NTP or the like thereof is incorporated into an oligonucleotide or a polynucleotide (such as a primer, an extension product, a complex formed by a primer and a nucleic acid molecule to be tested), and refers to a polynucleus.
  • a polynucleotide such as a primer, an extension product, a complex formed by a primer and a nucleic acid molecule to be tested
  • the 3' carbon atom of the nucleotide residue at the 3' end of the glycoside forms a phosphodiester bond with the 5' carbon atom of dNTP, NTP or the like, respectively.
  • the polymerase includes a DNA polymerase, an RNA polymerase, a reverse transcriptase, etc., the function or kind of which is known in the art; the DNA polymerase may or may not have a 3' to 5' end, for example.
  • Exonuclease activity for example, including E. coli DNA polymerase, Klenow large fragment, Phusion DNA polymerase, 9°N DNA polymerase, KOD polymerase, Therminator DNA polymerase, Taq DNA polymerase, Vent DNA polymerase, etc. .
  • the modification of the polymerase is used to achieve a desired rate of base incorporation (eg, 1-100 bases per second) upon sequencing in real time.
  • the polymerase is specifically engineered to have features that are more suitable for the application.
  • a polymerase capable of rapidly, continuously and accurately synthesizing a polymer is selected or obtained, and it is required that a single molecule DNA polymerase can synthesize 1-100 kb of DNA without being from a template strand. Dissociate.
  • the polymerase lacks exonucleotide activity and the error rate of incorporation (incorporation) of the base should be as low as 10 -5 -10 -6 /incorporation into the base.
  • the bias of the polymerase for each specific nucleotide can greatly increase the specificity of sequencing.
  • Polymerases have different measurable biases for DNA, RNA, and methylated bases that are beneficial for sequencing.
  • other properties include thermal stability, a stable buffer system, ability to work under conditions of polymer aggregation, and resistance to large amounts of by-products (pyrophosphate).
  • suitable mutants are screened in a pool of polymerases that are polymerized at a rate that matches the capabilities of the detection device to maximize true base incorporation and non-incorporation.
  • This single molecule/real-time polymerase method also requires polymerase to be incorporated Into the modified nucleotide. It is therefore necessary to select polymerase mutants and modified nucleotides that are compatible with the capabilities of the detection device.
  • the rate of incorporation into the base can be altered by buffer (eg, by pH and ions to slow or accelerate the rate of incorporation of a particular nucleotide).
  • buffer eg, by pH and ions to slow or accelerate the rate of incorporation of a particular nucleotide.
  • Polymerase mutants with higher or lower bias towards specific nucleotides can be selected by measuring the residence time of the nucleotides in the polymerase pocket. Enzyme kinetic measurements reduce the chance of false positive incorporation and distinguish between true incorporation signals and device noise.
  • the candidate is important for specific targeted mutations.
  • the residues help us find new, specific, stable interactions.
  • Another feature of the enzyme is the ability to release nucleic acid molecules upon reaching the end point to allow new primer-based template DNA to enter and begin sequencing.
  • a template of 1-10-100-1000 kb can be sequenced in one channel under the action of a sufficiently stable enzyme.
  • the microporous electrode of the present invention has the following advantages: the microporous electrode can comprise a plurality of nanochannels, thereby reducing the influence of electrode surface contamination; guiding the flow of the reagent by guiding the electric field and electroosmotic flow generated by the electrode in the nanochannel; Easy to integrate with microfluidics for reagent interaction; easy to fabricate electrode arrays on a large scale; compatible with a variety of signal detection mechanisms, including electronic signal detection (eg based on redox cycles, FETs, electrochemistry, electrical impedance)
  • the physical amplification of the signal is made possible; the monolithic CMOS can be integrated; the electric field can be used to control the movement of the nucleic acid molecule; the single substance detection of the chemical substance to be tested can be detected by detecting the electrical signal of the label molecule; the read length is greatly improved; the detection signal is sensitive.
  • the chemical substance analysis method of the present invention can be used for molecular detection and analysis, molecular diagnosis, disease detection, substance recognition, DNA detection and sequencing, and the like.
  • FIG. 1A is a top plan view of a microporous electrode in accordance with one embodiment of the present invention.
  • Figure 1B is a cross-sectional view taken along line B-B' shown in Figure 1A;
  • Fig. 1C is a cross-sectional view taken along C-C' shown in Fig. 1A.
  • FIG. 2A shows a working principle diagram of a microporous electrode according to an example of the present invention
  • 2B is a schematic view showing a correspondence relationship between different bases and detection voltages applied to the first electrode and the second electrode;
  • FIG. 3 is a schematic view showing a first electrode and a second electrode of a microporous electrode each including four segments according to an embodiment of the present invention
  • 4A shows a schematic diagram of a micropore electrode array composed of two microporous electrodes
  • Figure 4B shows a schematic diagram of a micropore electrode array consisting of four microporous electrodes
  • 5A, 5B, 5C, and 5D illustrate four arrangements of a plurality of microporous electrodes in a microwell electrode array
  • Figure 6 shows a schematic diagram of a sequencing system in accordance with one embodiment of the present invention.
  • FIG. 7 is a simplified flow diagram of a method of fabricating a microporous electrode in accordance with one embodiment of the present invention.
  • Figure 8A shows a cross-sectional view of a substrate structure in accordance with one embodiment of the present invention
  • Figure 8B is a plan view showing the structure of the substrate shown in Figure 8A;
  • 8C is a top plan view of a stage of a method of fabricating a microporous electrode in accordance with one embodiment of the present invention.
  • 8D is a top plan view of a stage of a method of fabricating a microporous electrode in accordance with one embodiment of the present invention.
  • 8E is a cross-sectional view showing a stage of a method of manufacturing a microporous electrode according to an embodiment of the present invention.
  • 8F is a step of a method of fabricating a microporous electrode according to another embodiment of the present invention. Sectional view of the segment;
  • 8G is a cross-sectional view showing a stage of a method of manufacturing a microporous electrode according to another embodiment of the present invention.
  • 8H is a cross-sectional view showing a stage of a method of manufacturing a microporous electrode according to still another embodiment of the present invention.
  • Figure 8I is a cross-sectional view showing a stage of a method of manufacturing a microporous electrode according to still another embodiment of the present invention.
  • 8J is a cross-sectional view showing a stage of a method of manufacturing a microporous electrode according to still another embodiment of the present invention.
  • 8K is a cross-sectional view showing a stage of a method of manufacturing a microporous electrode according to still another embodiment of the present invention.
  • 9A is a cross-sectional view showing a stage of forming a substrate structure in accordance with an embodiment of the present invention.
  • 9B is a cross-sectional view showing a stage of forming a substrate structure in accordance with an embodiment of the present invention.
  • 9C is a cross-sectional view showing a stage of forming a substrate structure in accordance with an embodiment of the present invention.
  • Figure 9D is a cross-sectional view showing a stage of forming a substrate structure in accordance with one embodiment of the invention.
  • Figure 9E is a cross-sectional view showing a stage of forming a substrate structure in accordance with one embodiment of the invention.
  • Figure 9F is a cross-sectional view showing a stage of forming a substrate structure in accordance with one embodiment of the invention.
  • Figure 9G is a cross-sectional view showing a stage of forming a substrate structure in accordance with one embodiment of the invention.
  • Figure 9H is a cross-sectional view showing a stage of forming a substrate structure in accordance with one embodiment of the invention.
  • Figure 9I is a cross-sectional view showing a stage of forming a substrate structure in accordance with one embodiment of the invention.
  • 9J is a cross-sectional view showing a stage of forming a substrate structure in accordance with an embodiment of the present invention.
  • 10A and 10B respectively show detection results of detecting different free label molecules using an exemplary micropore electrode of the present invention
  • Figure 11 shows the distribution of the number of collisions of a single tag molecule with an electrode obtained by simulation calculation in an exemplary micropore electrode of the present invention.
  • FIG. 1A is a top plan view of a microporous electrode in accordance with one embodiment of the present invention.
  • Fig. 1B is a cross-sectional view taken along line B-B' shown in Fig. 1A.
  • Fig. 1C is a cross-sectional view taken along C-C' shown in Fig. 1A.
  • the microvia electrode 100 may include:
  • first electrodes 301 may be spaced apart, for example, a gap may be formed between the different first electrodes 301, or the gap may be filled with an insulating layer.
  • the thickness d1 of the first electrode 301 may be about 1-1000 nm, for example, 30 nm, 50 nm, 200 nm, 600 nm, 800 nm, or the like.
  • the material of the first electrode 301 may include: platinum, gold, silver, indium tin oxide, carbon-based materials (such as diamond, graphite, amorphous carbon, carbon nanotubes, etc.), silicon, or a combination of the foregoing materials. Wait.
  • the micropore electrode 100 further includes a second electrode 303 disposed opposite each of the first electrodes 301.
  • a channel 601 is defined between each of the first electrodes 301 and the second electrode 303 opposite thereto, and at least one end of the channel 601 is connected to the cavity 401.
  • the thickness d2 of the second electrode 303 may be about 1-1000 nm, for example, 30 nm, 50 nm, 200 nm, 600 nm, 800 nm, or the like.
  • the material of the second electrode 303 may include: platinum, gold, silver, indium tin oxide, carbon-based materials (eg, diamond, graphite, amorphous carbon, carbon nanotubes, etc.), silicon, or a combination of the foregoing materials. Wait.
  • the bottom surface of cavity 401 and the bottom surface of 601 may be in the same plane. In another embodiment, the bottom surface of the cavity 401 and the bottom surface of the 601 may be in different planes.
  • the microvia electrode 100 also includes one or more lead electrodes 501 located in the cavity 401.
  • the lead electrode 501 is capable of directing charged species into the channel 601.
  • the guiding electrode 501 is also capable of controlling the movement of the charged substance in the channel 601.
  • a guiding electric field generated between the guiding electrodes 501 can control a charged substance (for example, a redox active molecule) to enter the channel 601, and can control a charged substance in the channel 601 ( For example, the movement of the redox active molecule does not allow the charged species to move outside of the channel 601.
  • the guiding electrode 501 can be used to control the movement of the charged substance in the one or more channels 601 communicating with the cavity 401; if there are a plurality of guiding electrodes 501 in the cavity 401, each The guiding electrodes 501 can respectively control the movement of the charged substance in the channel 601 adjacent thereto.
  • the material of the guiding electrode 501 may include: silicon, platinum, gold, indium tin oxide, a carbon-based material, or a combination of the foregoing.
  • the guiding electrode 501 may be substantially perpendicular to the first electrode 301 or the second electrode 303 (as shown in FIG. 1A).
  • the guiding electrode 501 and the first electrode 301 or the second electrode 303 do not necessarily have to be perpendicular, and may have other angles.
  • the microporous electrode provided by the present invention can be used for, but not limited to, nucleic acid sequencing, and when used for nucleic acid sequencing, the cavity 401 can be a microfluidic cavity.
  • the manufacturing process of the microporous electrode can be compatible with the CMOS process and is easy to manufacture on a large scale; in addition, various signal mechanisms can be employed for nucleic acid sequencing and the read length is longer.
  • the micropore electrode can include multiple channels at the same time, this can reduce the influence of electrode contamination on the sequencing result, and the sequencing result is more accurate.
  • Fig. 2A shows a working principle diagram of a microporous electrode according to an example of the present invention.
  • the micropore electrode includes a set of detecting electrodes (a first electrode 301 and a second electrode 303) and a set of guiding electrodes 501.
  • the voltage applied to the first electrode 301 is V 1
  • the voltage applied to the second electrode 303 is V 2
  • the voltage difference between the two guiding electrodes is V g .
  • the guiding electric field between the two guiding electrodes 501 can draw a molecule to be detected (for example, a tag molecule having redox activity) into a channel between the two detecting electrodes.
  • the label molecule undergoes a redox cycle reaction between the two detection electrodes, and the redox cycle reaction produces a corresponding current signal that can be detected by the two detection electrodes.
  • Different tag molecules correspond to different current signals, so the type of tag molecules can be determined by the detected current signals.
  • Fig. 2B is a diagram showing the correspondence relationship between different bases and detection voltages applied to the first electrode and the second electrode.
  • the current signal of the molecule to be detected can be detected only when the voltages V 1 and V 2 are located in the oxidation-reduction potential window corresponding to the molecule to be detected.
  • a plurality of times by changing the voltage V 1 and V 2 such that the size reduction and the voltage V 1 is V 2 corresponding to the molecule to be detected is located in the oxidation potential window, and thus can identify the type of the molecule to be detected .
  • current signals of different molecules to be detected can be detected at different voltages V 1 and V 2 . As shown in FIG.
  • bases A, T, C, and G correspond to different voltages V 1 and V 2 , respectively .
  • the voltage corresponding to the base A is V 1A and V 2A
  • the voltage corresponding to the base T is V 1T and V 2T
  • the voltage corresponding to the base C is V 1C and V 2C
  • the voltage corresponding to the base G is V. 1G and V 2G .
  • the detection electrode can detect the tag molecule corresponding to base A in the redox reaction.
  • V 1 and V 2 can be reduced and so that the voltage V 1 is located in the V-base T, C or G corresponding tag molecule oxidation potential window 2, which can identify the type of label molecule, thereby identifying For the base type corresponding to the tag molecule.
  • the microvia electrode may further include a substrate 101 and an insulating layer 102 on the substrate 101.
  • the first electrode 301, the second electrode 303, and the guiding electrode 501 may be located on the insulating layer 102.
  • the substrate 101 may be, for example, a silicon substrate, a substrate of a III-V semiconductor material, a silicon-on-insulator substrate (SOI), or may be oxidized such as ZnO, CdO, TiO 2 , Al 2 O 3 , SnO, or the like.
  • the semiconductor substrate or may be a substrate of an insulating material such as quartz glass or soda glass.
  • the insulating layer 102 may typically be an oxide of silicon (eg, silicon dioxide), a nitride of silicon (eg, SiN), an oxynitride of silicon, and the like.
  • the first electrode 301 and the second electrode 303 may be supported by the supporting component.
  • the microwell electrode may further include a first support member 201 for supporting the first electrode 301.
  • the microvia electrode can include a plurality of first electrodes 301, all of which can be supported by the first support member 201.
  • the microporous electrode may include a plurality of first electrodes 301, and the first support member 201 may include a plurality of first support members 201, each of which may be respectively associated with a corresponding first support member 201 support.
  • the microporous electrode can include a plurality of second electrodes 303 and include a plurality of second support members 304, each of which can be supported by a respective second support member 304, respectively.
  • the first support member 201 and the second support member 304 described above may be conductive members or non-conductive members, preferably conductive members.
  • the first support member 201 and the second support member 304 mainly function to support the first electrode 301 and the second electrode 303.
  • a voltage may be applied to the first electrode 301 and the second electrode 303 by applying a voltage to the first support member 201 and the second support member 304.
  • the material of the conductive element may include silicon (such as polysilicon), platinum, gold, silver, indium tin oxide or carbon-based materials, etc.; the material of the non-conductive element may include silicon oxide, silicon nitride, nitrogen. Silicon oxide, borophosphosilicate glass, and the like.
  • the cross-sectional shape of the first support member 201 in a direction parallel to the surface of the substrate 101 may be elliptical, circular, polygonal, square or gear-shaped. It should be understood that the present invention is not limited to the above-described exemplary shapes.
  • the material of the first electrode 301 and the material of the second electrode 303 may be the same or different.
  • one of the first electrode 301 and the second electrode 303 may be an electrode material that is susceptible to oxidation, and the other electrode may be an electrode material that is easily reduced.
  • the micropore electrode 100 may further include a first electrode 301, a second electrode 303, a first support member 201 (if any), A passivation layer 701 on the surface of at least one of the two support members 304, if any.
  • the passivation layer 701 may be a nitride of silicon (eg, SiN), an oxide of silicon (eg, SiO 2 ), or the like.
  • the passivation layer 701 can function to protect the micropore electrode. Specifically, when nucleic acid sequencing is performed using the micropore electrode, only the region not covered by the passivation layer 701 can contact the reaction liquid, which can avoid the micropore electrode. Some parts are damaged. In addition, the passivation layer 701 can also reduce the noise generated during the sequencing process.
  • the microwell electrode 100 may further comprise: a nanostructure 801 (eg, a nanodots) capable of immobilizing an enzyme or a chemical to be detected 802.
  • the nanostructures 801 can be located at the bottom of the cavity 401 (as shown in FIGS. 1A and 1B) or sidewalls, or at the bottom (not shown) or sidewalls of the channel 601, or on the guiding electrode 211.
  • the nanostructures 801 may have a size of 1-100 nm, such as 8 nm, 20 nm, 60 nm, and the like.
  • the material of the nanostructure 801 may include a metal oxide, preferably, the metal oxide is a transition metal oxide such as zirconium dioxide (ZrO 2 ) or hafnium oxide (HfO 2 ); or, the nanostructure 801
  • the material may also include a metal, preferably the metal is an inert metal such as gold or platinum; or the material of the nanostructure 801 may also be an inorganic polymer, an organic polymer, or a combination of the foregoing.
  • the first electrode 301 and/or the second electrode 303 can include spaced apart segments, such as two or more segments, as described below in connection with different implementations.
  • the first electrode 301 includes a plurality of spaced apart segments and the second electrode 303 includes only one segment.
  • the microporous electrode may include a plurality of first support members 201, each of which may be supported by a corresponding first support member 201, respectively.
  • a certain voltage may be applied to the second electrode 303, and a voltage corresponding to a different molecule to be detected (for example, a redox tag molecule) may be applied to each of the first electrodes 301.
  • a voltage corresponding to a different molecule to be detected for example, a redox tag molecule
  • the first electrode 301 includes a plurality of spaced apart segments
  • the second electrode 303 also includes a plurality of spaced apart segments, each of the first electrodes 301 being disposed opposite each of the second electrodes 303.
  • Each of the first electrodes 301 may be supported by a corresponding first support member 201
  • each of the second electrodes 303 may be supported by a corresponding second support member 304, respectively.
  • a different voltage may be applied to each of the first electrode 301 and a second electrode 303 disposed opposite to the first electrode 301, and the molecules to be detected controlled by the electric field are sequentially passed through each of the electrodes. Only the voltage corresponding to the redox potential window applied to the tag molecule A segment of the electrode can detect the current signal. Therefore, different molecules to be detected can be detected by the first electrode 301 and the corresponding second electrode 303 of different segments.
  • the first electrode 301 can include a segment and the second electrode 303 can include a plurality of segments that are spaced apart.
  • a certain voltage may be applied to the first electrode 301, and a voltage corresponding to a redox window of a different molecule to be detected (for example, a redox tag molecule) may be applied to each of the second electrodes 303.
  • a voltage corresponding to a redox window of a different molecule to be detected for example, a redox tag molecule
  • time information of moving the molecules to be detected from one segment of the first electrode 303 to the other of the first electrodes 303 can be obtained. It is beneficial to improve the detection resolution of different molecules to be detected.
  • the first electrode 301 includes a first segment first electrode 311, a second segment first electrode 321, a third segment first electrode 331 and a fourth segment first electrode 341, and the first segment first electrode 311
  • the voltages applied to the first electrode 341 to the fourth stage are V 11 , V 21 , V 31 and V 41 , respectively .
  • the second electrode 303 includes a first segment second electrode 313, a second segment second electrode 323, a third segment second electrode 333, and a fourth segment second electrode 343, the first segment second electrode 313 to the fourth segment second
  • the voltages applied to the electrodes 343 are V 12 , V 22 , V 32 and V 42 , respectively .
  • Each of the first electrode 301 and the second electrode 303 are disposed opposite each other, and each of the first electrodes 301 and a second electrode 303 disposed opposite thereto are used as a group of detecting electrodes.
  • the first segment of the first electrode 311 is disposed opposite to the first segment of the second electrode 313, and the two segments of the electrode can serve as a group of detecting electrodes.
  • the detection voltages applied to each set of detection electrodes correspond to one type of label molecule to be detected, respectively.
  • the voltage difference applied across the two lead electrodes 501 is V g .
  • the type of the tag molecule to be detected can be determined based on the detection electrode that detects the current signal. For example, if the detection voltage applied on the first group of detection electrodes corresponds to the redox window of a certain tag molecule to be detected, then the first group of detection electrodes can detect that the tag molecule to be detected is detected after detecting the current signal.
  • the present invention also provides a micropore electrode array comprising any one of the above embodiments One or more microporous electrodes.
  • a plurality of microporous electrodes may share a single lead electrode.
  • FIG. 4A shows a schematic of an array of microporous electrodes consisting of two microporous electrodes.
  • the micropore electrode includes a first micropore electrode and a second micropore electrode, and the first micropore electrode and the second micropore electrode share an intermediate guiding electrode 501.
  • the voltages applied to the first electrode 301 and the second electrode 303 of the first microporous electrode are V 11 and V 12 , respectively, and the voltages applied to the first electrode 301 and the second electrode 303 of the second microporous electrode are V 21 , respectively.
  • V 22 shows a schematic of an array of microporous electrodes consisting of two microporous electrodes.
  • the micropore electrode includes a first micropore electrode and a second micropore electrode, and the first micropore electrode and the second micropore electrode share an intermediate guiding electrode 501.
  • the voltages applied to the first electrode 301 and the second electrode 303 of the first microporous electrode are V 11 and V 12 , respectively, and the voltages applied to the first
  • the voltage difference applied across the two guiding electrodes 501 of the first microporous electrode is Vg1
  • the voltage difference applied across the two guiding electrodes 501 of the second microporous electrode is Vg2 .
  • the negatively charged tag molecule is drawn into the channel of the first microporous electrode
  • the positively charged tag molecule is drawn into the channel of the second microporous electrode, thereby enabling screening of the tag molecule.
  • a micropore electrode array composed of two microporous electrodes can recognize the charge polarity of the tag molecule, and the type of the tag molecule can be determined based on the current signal.
  • FIG. 4B shows a schematic of a micropore electrode array consisting of four microporous electrodes.
  • the micropore electrode array includes a first micropore electrode, a second micropore electrode, a third micropore electrode, and a fourth micropore electrode.
  • the four micropore electrodes share the intermediate guiding electrode 501.
  • the voltages applied to the first electrode 301 and the second electrode 303 of the first microporous electrode are V 11 and V 12 , respectively, and the voltages applied to the first electrode 301 and the second electrode 303 of the second microporous electrode are V 21 , respectively.
  • the voltages applied to the first electrode 301 and the second electrode 303 of the third microporous electrode are V 31 and V 32 , respectively, and the voltage applied to the first electrode 301 and the second electrode 303 of the fourth microporous electrode V 41 and V 42 respectively .
  • the voltage differences applied to the leading electrodes 501 of the first to fourth micropore electrodes are V g1 , V g2 , V g3 , and V g4 , respectively .
  • a schematic representation of Vg1 , Vg2 , Vg3, and Vg4 as a function of time is shown in the dashed box.
  • micropore electrode array a plurality of micropore electrodes may be arranged in different shapes.
  • FIG. 5A, 5B, and 5C show three of the plurality of microporous electrodes in the micropore electrode array, respectively Arrangement.
  • a plurality of microporous electrodes are arranged in a ring shape.
  • a plurality of microporous electrodes are arranged in a square shape, such as a rectangle or a square.
  • FIG. 5C a plurality of microporous electrodes are arranged in a matrix of matrix.
  • the invention is not limited to the above several arrangements.
  • the plurality of microporous electrodes may also be arranged in an elliptical shape, a circular shape, a sector shape, a zigzag shape, a gear shape, or the like.
  • the arrangement of the plurality of microporous electrodes is also not limited to the arrangement of two-dimensional planes, for example, the plurality of microporous electrodes may also be arranged in a three-dimensional arrangement of upper and lower laminate shapes.
  • the plurality of microporous electrodes in the microwell electrode array may also be connected in series, i.e., the channels of each microwell electrode are connected in series as shown in Fig. 5A or Fig. 5B.
  • the plurality of microporous electrodes may also be independent of one another, as shown in Figure 5C, each of which may be used to detect different tag molecules, respectively.
  • the plurality of microporous electrodes may also be connected in parallel, that is, the channels of each of the microporous electrodes are connected in parallel, as shown in FIG.
  • the voltage difference applied across the guiding electrodes of the plurality of (for example, four) microporous electrodes All of V g , a plurality of microporous electrodes can detect the tag molecules in parallel under respective detection voltages (ie, voltages applied on the first electrode and the second electrode).
  • the invention also provides a sensor chip comprising the micropore electrode array described in the above embodiments.
  • a micropore electrode array may be included, and a circuit for applying a voltage to an electrode (eg, a first electrode, a second electrode, or a guiding electrode), an amplifying device, a current sensing circuit, a voltage sensing circuit, etc. may be included. Wait.
  • the invention also provides a sequencing system comprising the sensor chip described in the above embodiments.
  • Figure 6 shows a schematic diagram of a sequencing system in accordance with one embodiment of the present invention.
  • the sequencing system includes a flow cell, a sensor chip, a printed circuit board PCB, a field programmable gate array (FPGA) card, and a computer/mobile device.
  • the flow cell can supply microfluidics to the sensor chip, and the packaged sensor chip can be fixed on the PCB through a socket on the PCB.
  • the PCB can include current and/or voltage sensing circuit chips, current and/or voltage amplifying circuit chips, data storage chips, system control chips, and the like.
  • the FPGA card can be integrated with the PCB and can be interfaced to a computer/mobile device to develop application control software on a computer or mobile device for control of the sequencing system.
  • sequencing systems can be designed in different styles, such as palm, portable, desktop or mainframe.
  • the micropore electrode array can be integrated into the sensor chip of the sequencing system, and can be software programmed through the FPGA card to realize the whole of the hardware and software. Hehe.
  • microporous electrode proposed by the present invention can be compatible with a CMOS process, and an exemplary manufacturing method of the microporous electrode will be described below.
  • FIG. 7 is a simplified flow diagram of a method of fabricating a microporous electrode in accordance with one embodiment of the present invention.
  • 8A-8K illustrate various stages of a method of fabricating a microvia electrode in accordance with one embodiment of the present invention. The method of manufacturing the microporous electrode will be described in detail below with reference to FIG. 7 and FIGS. 8A to 8K.
  • step 702 a substrate structure is provided.
  • Figure 8A shows a cross-sectional view of a substrate structure in accordance with one embodiment of the present invention.
  • Fig. 8B shows a plan view of the substrate structure shown in Fig. 8A.
  • the substrate structure includes a substrate 101 having an insulating layer 102 on its surface, and a first support member material layer 201 on the insulating layer 102, and a sidewall of the first support member material layer 201.
  • the first electrode material layer 301, the sacrificial material layer 302, the second electrode material layer 303, and the second support member material layer 304 are sequentially disposed (for example, sidewalls on one or both sides of the first support member material layer 201). Note that, in the substrate structure shown in FIGS.
  • the side wall on the side of the first support member material layer 201 has the first electrode material layer 301, the sacrificial material layer 302, the second electrode material layer 303, and the second support. Element material layer 304.
  • the sidewall of the other side of the first support member material layer 201 may also have a first electrode material layer 301, a sacrificial material layer 302, a second electrode material layer 303, and a second support member material layer 304. .
  • the first electrode material layer 301 may have a thickness of about 1 to 1000 nm, such as 30 nm, 50 nm, 200 nm, 600 nm, 800 nm, and the like.
  • the thickness of the sacrificial material layer 302 may be about 0.5-100 nm, such as 5 nm, 10 nm, 20 nm, 40 nm, 80 nm, and the like.
  • the second electrode material layer 303 may have a thickness of about 1 to 1000 nm, for example, 30 nm, 50 nm, 200 nm, 600 nm, 800 nm, or the like.
  • the constituent materials of the first electrode material layer 301 and the second electrode material layer 303 may be the same or different.
  • the material of the sacrificial material layer 302 may be selected according to the first electrode material layer 301 and the second electrode material layer 303.
  • materials of the first electrode material layer 301 and the second electrode material layer 303 may include: silicon, platinum, gold, silver, indium tin oxide, carbon-based materials (eg, diamond, graphite, amorphous carbon, etc.) or the foregoing Combination of materials; sacrificial materials
  • the material of layer 302 may include: chromium, tungsten, aluminum, an oxide of aluminum, silicon, an oxide of silicon, a nitride of silicon, or a combination of the foregoing.
  • the material of the first electrode material layer 301 and the second electrode material layer 303 may be platinum, and the material of the sacrificial material layer 302 may be chromium or silicon oxide.
  • the material of the first electrode material layer 301 and the second electrode material layer 303 may be gold, and the material of the sacrificial material layer 302 may be tungsten.
  • the first support member material layer 201, the first electrode material layer 301, the sacrificial material layer 302, the second electrode material layer 303, and the second support member material layer 304 are patterned to form one or more a cavity 401 and forming a first support member 201, and a first electrode 301, a sacrificial layer 302, a second electrode 303, and a second support member 304, which are sequentially located on the sidewall of the first support member 201, as shown in FIG. 8C. .
  • the first supporting member 201, the first electrode 301, the sacrificial layer 302, the second electrode 303, and the second supporting member 304 are respectively composed of the aforementioned first supporting member material layer, the first electrode material layer, the sacrificial material layer, and the second An electrode material layer and a second support member material layer are formed.
  • the cavity 401 formed is in communication with the channel 601 formed later.
  • the first electrode material layer 301 and/or the second electrode material layer 303 may also be divided into spaced apart segments such that the formed first electrode 301 and/or second electrode 303 comprise spaced apart segments. .
  • one or more guiding electrodes 501 are formed in the cavity 401 as shown in FIG. 8D.
  • the guiding electrode 501 may be formed by a process such as photolithography, deposition, lift-off, or the like, and the guiding electrode 501 may be formed by a process such as deposition, planarization, etching, or the like.
  • the guiding electrode 501 may be substantially perpendicular to the first electrode 301; or the guiding electrode 501 may also be substantially perpendicular to the second electrode 303.
  • the direction of the electric field between the guiding electrodes 501 and the direction of the electric field between the first electrode 301 and the second electrode 303 may be substantially vertical.
  • step 708 the sacrificial layer 302 on the sidewall of the first support member 201 is removed to form a channel 601 between the first electrode 301 and the second electrode 303, wherein at least one end of the channel 601 is connected to the cavity 401 As shown in Figure 8E.
  • At least one of the first support member 201, the second support member 304, the first electrode 301, and the second electrode 303 may be before removing the sacrificial layer 302 on the sidewall of the first support member 201.
  • a passivation layer 701 is formed on the surface of the component, as shown in FIG. 8F. Show.
  • the material of the passivation layer 701 may be a nitride of silicon, an oxide of silicon, or the like.
  • a passivation material may be deposited on top of the first support member 201, the first electrode 301, the second electrode 303, and the second support member 304, and then the deposited passivation material is patterned to be on the first support member 201.
  • a passivation layer 701 is formed on a surface of at least one of the second support member 304, the first electrode 301, and the second electrode 303. Thereafter, the sacrificial layer 302 may be removed by a selective etching process to form a via 601 between the first electrode 301 and the second electrode 303, as shown in FIG. 8G.
  • a portion of the top of the first support member 201 and the second support member 304 may be removed prior to removing the sacrificial layer 302 on the sidewall of the first support member 201 to expose the first electrode 301,
  • the sacrificial layer 302 and a portion of the second electrode 303 are as shown in FIG. 8H.
  • a passivation layer 701 is deposited on the remaining first support member 201 and second support member 304, and the exposed first electrode 301, sacrificial layer 302, and second electrode 303, as shown in FIG.
  • the deposited passivation layer 701 is planarized to form a passivation layer 701 on the remaining first support member 201 and second support member 304 and expose the sacrificial layer 302 as shown in FIG. 8J.
  • the planarization process may stop at the upper surfaces of the first electrode 301, the sacrificial layer 302, and the second electrode 303.
  • the planarization process may also remove portions of the first electrode 301, the sacrificial layer 302, and the second electrode 303 as long as a portion of the passivation layer 701 remains on the remaining first support member 201 and second support member 304.
  • the exposed sacrificial layer 302 may be removed by a selective etching process to form a via 601 between the first electrode 301 and the second electrode 303, as shown in FIG. 8K.
  • the width W of the formed channel 601 may be about 0.5-100 nm (eg, 1 nm, 2 nm, 10 nm, 50 nm, 80 nm, etc.), and the length L of the channel 601 may be about 50 nm-100 ⁇ m ( For example, 100 nm, 500 nm, 5 ⁇ m, 10 ⁇ m, 30 ⁇ m, etc.), the depth H of the channel 601 may be about 0-10 ⁇ m (for example, 100 nm, 300 nm, 1 ⁇ m, 2 ⁇ m, 8 ⁇ m, etc.).
  • the width of the channel 601 is the distance between the first electrode 301 and the second electrode 303
  • the length of the channel 601 is the length extending around the first support member 201
  • the depth of the channel 601 is the first electrode 301 and the second electrode 303. The distance between the upper surface and the insulating layer 102.
  • the microporous electrode shown in FIGS. 1A, 1B, and 1C was formed by the above method. However, it should be understood that the present invention is not limited to the use of the above method to form a microporous electrode. In practical applications, the corresponding manufacturing method may be adopted according to the specific structure of the microporous electrode. For example, some steps may be added or some steps of the above methods may be added based on the above method. For example, when the first electrode material layer is formed on the sidewall of the first support member, it may be determined whether the first electrode is formed on one side or the side walls of the first support member according to the number of the first electrodes required. Material layer.
  • a nanostructure 801 capable of immobilizing an enzyme or a chemical to be detected 802 may also be formed on the bottom or side wall of the cavity 401, or the bottom or side wall of the channel 601, or the guiding electrode 501.
  • FIG. 1C shows the case where the nanostructures are located at the bottom of the cavity 401.
  • the nanostructures 801 can be nanodots.
  • the nanostructures 801 may have a size of 1-100 nm, such as 8 nm, 20 nm, 50 nm, 80 nm, and the like.
  • the material of the nanostructure 802 may include a transition metal oxide such as zirconium dioxide (ZrO 2 ) or hafnium oxide (HfO 2 ), an inert metal, an inorganic polymer, an organic polymer, or a combination of the foregoing.
  • a transition metal oxide such as zirconium dioxide (ZrO 2 ) or hafnium oxide (HfO 2 )
  • an inert metal such as zirconium dioxide (ZrO 2 ) or hafnium oxide (HfO 2 )
  • an inert metal such as zirconium dioxide (ZrO 2 ) or hafnium oxide (HfO 2 )
  • an inert metal such as zirconium dioxide (ZrO 2 ) or hafnium oxide (HfO 2 )
  • an inert metal such as zirconium dioxide (ZrO 2 ) or hafnium oxide (HfO 2 )
  • an inert metal such as zirconium dioxide
  • the substrate structure shown in Figures 8A and 8B can be implemented in different ways. A specific implementation of forming a substrate structure is described below in conjunction with Figures 9A-9J.
  • the substrate 101 may be, for example, a silicon substrate, a substrate of a III-V semiconductor material, a silicon-on-insulator substrate (SOI), or may be oxidized such as ZnO, CdO, TiO 2 , Al 2 O 3 , SnO, or the like.
  • the semiconductor substrate or may be a substrate of an insulating material such as quartz glass or soda glass.
  • the insulating layer 102 may be formed on the substrate 101 by thermal oxidation, or may be formed on the substrate by deposition (for example, physical vapor deposition PVD, chemical vapor deposition CVD, etc.). Typically, the insulating layer 102 can be silicon dioxide.
  • a first support member material layer 201 is formed on a portion of the insulating layer 102.
  • the material of the first support member material layer 201 may be a conductive material or a non-conductive material, preferably a conductive material.
  • a conductive material may be deposited on the insulating layer 102 by deposition (eg, PVD or CVD), and then the conductive material may be patterned to form the first support member material layer 201.
  • the cross section of the first support member material layer 201 in a direction parallel to the surface of the substrate 101 is elliptical, square, circular or polygonal. However, it should be understood that the present invention is not limited thereto, and the first support member material layer 201 may be other suitable shapes.
  • a first electrode material layer 301 is deposited to cover the upper surface and sidewalls of the first support member material layer 201.
  • the first electrode material layer 301 may also cover a part or all of the exposed insulating layer 102.
  • the first surface on the upper surface of the first support member material layer 201 may be removed by anisotropic dry etching such as reactive ion etching (RIE), ion beam etching (IBE), or the like.
  • the electrode material layer 301 retains only the first electrode material layer 301 on the sidewall of the first support member material layer 201.
  • the first electrode material layer 301 is also deposited on the insulating layer 102, the first electrode material layer 301 on the insulating layer 102 is removed.
  • a sacrificial material layer 302 is deposited to cover the upper surface of the first support member material layer 201, and the remaining first electrode material layer 301 (ie, the sidewall of the first support member material layer 201). The upper surface and side walls of the first electrode material layer 301).
  • the sacrificial material layer 302 may also cover a portion or all of the exposed insulating layer 102.
  • the upper surface of the first support member material layer 201, the sacrificial material layer 302 on the upper surface of the remaining first electrode material layer 301 is removed, leaving only the remaining first electrode material layer 301 A layer of sacrificial material 302 on the sidewall.
  • sacrificial material layer 302 is also deposited on insulating layer 102, sacrificial material layer 302 on insulating layer 102 is removed.
  • a second electrode material layer 303 is deposited to cover the upper surface of the first support member material layer 201, the upper surface of the remaining first electrode material layer 301, and the remaining sacrificial material layer 302. Surface and side walls.
  • the second electrode material layer 303 may also cover a part or all of the exposed insulating layer 102.
  • the upper surface of the first support member material layer 201, the upper surface of the remaining first electrode material layer 301, and the second electrode material layer 303 on the upper surface of the remaining sacrificial material layer 302 are removed, The second electrode material layer 303 on the sidewalls of the remaining sacrificial material layer 302 is retained.
  • the second electrode is also deposited on the insulating layer 102
  • the material layer 303 removes the second electrode material layer 303 on the insulating layer 102.
  • a second support member material layer 304 is deposited to cover the first support member material layer 201, the first electrode material layer 301 on the sidewall of the first support member material layer 201, and the sacrificial material layer 302. a second electrode material layer 303, and an exposed insulating layer 102.
  • the deposited second support member material layer 304 is planarized to expose the sacrificial layer 302 on the sidewalls of the first support member material layer 201, thereby forming a substrate structure.
  • the planarization process may cause the first support material material layer 201 and the first electrode material layer 301, the sacrificial layer 302, and the upper surface of the second electrode material layer 303 and the second support member on the sidewalls thereof The upper surface of material layer 304 is substantially flush.
  • the subsequent steps 704 to 708 can be performed in the manner given above, and will not be described herein.
  • the tag molecule for the redox cycle reaction can be selected, for example, from:
  • the tag molecule modified dNTP or analog thereof can be synthesized, for example, by the following method:
  • different label molecules can be designed to modify four different dNTPs, NTP molecules or the like, so that the free label molecules have different redox potentials, thereby being able to distinguish different dNTPs, NTP molecules or the like.
  • the different tag molecules can be as follows:
  • the charges carried by different tag molecules are as follows:
  • the uncharged or negatively charged tag molecule modified dNTP becomes a positively charged redox active under the action of DNA polymerase and alkaline phosphatase as follows:
  • the principle of the redox cycle reaction of the first electrode and the second electrode is as follows:
  • FIGS. 10A and 10B show the results of detection of different free tag molecules using an exemplary micropore electrode of the present invention, respectively.
  • 10A is a redox current curve of the hexacyanoferrate molecule detected using the microporous electrode of the present invention
  • FIG. 10B is a ferrocene molecular redox current curve detected using the microporous electrode of the present invention.
  • the results of FIGS. 10A and 10B show that there is a significant difference in the redox potential window and the curve shape of the hexacyanoferrate molecule and the ferrocene molecule. This result indicates that the current signals detected by the microporous electrodes of the present invention can be utilized to distinguish and distinguish various free tag molecules.
  • the microporous electrode of the present invention can distinguish and discriminate by the unique electrical signal detected.
  • the type of the tag molecule and further distinguishes and discriminates the type of basic unit (for example, dNTP, NTP, or the like) involved in the reaction in the reaction solution, and finally realizes analysis of the chemical substance to be tested (for example, nucleic acid).
  • Figure 11 shows the distribution of the number of collisions of a single tag molecule with an electrode obtained by simulation calculation in an exemplary micropore electrode of the present invention.
  • Neglecting non-ideal conditions such as molecular adsorption if the electrochemically active molecule exchanges an electron with each electrode collision, the number of collisions per unit time can be directly converted into the maximum theoretical current value generated. Therefore, by further reducing the minimum size of the channel, changing the chemical structure of the label molecule, increasing the number of electron exchanges per collision, and treating the surface of the electrode to reduce molecular adsorption, etc., the current signal can be further amplified, thereby improving the accuracy of the electrical signal detection. degree.

Abstract

一种微孔电极，所述微孔电极包括：一个或多个第一电极（301）；与每个第一电极（301）相对设置的第二电极（303），每个第一电极（301）和与其相对的第二电极（303）之间具有通道（601），通道（601）的至少一端连通有腔（401）；位于所述腔（401）中的一个或多个引导电极（501），该微孔电极检测信号灵敏，读长大大提升。还涉及该微孔电极的制造方法、微孔电极阵列、传感器芯片及测序系统，以及基于该微孔电极的分析化学物质和核酸分子的方法。

Description

微孔电极及分析化学物质的方法 技术领域
本发明涉及半导体技术领域及化学物质分析领域，尤其涉及一种微孔电极及其制造方法、微孔电极阵列、传感器芯片及测序系统，以及基于该微孔电极、微孔电极阵列、传感器芯片或测序系统的分析化学物质和核酸分子的方法。
背景技术
在过去的十年中，第二代DNA测序技术的不断改进使得千元基因组($1,000Genome)时代离我们越来越近。然而，真正达到千元基因组测序并且推进DNA测序技术用于个体化医疗，还需要取得实质性的进展。以下是第二代测序平台目前面临的四个主要问题：1)由于固有的移相问题导致的读长短；2)由于在碱基并入时需要洗脱步骤导致读速慢；3)扩增时样品准备的工作量大，成本高；4)昂贵的光学系统。而单分子测序技术被认为是最有希望同时解决上述问题的测序方法。
一种新的测序技术是基于纳米孔的测序技术。该技术的基本思想是，当单个DNA分子通过纳米孔时，该纳米孔结构同时作为限制位点和并入位点。牛津纳米孔技术公司(Oxford Nanopore Technology，ONT)作为纳米孔测序的领先企业，最近发布了其第一台蛋白纳米孔测序仪，该仪器的读长可以达到一万个碱基，读取速度可以达到100个碱基/秒。与单分子测序的领先公司Pacific Biosciences(PacBio)相比，ONT公司的技术由于不需要光学设备而使得仪器成本和占用空间大大降低了。
虽然大家普遍认为固态纳米孔在稳定性和可扩展性方面更有优势，而稳定性和可扩展性对于一台测序设备的耐用和低成本来说至关重要，但目前基于蛋白纳米孔测序技术的发展更快。与蛋白纳米孔相比，固态纳米孔仍然缺少原子级的准确性和化学的特异性。通过不同的表面修饰 技术可以基本达到化学的特异性，但重复生产大的纳米孔阵列在制造工艺上仍然有难度。目前大多数基于纳米孔的测序方法依赖于3-D的纳米级大小的结构，不仅孔的直径足够小，而且孔或者电极的厚度也要与相邻碱基之间的距离一样小。要形成这样的纳米孔，最常用的方法是在氮化硅或石墨烯等薄绝缘材料上用离子束蚀刻或电子束钻孔，但这种方法并非传统方法，并且与标准的半导体制造工艺不相容。这就使得纳米孔制备工艺非常昂贵并且不可重复。但从商业应用上看，基于纳米孔的技术能够提供很长的读长，在读长上优于其它现有的技术，并且能够大大降低测序成本。
有两种方法可以绕开固态纳米孔对准确、有效的制造工艺的要求。一种方法是对DNA样品进行修饰以提高碱基间的信号差别，从而减弱对纳米孔准确性和尺寸的工艺要求。有一些纳米孔测序公司，如Genia Technologies和Stratos Genomics，正在进行这方面的研究。例如，在利用扩增技术的Stratos Genomics测序中，利用专有分子扩增方法，每个碱基都用一个大的、具有强信号的报告分子所替代。Genia Technologies利用DNA聚合酶对模板DNA进行测序，纳米孔可以捕获并识别用酶切割后产生的碱基特异性标签。虽然这些公司目前的研究都集中在蛋白纳米孔，但将这些相同的想法运用到固态纳米孔中应该没有什么技术障碍。另一种方法是将纳米结构从3D减少为2D，因为标准的半导体制造工艺已经能够适用于常规的2D纳米结构，例如纳米线、纳米通道和纳米间隙。当一维的纳米结构不再是个位数时，挑战已经变成如何达到单个碱基的分辨率。目前已有一些方法用于解决这个问题。Nabsys正在研发一种定位测序平台，当DNA模板通过一个纳米通道时(～100nm)，通过检测结合于DNA模板的序列特异性标签，来产生短探针序列的长距离测序图谱。
总之，以纳米孔为基础的DNA测序仍然面临诸多技术难题的挑战。因此，如何攻克上述问题，提出可实际操作的低成本、高通量纳米孔DNA测序方案就成为了世界范围内的科技焦点，开发具有自主知识产权的新一代单分子水平的DNA检测技术，对我国未来的高新科技产业 布局将起到重要作用。而且其解决方案的集成化和便携化更将对我国的许多领域，如疾病诊断，食品检测，环境监测产生积极的促进作用。
发明内容
根据本发明的一方面，提供一种微孔电极，包括：一个或多个第一电极；与每个第一电极相对设置的第二电极，每个第一电极和与其相对的第二电极之间具有通道，通道的至少一端连通有腔；位于所述腔中的一个或多个引导电极。
在一个实施例中，所述引导电极能够引导带电物质进入通道和/或控制带电物质在通道中的运动。
在一个实施例中，所述微孔电极还包括：用于支撑所述第一电极的第一支撑元件。优选地，每个第一电极位于所述第一支撑元件的侧壁上。
在一个实施例中，所述微孔电极包括多个第一电极，所述第一支撑元件包括多个第一支撑元件，每个第一电极分别由对应的第一支撑元件支撑。
在一个实施例中，所述微孔电极包括多个第二电极，所述微孔电极还包括：多个第二支撑元件，每个第二电极分别由对应的第二支撑元件支撑。优选地，每个第二电极位于所述第二支撑元件的侧壁上。
在一个实施例中，所述第一电极包括间隔开的多段。
在一个实施例中，所述第二电极包括间隔开的多段。
在一个实施例中，所述第一电极包括间隔开的多段，并且所述微孔电极还包括多个第一支撑元件，每段第一电极分别由对应的第一支撑元件支撑。
在一个实施例中，所述第二电极包括间隔开的多段，并且所述微孔电极还包括多个第二支撑元件，每段第二电极分别由对应的第二支撑元件支撑。
在一个实施例中，所述第一支撑元件为导电元件。在此类实施例中，通过所述第一支撑元件能够向所述第一电极施加电压。
在一个实施例中，所述第一支撑元件为非导电元件。在此类实施例 中，优选地，第一支撑元件主要起到支撑第一电极的作用，并且，优选地，可以通过内嵌在第一支撑元件中的导线向所述第一电极施加电压。
在一个实施例中，所述第二支撑元件为导电元件。在此类实施例中，通过所述第二支撑元件能够向所述第二电极施加电压。
在一个实施例中，所述第二支撑元件为非导电元件。在此类实施例中，优选地，第二支撑元件主要起到支撑第二电极的作用。并且，优选地，可以通过内嵌在第二支撑元件中的导线向所述第二电极施加电压。
在一个实施例中，所述微孔电极还包括能够固定酶或待检测化学物质的纳米结构。在一个实施例中，所述纳米结构位于所述腔的底部或侧壁，或者位于所述通道的底部或侧壁，或者位于所述引导电极上。
在一个实施例中，所述纳米结构的材料包括金属、金属氧化物、无机聚合物、有机聚合物或前述材料的组合。
在一个实施例中，所述通道的宽度为0.5-100nm，例如1nm、2nm、10nm、50nm、80nm等；和/或所述通道的长度为50nm-100μm，例如100nm、500nm、5μm、10μm、30μm等；和/或所述通道的深度为0-10μm，例如100nm、300nm、1μm、2μm、8μm等。
在一个实施例中，所述第一电极的厚度为1-1000nm，例如30nm、50nm、200nm、600nm、800nm等。
在一个实施例中，所述第二电极的厚度为1-1000nm，例如30nm、50nm、200nm、600nm、800nm等。
在一个实施例中，所述第一电极的材料与所述第二电极的材料相同，
在一个实施例中，所述第一电极的材料与所述第二电极的材料不同。
在一个实施例中，所述第一支撑元件的材料与所述第一电极的材料以及所述第二电极的材料可以相同，也可以不同。
在一个实施例中，所述第一电极的材料包括：铂、金、氧化铟锡、碳基材料、硅或其它导电材料；和/或所述第二电极的材料包括：铂、金、氧化铟锡、碳基材料、硅或其它导电材料；和/或所述引导电极的材料包括：硅、铂、金、氧化铟锡或碳基材料。
在一个实施例中，所述导电元件的材料包括：硅、铂、金、银、氧 化铟锡、碳基材料或其它导电材料。
在一个实施例中，所述非导电元件的材料包括：氧化硅、氮化硅、氮氧化硅、硼磷硅玻璃等。
在一个实施例中，所述第一支撑元件沿着与衬底表面平行的方向的截面为椭圆形、圆形、多边形或齿轮形。
在一个实施例中，所述腔的底面与所述通道的底面位于同一平面或不同平面。
在一个实施例中，所述微孔电极还包括：衬底和位于衬底上的绝缘层；所述第一电极、所述第二电极、以及所述引导电极位于所述绝缘层上。
在一个实施例中，所述引导电极与所述第一电极或所述第二电极基本垂直。也即，对引导电极、第一电极、以及第二电极分别施加电压后，引导电极之间的电场的方向与第一电极和第二电极之间的电场的方向可以基本垂直。
在一个实施例中，所述微孔电极还包括：位于所述第一电极和/或所述第二电极的表面上的钝化层。
根据本发明的另一方面，提供一种微孔电极阵列，包括：一个或多个上述任意一个实施例所述的微孔电极。
在一个实施例中，所述微孔电极阵列包括多个所述微孔电极。例如，微孔电极的数量可以是100个、10000个、106个或108个等等。
在一个实施例中，多个所述微孔电极在微孔电极阵列中被布置为椭圆形、圆形、环形、扇形、矩形、正方形、锯齿形、齿轮形、行列矩阵形或上下叠层形等等。
在一个实施例中，多个所述微孔电极是相互独立的或串联的或并联的。
在一个实施例中，所述微孔电极阵列中的多个微孔电极共用一个引导电极。
根据本发明的又一方面，提供一种传感器芯片，包括：上述实施例所述的微孔电极阵列。
本发明可以采用与CMOS工艺相匹配的工艺来制造传感器芯片以及相应的集成电路。在具体应用中，可以根据微孔电极大小、待检测分子的性质、成本等因素，确定传感器芯片上的微孔电极阵列所包含的微孔电极的个数，例如微孔电极阵列可以示例性的为10×10的阵列、100×100的阵列、1000×1000的阵列或104×104的阵列等等。
根据本发明的再一方面，提供一种测序系统，包括：上述实施例所述的传感器芯片。
根据本发明的还一方面，提供一种微孔电极的制造方法，包括：提供衬底结构，所述衬底结构包括表面具有绝缘层的衬底、位于绝缘层上的第一支撑元件材料层，所述第一支撑元件材料层的侧壁上依次具有第一电极材料层、牺牲材料层、第二电极材料层和第二支撑元件材料层；图案化所述第一支撑元件材料层、所述第一电极材料层、所述牺牲材料层、所述第二电极材料层和所述第二支撑元件材料层，从而形成一个或多个腔，并形成第一支撑元件、以及依次位于所述第一支撑元件的侧壁上的第一电极、牺牲层、第二电极和第二支撑元件；在所述腔中形成一个或多个引导电极；去除所述第一支撑元件的侧壁上的牺牲层，以在所述第一电极和所述第二电极之间形成通道；其中，所述通道的至少一端连通有所述腔。
在一个实施例中，所述提供衬底结构的步骤包括：提供表面具有绝缘层的衬底；在所述绝缘层的一部分上形成第一支撑元件材料层；沉积第一电极材料层，以覆盖所述第一支撑元件材料层的上表面和侧壁；去除所述第一支撑元件材料层的上表面上的第一电极材料层；沉积牺牲材料层，以覆盖所述第一支撑元件材料层的上表面、剩余的第一电极材料层的上表面和侧壁；去除所述第一支撑元件材料层的上表面和剩余的第一电极材料层的上表面上的牺牲材料层；沉积第二电极材料层，以覆盖所述第一支撑元件材料层的上表面、剩余的第一电极材料层的上表面、以及剩余的牺牲材料层的上表面和侧壁；去除所述第一支撑元件材料层的上表面、剩余的第一电极材料层的上表面和剩余的牺牲材料层的上表面上的第二电极材料层；沉积第二支撑元件材料层，以覆盖所述第一支 撑元件材料层、所述第一支撑元件材料层的侧壁上的第一电极材料层、牺牲材料层和第二电极材料层、以及暴露的绝缘层；对沉积的第二支撑元件材料层进行平坦化，以暴露所述第一支撑元件材料层的侧壁上的牺牲材料层。
在一个实施例中，所述引导电极与所述第一电极基本垂直；和/或所述引导电极与所述第二电极基本垂直。
在一个实施例中，在去除所述第一支撑元件的侧壁上的牺牲层之前，还包括：在所述第一支撑元件、所述第二支撑元件、所述第一电极和所述第二电极中的至少一个部件的表面上形成钝化层。
在一个实施例中，在去除所述第一支撑元件的侧壁上的牺牲层之前，还包括：去除所述第一支撑元件和所述第二支撑元件的顶部的一部分，以露出所述第一电极、所述牺牲层和所述第二电极的一部分；在剩余的第一支撑元件和第二支撑元件上、以及露出的所述第一电极、所述牺牲层和所述第二电极上沉积钝化层；对沉积的钝化层进行平坦化，以在剩余的第一支撑元件和第二支撑元件上形成钝化层并露出所述牺牲层。
在一个实施例中，所述图案化所述第一支撑元件材料层、所述第一电极材料层、所述牺牲材料层、所述第二电极材料层和所述第二支撑元件材料层的步骤包括：将所述第一电极材料层和/或所述第二电极材料层分成间隔开的多段，从而使得形成的第一电极和/或第二电极包括间隔开的多段。
在一个实施例中，所述方法还包括：在所述腔的底部或侧壁，或者所述通道的底部或侧壁，或者所述引导电极上形成能够固定酶或待检测化学物质的纳米结构。
在一个实施例中，所述纳米结构的材料包括金属、金属氧化物、无机聚合物、有机聚合物或前述材料的组合。
在一个实施例中，所述通道的宽度为0.5-100nm，例如1nm、2nm、10nm、50nm、80nm等；和/或所述通道的长度为50nm-100μm，例如100nm、500nm、5μm、10μm、30μm等；和/或所述通道的深度为0-10μm，例如100nm、300nm、1μm、2μm、8μm等。
在一个实施例中，所述第一电极的厚度为1-1000nm，例如30nm、50nm、200nm、600nm、800nm等。
在一个实施例中，所述第一电极的材料和所述第二电极的材料相同。
在一个实施例中，所述第一电极的材料和所述第二电极的材料不同。
在一个实施例中，所述第一电极的材料包括：硅、铂、金、银、氧化铟锡或碳基材料；和/或所述牺牲层的材料包括：铬、钨、铝、铝的氧化物、硅、硅的氧化物或硅的氮化物；和/或所述第二电极的材料包括：硅、铂、金、银、氧化铟锡或碳基材料；和/或所述引导电极的材料包括：硅、铂、金、银、氧化铟锡或碳基材料。
在一个实施例中，所述第一支撑元件和所述第二支撑元件包括导电元件。
在一个实施例中，所述导电元件的材料包括：硅、铂、金、银、氧化铟锡或碳基材料。
在一个实施例中，所述第一支撑元件和/或所述第二支撑元件沿着与衬底表面平行的方向的截面为椭圆形、圆形、矩形、正方形或齿轮形。
本发明还提供一种分析化学物质的方法，所述方法包括以下步骤：
(1)提供本发明任意一项的微孔电极或微孔电极阵列；
(2)将含有待测化学物质的反应液加入微孔电极或微孔电极阵列中，并使所述反应液发生反应，以生成带电分子；
(3)使带电分子在引导电极和/或流体力学的作用下进入通道，或者在引导电极的作用下聚集在通道中；和
(4)利用第一电极、第二电极和/或引导电极确定带电分子的种类，进而获得待测化学物质的信息。
在一个实施例中，其中步骤(4)中，通过选自下列的一种或多种方法，利用第一电极、第二电极和/或引导电极来确定带电分子的种类：氧化还原效应、电阻效应、电容效应、场效应、和隧穿效应。
在一个实施例中，所述待测化学物质选自生物分子(例如核酸、蛋白质、脂类、多糖等)、化合物、和有机聚合物等。在一个实施例 中，所述待测化学物质是核酸，例如DNA或RNA。
在一个实施例中，所述待测化学物质包含或由一种或多种基本单位(例如核苷酸、氨基酸、聚合物单体等)组成。在一个实施例中，所述待测化学物质的基本单位是未经修饰的。在另一个实施例中，所述待测化学物质的基本单位是经标签分子修饰的。
在一个实施例中，所述反应液中含有游离的、经标签分子修饰的基本单位，其在所述反应后生成游离的标签分子。优选地，所述游离的标签分子带电荷(即为带电分子)，并且能够在引导电极的作用下进入通道，或者在引导电极的作用下聚集在通道中。由此，可利用第一电极、第二电极和/或引导电极来确定标签分子的种类，并进而获得待测化学物质的信息。
在一个实施例中，所述反应液中含有一种或多种游离的、经标签分子修饰的基本单位。在一个实施例中，所述反应液中含有至少两种(例如三种，四种，或者更多种)游离的、经标签分子修饰的基本单位。在一个实施例中，不同的基本单位被相同的标签分子所修饰。在另一个实施例中，不同的基本单位被不同的标签分子所修饰。
在一个实施例中，游离的标签分子为氧化还原活性物质，并且利用第一电极和第二电极作为检测电极，通过氧化还原效应来确定标签分子的种类。
在一个实施例中，除了上述的氧化还原效应外，在步骤(4)中，还通过电阻效应、电容效应、场效应、隧穿效应中的一种或多种，利用第一电极、第二电极和/或引导电极来确定带电分子的种类。在此类实施例中，可以利用一种或多种检测原理来对带电分子进行检测，以提高检测结果的准确性。例如，当引导电极之间产生电场时，在通道中出现的任何分子都将对离子流会产生物理阻挡，由此可以产生可检测到的离子电流下降。通过选择合适大小的标签分子和合适长度的通道，通过电阻效应产生的信号特征将会进一步提高对带电分子的检测的准确性。因此，本领域技术人员可以根据实际需要来选择用于确定带电分子的种类的方法的组合。例如，可以将场效应与氧化还原效应 结合、或者将电容和氧化还原效应结合、或者将电阻与氧化还原效应结合、或将氧化还原效应、电阻效应与场效应结合等，从而确定带电分子的种类。
在一个实施例中，所述通道底部的绝缘层表面还可以有修饰膜。优选地，将第一电极与第二电极分别作为源电极与漏电极，将修饰膜作为两电极间的导电通道。进一步优选地，可以在导电通道上形成标签分子的识别点作为门电极，从而形成一个场效应管。当不同的标签分子附着在修饰膜上时，导电通道将产生不同的导电性能，从而在源电极与漏电极之间将产生不同的电流强度。由此，根据电流强度的差异，将能够区分不同的标签分子。
在系统的可靠性方面，由于各种检测方式之间不存在相互交叉，因此可以互相作为备份，而不会导致整个系统的全面崩溃。
在一个实施例中，第一电极和/或第二电极各自可以被间隔为多段，也即两段或更多段(例如3-4段)。在一个实施例中，分段后，每段电极具有不同的电压。在此类实施例中，特别优选地，每段电极的电压分别对应于一种标签分子的响应电压(氧化还原电位窗口)。由此，对于一种标签分子来说，只有其电压匹配标签分子的响应电压的一段电极才能响应该标签分子，产生信号；而其它段电极则不能响应该分子，不产生信号。因此，通过一段电极的信号的存在与否，可以判断对应于该段电极的标签分子的存在与否。这种检测方式有利于将信号与噪声区分开。这种能够提供动态信息的电极设计方式也明显区别于传统的纳米孔方案。在一个实施例中，在同一通道中将第一电极和/或第二电极间隔为多段例如2-4段横向电极，并且每段单独的电极都可以单独控制，以产生不同的电压。
此外，在一个实施例中，每个微孔电极可以具有多个通道。在一个微孔电极中设置多个通道可以极大地增加检测表面积，这样不仅可以提高信号强度，还能够减小潜在的污染效应。微孔电极阵列和腔的结合也提供了更多可控的样品注入模式。
在一个实施例中，通道可以是开放式的。开放式通道的制造工艺 更为简易，且便于样品及液体注入，能够更好地在速度和准确性之间寻找平衡。在一个实施例中，通道可以是封闭式的。封闭式通道有利于控制并减少外部杂质信号的干扰。在实际应用中，可根据具体情况选择开放式、封闭式或者两者组合的通道结构。在一个实施例中，所述微孔电极具有开放式通道，或者封闭式通道，或者开放式通道和封闭式通道。
在一个实施例中，所述待测化学物质为核酸分子，并且，在步骤(2)中，使所述反应液进行核苷酸的聚合反应。
在一个实施例中，所述方法用于分析化学物质的组成、序列、电荷、大小或浓度等。
本发明还提供一种分析核酸分子的方法，所述方法包括以下步骤：
(1)提供本发明任意一项的微孔电极或微孔电极阵列；
(2)将聚合酶(例如DNA聚合酶或RNA聚合酶)固定于微孔电极或微孔电极阵列的腔或通道中或引导电极上；
(3)在所述微孔电极或微孔电极阵列中加入反应液，所述反应液包含待测核酸分子，引物，以及至少一种(例如一种、两种、三种、四种)脱氧核糖核苷三磷酸(dNTP)分子、核糖核苷三磷酸(NTP)分子或它们的类似物，其中，所述引物能够与所述待测核酸分子的部分序列杂交或退火，并且所述至少一种dNTP、NTP分子或它们的类似物的每一种各自用标签分子修饰；随后，在适当的条件下，使所述待测核酸分子与所述引物杂交形成复合物；
(4)在聚合酶的催化作用下，所述用标签分子修饰的dNTP、NTP分子或它们的类似物中的一种被掺入(或并入)所述引物，形成与待测核酸分子互补的延伸产物，并且掺入所述引物的dNTP、NTP分子或它们的类似物所携带的标签分子被去除而成为游离的标签分子，所述游离的标签分子带有电荷；
(5)使所述游离的标签分子在引导电极和/或流体力学的作用下进入通道，或者在引导电极的作用下聚集在通道中；优选地，利用电 荷极性或反应和释放顺序来控制标签分子进入或聚集在不同的微孔电极通道中；
(6)利用第一电极和第二电极，确定标签分子的种类；进而，通过标签分子与dNTP、NTP分子或它们的类似物之间的对应性，确定掺入所述引物的dNTP、NTP分子或它们的类似物的种类；进而，通过碱基互补配对原则，确定待测核酸分子相应位置的碱基信息；
(7)重复进行步骤(4)、(5)和(6)，直至所述复合物的延伸反应结束。
在一个实施例中，在步骤(6)中，通过氧化还原效应、电阻效应、电容效应、场效应、隧穿效应中的一种或多种来确定标签分子的种类。
在一个实施例中，通过氧化还原效应来确定标签分子的种类。在一个实施例中，所述游离的标签分子为可发生氧化还原循环反应的氧化还原活性物质，或者可以转换为可发生氧化还原循环反应的氧化还原活性物质。在一个实施例中，所述游离的标签分子通过物理或化学作用转换为可发生氧化还原循环反应的氧化还原活性物质。优选地，所述氧化还原活性物质在第一电极和第二电极之间发生氧化还原循环反应，并产生可检测的电流。由此，可根据该可检测的电流来确定标签分子的种类。在一个实施例中，除了前述的氧化还原效应之外，还通过电阻效应、电容效应、场效应、隧穿效应中的一种或多种来确定标签分子的种类。
在一个实施例中，利用引导电极或其它手段引导和/或控制游离的标签分子(例如具有氧化还原活性的标签分子)进入通道，并在通道的第一电极和第二电极上分别施加电位。当第一电极与第二电极的电位匹配该标签分子的氧化还原电位窗口时，所述标签分子能够在第一电极和第二电极之间发生氧化还原循环反应，并产生可检测的氧化还原电流脉冲信号。利用该可检测的氧化还原电流脉冲信号，可实现对标签分子的特异性识别和检测。当第一电极和第二电极之间设置了匹配电位但没有检测到该脉冲信号时，即表明没有该标签分子存在。
在一个实施例中，所述至少一种dNTP、NTP分子或它们的类似 物的每一种各自携带具有不同氧化还原电位窗口的标签分子。在一个优选的实施例中，通过改变第一电极和/或第二电极的电位，并测量在各种电位条件下是否产生氧化还原电流脉冲信号，以及任选地测量脉冲信号的信号幅度等信息，可以分辨出通道内的标签分子种类。
在一个实施例中，所述至少一种dNTP、NTP分子或它们的类似物的每一种各自携带具有不同氧化还原电位窗口的标签分子。在一个优选的实施例中，将通道中的第一电极和/或第二电极分为多段，并且每段分别施加与不同标签分子的氧化还原电位窗口相匹配的电位。由此，当标签分子通过通道时，仅在对应该标签分子的氧化还原电位窗口的电极段可检测到氧化还原电流脉冲信号，从而可分辨出通道内的标签分子种类。
在一个实施例中，所述反应液中还含有磷酸酶。
在一个实施例中，在所述方法的步骤(4)中，所述游离的标签分子在磷酸酶的作用下进一步脱去磷酸基团。
在一个实施例中，标签分子修饰的dNTP、NTP或其类似物的净电荷为中性或具有负电荷。
在一个实施例中，所述游离的标签分子具有正电荷或负电荷。
在一个实施例中，所述用标签分子修饰的dNTP、NTP或其类似物的净电荷为中性或具有负电荷，并且所述游离的标签分子具有正电荷。在此类实施例中，带有正电荷的游离的标签分子可以在电场的作用下沿着通道移动，而标签分子修饰的dNTP、NTP分子由于为中性或带负电荷而不会在腔或通道中移动。
在一个实施例中，所述用标签分子修饰的dNTP、NTP或其类似物为具有负电荷，并且所述游离的标签分子具有负电荷。在此类实施例中，带有负电荷的游离的标签分子、用标签分子修饰的dNTP、NTP分子和未经修饰的dNTP、NTP分子可以在电场的作用下共同沿着通道移动。
在一个实施例中，只有游离的标签分子具有氧化还原活性，而标签分子修饰的dNTP、NTP分子和未经修饰的dNTP、NTP分子不具 有氧化还原活性。在此类实施例中，氧化还原电流信号只能来自于游离的标签分子。
在一个实施例中，所述的标签分子连接于dNTP、NTP分子或其类似物的磷酸基团、碱基或糖基团。优选地，所述的标签分子选自以下的一种或多种：氨基酸、肽、碳水化合物、金属化合物、染料、化学发光化合物、核苷酸、脂肪族酸、芳香族酸、醇、氨基苯基、羟苯基、萘基、硫醇基、氰基、硝基、烷基、烯基、炔基、叠氮基或上述基团的衍生物。优选地，所述标签分子选自以下的一种或多种：氨基苯基、羟苯基、萘基、变价态的金属化合物(如二茂铁、六氰合铁、六氰合亚铁)、蒽醌、次甲基蓝等分子以及上述基团的衍生物。在一个实施例中，标签分子连接于dNTP、NTP分子或其类似物的γ-磷酸基团，并且，优选地，所述标签分子选自氨基苯基、羟苯基、萘基基团以及上述基团的衍生物。在一个实施例中，标签分子连接于dNTP、NTP分子或其类似物的碱基或糖基团，并且，优选地，所述标签分子选自变价态的金属化合物(例如二茂铁、六氰合铁、六氰合亚铁)、蒽醌、次甲基蓝等分子以及上述基团的衍生物。
在一个实施例中，每种类型的dNTP(例如dATP、dTTP、dCTP、dGTP、dUTP)或NTP(例如ATP、TTP、CTP、GTP、UTP)分子用一种特异的具有氧化还原活性的标签分子标记，所述特异的标签分子在发生氧化还原循环反应时能够产生特异的氧化还原电信号。
在一个实施例中，在步骤(3)中同时加入所有四种类型的dNTP(例如选自dATP、dTTP/dUTP、dCTP、dGTP)或NTP(例如ATP、TTP、CTP、GTP、UTP)分子。在此类实施例中，可以省去每种碱基并入后的洗涤步骤，由此可以大大降低试剂成本并加快检测速度。
在一个实施例中，通过选择标签分子来调整游离的标签分子携带的电荷数量，进而调整游离的标签分子在引导电极作用下的移动速度。
在一个实施例中，其中步骤(1)中所述的聚合酶固定于腔或通道内的底部绝缘层上，或者固定于引导电极上。优选地，所述聚合酶固定于腔的底部靠近通道入口处的部位；优选地，所述通道的入口处可 以设计为多种形状(例如漏斗形)，以容纳聚合酶。
在一个实施例中，每个腔或通道内固定一个聚合酶。
在一个实施例中，所述反应液中的待测核酸分子为单链核酸分子。
在一个实施例中，每个聚合酶可以捕获一条单链核酸分子或者由核酸分子和引物杂交形成的一个复合物。
在本发明中，将聚合酶固定于腔或通道内的底部的方法为本领域所公知。
在一个实施例中，所述绝缘层的材料选自二氧化硅、氮氧化硅、氮化硅或其他绝缘材料(例如碳掺杂氧化物(CDO)、碳化硅、诸如聚酰亚胺、八氟环丁烷或聚四氟乙烯的有机聚合物、氟硅酸盐玻璃(FSG)和诸如倍半硅氧烷、硅氧烷或有机硅酸盐玻璃之类的有机硅酸盐)。
在一个实施例中，在所述绝缘层与聚合酶之间还包括可功能化区和/或分子结合区。在一个实施例中，所述可功能化区包含二氧化硅、氧化铪、氧化铝、氧化钽、和/或氧化锆等可功能化材料。例如，所述可功能化材料可以使用选自以下的链接分子进行功能化：硅烷(例如氨丙基三乙氧基硅烷)、硫醇(-SH)、二硫化物(-S-S-)、异硫氰酸盐、烯烃和炔烃。在一个实施例中，所述分子结合区包括探针分子。优选地，所述探针分子例如选自生物素、亲和素、抗体、抗原、受体、配体、DNA序列、RNA序列、蛋白及其配体。
在一个实施例中，可以通过选择结合分子使聚合酶在合适的方向上固定。
在一个实施例中，所述方法用于分析核酸分子的序列、组成、电荷、大小或浓度等。
在本发明中，电解液和反应液可以加在微孔电极的表面，以使所有的腔和通道充满电解液和反应液。不同电极或同一电极的不同段的电压可以独立设置，以独立地控制或检测反应分子。
在氧化还原检测模式中，所述引导电极控制电解液和氧化还原活性 物质进入通道，第一电极和第二电极作为氧化还原反应检测装置，用于检测氧化还原活性物质。
在本发明中，引导电极形成的电场有利于带电分子(例如带正电荷分子)进入通道中，并且在通道中聚集，以减少这些分子从通道扩散出去的可能性，这样可以提高检测到的信号的强度。
为了保证单个核苷酸的分辨率，可以调整引导电极的电压以控制带电分子转运的速度。此外，也可以通过改造聚合酶使其最佳的合成速度与分子转运的速度相匹配。此外，还可以通过微米或者纳米尺度的流体力学来控制分子在通道中的运动。
在本发明中，所述核酸分子包括脱氧核糖核苷酸(DNA)和核糖核苷酸(RNA)以及被磷酸二酯键链接在一起的其它类似物的聚合物。多核苷酸可以是基因组的片段、基因或其一部分、cDNA或合成聚脱氧核糖核酸序列。包括多核苷酸的核苷酸是诸如被链接到2’-脱氧核糖的腺嘌呤、胞嘧啶、鸟嘌呤或胸腺嘧啶之类的自然发生的脱氧核糖核苷酸，或者诸如被链接到核糖的腺嘌呤、胞嘧啶、鸟嘌呤或尿嘧啶之类的核糖核苷酸。然而，多核苷酸或低聚核苷酸(例如探针或引物)还可以包含核苷酸类似物，其包括非自然发生的合成核苷酸或修饰性自然发生的核苷酸。
在本发明中，所述氧化还原循环是指，其中能够被可逆地氧化和/或还原的分子(即氧化还原活性分子)在被独立地偏置的至少两个电极之间移动的电化学方法，所述至少两个电极中的一个低于被检测的氧化还原活性分子的还原电位且另一个高于氧化电位，使电子在独立偏置电极之间穿梭往返(即，分子在第一电极处被氧化，并且扩散至第二电极，其在那里被还原，或者反之亦然，其首先被还原然后被氧化，这取决于分子和电极被偏置时的电位)。在氧化还原循环中，同一分子能够因此向被记录的电流贡献多个电子，导致信号的净放大。来自氧化还原活性物质的信号潜在地能够被放大100倍以上，这取决于诸如氧化还原活性物质的稳定性和氧化还原活性物质扩散到感测区域的能力等因素。在本发明中，引导电极的设置可以防止氧化还原活性物质扩散出通道，从而 增加氧化还原活性物质的有效浓度。
在本发明中，所述氧化还原活性物质(或氧化还原活性分子)具有本领域的一般含义，是能够多次可逆地通过氧化和/或还原的状态循环的分子。
在本发明中，所述磷酸酶例如选自碱性磷酸酶、酸性磷酸酶、蛋白质磷酸酶、多磷酸磷酸酶、糖磷酸酶以及焦磷酸酶。
在本发明中，在合成延伸产物的过程中，标签分子修饰的dNTP、NTP或其类似物的并入将标签分子焦磷酸盐(PPi)释放到溶液中。磷酸酶的作用是从标签分子中去除焦磷酸盐。磷酸基团的去除使氧化还原活性物质进一步活化，继而可以用电化学方式来检测氧化还原活性物质的存在。
在本发明中，所述硅烷分子可以具有化学方程式X3-Si-YR”，X2-Si-(N)YR”和X-Si-(N2)YR”，其中X是离去基团，例如-Cl、-OCH3或者-OCH2CH3，R”是反应耦合基团，诸如，-NH2、-COOH、-COH、-CHCH2或-SH，并且R是非反应基团，例如，烷基基团。用于耦合的表面附着的硅烷分子呈现的有机基团可以例如是羧基基团、醛、酯、烯烃、炔烃、硫醇、异硫氰酸盐、异氰酸盐、取代胺、环氧化物，诸如生物素之类的小分子或乙醇。一般地，Y是非反应基团，诸如，具有1到16个碳原子的碳氢化合物。-YR”的示例包括-(CH2)3NH2、-(CH2)2COOH以及-(CH2)2SH。一些示例性硅烷包括3-氨丙基三乙氧基硅烷(APTS)、巯基硅烷和环氧丙氧基三甲氧基硅烷(具有环氧化物反应耦合基团)。要硅烷化的表面可以与例如溶液中或作为硅烷气体的硅烷分子反应。
在本发明中，所述碱基例如选自腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶、尿嘧啶、7-脱氮鸟嘌呤、7-脱氮腺嘌呤和5-甲基胞嘧啶。
在本发明中，所述引物(引物序列)是具有适当长度(例如约18-24个碱基)的短的通常以化学方式合成的寡核苷酸，其足以与标靶核酸(例如单链DNA)杂交并且允许在本领域公知的适合条件下向其中加入核苷酸残基或由其合成寡核苷酸或聚核苷酸。在一个实施例中，引物是 DNA引物，即由脱氧核糖核苷酸残基组成或主要由脱氧核糖核苷酸残基组成的引物。引物被设计成具有与所述引物杂交的模板/标靶核酸(例如单链DNA)的区域的反向互补序列。核苷酸残基通过形成磷酸二酯键而加入到引物的3’端产生了延伸产物；核苷酸残基通过形成磷酸二酯键而加入到延伸产物的3’端产生了另一种延伸产物。
在本发明中，所述dNTP、NTP或其类似物并入寡核苷酸或聚核苷酸(如引物、延伸产物、引物与待测核酸分子形成的复合物)中是指，在聚核苷酸的3’末端的核苷酸残基的3’碳原子分别与dNTP、NTP或其类似物的5’碳原子之间形成磷酸二酯键。
在本发明中，所述聚合酶包括DNA聚合酶、RNA聚合酶、逆转录酶等，其功能或种类为本领域所公知；所述DNA聚合酶例如可以具有或不具有3’至5’端核酸外切酶活性，例如包括E.coli DNA聚合酶，Klenow大片段，Phusion DNA聚合酶，9°N DNA聚合酶，KOD聚合酶，Therminator DNA聚合酶，Taq DNA聚合酶，Vent DNA聚合酶等。
在一个实施例中，通过聚合酶的修饰，以在实时测序时达到理想的碱基并入速度(例如1-100个碱基/秒)。
在一个实施例中，聚合酶经过特异性改造以具有更适于应用的特征。例如，从DNA聚合酶的克隆库中，选择或得到能够快速、连续和准确地合成聚合物的聚合酶，要求是单个分子的DNA聚合酶能够合成1-100kb的DNA，而不会从模板链上解离。优选地，聚合酶缺少外切核苷酸活性，并且并入(掺入)碱基的错误率应低至10-5-10-6/掺入碱基。聚合酶对每种特异性核苷酸的偏向性能够大大提高测序的特异性。聚合酶对DNA、RNA和甲基化碱基具有不同的可测量的偏向性对测序很有益处。除了提到的物理化学和酶学特性，其它特性还包括热稳定性、稳定的缓冲体系，在高分子聚集的条件下的工作能力，以及对大量副产物(焦磷酸盐)的耐受能力。
在一个实施例中，在聚合酶的克隆库中筛选出合适的突变体，其聚合速度与检测设备的能力相匹配，这样能最大程度地区分真正的碱基掺入和未掺入。这种单分子/实时聚合酶方法同样要求聚合酶能够掺 入修饰的核苷酸。因此需要挑选与检测设备能力相符的聚合酶突变体和修饰的核苷酸。
在一个实施例中，除了选择最适合的聚合酶突变体，还可以通过缓冲液来改变掺入碱基的速度(例如通过pH和离子来减慢或加快特定核苷酸的掺入速度)。可以通过测量核苷酸在聚合酶口袋中的停留时间来挑选具有更高或更低的对特定核苷酸的偏向性的聚合酶突变体。酶动力学的测量可以降低假阳性掺入的机率，能够区分真正的掺入信号和设备噪声。
在一个实施例中，通过分析和比较聚合酶突变体的二元复合物和三元复合物的晶体结构并且与可测量的掺入事件相匹配，可以找到候选物中对特异性靶向突变重要的残基，进而帮助我们找到新的特异、稳定的相互作用。
酶的另一个特征是在到达终点时能够释放核酸分子，以使新的带引物的模板DNA进入并开始测序。在一个实施例中，在足够稳定的酶的作用下，能够在一个通道中对1-10-100-1000kb的模板进行测序。
发明的有益效果
本发明的微孔电极具有以下优点：微孔电极可包含多个纳米通道，从而可减少电极表面污染带来的影响；通过引导电极在纳米通道中产生的电场和电渗流来引导试剂的流动；易于与微流体进行整合达到试剂交互的目的；易于大规模制造电极阵列；兼容多种信号检测机制，包括电子信号检测(例如基于氧化还原循环、FET(场效应管)、电化学、电阻抗)；使信号的物理放大成为可能；可以集成单片CMOS；可使用电场控制核酸分子的移动；通过检测标签分子的电信号对待测化学物质进行单分子检测；读长大大提升；检测信号灵敏。
本发明的化学物质分析方法可用于分子检测和分析、分子诊断、疾病检测、物质识别以及DNA检测和测序等。
通过以下参照附图对本发明的示例性实施例的详细描述，本发明 的其它特征、方面及其优点将会变得清楚。
附图说明
附图构成本说明书的一部分，其描述了本发明的示例性实施例，并且连同说明书一起用于解释本发明的原理，在附图中：
图1A是根据本发明一个实施例的微孔电极的俯视图；
图1B是沿着图1A所示的B-B’截取的截面图；
图1C是沿着图1A所示的C-C’截取的截面图。
图2A示出了根据本发明一个例子的微孔电极的工作原理图；
图2B示出了不同的碱基与第一电极和第二电极上施加的检测电压的对应关系的示意图；
图3示出了根据本发明一个实施例的微孔电极的第一电极和第二电极均包括四段的示意图；
图4A示出了由两个微孔电极组成的微孔电极阵列的示意图；
图4B示出了由四个微孔电极组成的微孔电极阵列的示意图；
图5A、5B、5C和5D示出了微孔电极阵列中的多个微孔电极的四种布置方式；
图6示出了根据本发明的一个实施例的测序系统的示意图；
图7是根据本发明一个实施例的微孔电极的制造方法的简化流程示意图；
图8A示出了根据本发明一个实施例的衬底结构的截面图；
图8B示出了图8A所示衬底结构的俯视图；
图8C是根据本发明一个实施例的微孔电极的制造方法的一个阶段的俯视图；
图8D是根据本发明一个实施例的微孔电极的制造方法的一个阶段的俯视图；
图8E是根据本发明一个实施例的微孔电极的制造方法的一个阶段的截面图；
图8F是根据本发明另一个实施例的微孔电极的制造方法的一个阶 段的截面图；
图8G是根据本发明另一个实施例的微孔电极的制造方法的一个阶段的截面图；
图8H是根据本发明又一个实施例的微孔电极的制造方法的一个阶段的截面图；
图8I是根据本发明又一个实施例的微孔电极的制造方法的一个阶段的截面图；
图8J是根据本发明又一个实施例的微孔电极的制造方法的一个阶段的截面图；
图8K是根据本发明又一个实施例的微孔电极的制造方法的一个阶段的截面图；
图9A是根据发明一个实施例的形成衬底结构的一个阶段的截面图；
图9B是根据发明一个实施例的形成衬底结构的一个阶段的截面图；
图9C是根据发明一个实施例的形成衬底结构的一个阶段的截面图；
图9D是根据发明一个实施例的形成衬底结构的一个阶段的截面图；
图9E是根据发明一个实施例的形成衬底结构的一个阶段的截面图；
图9F是根据发明一个实施例的形成衬底结构的一个阶段的截面图；
图9G是根据发明一个实施例的形成衬底结构的一个阶段的截面图；
图9H是根据发明一个实施例的形成衬底结构的一个阶段的截面图；
图9I是根据发明一个实施例的形成衬底结构的一个阶段的截面图；
图9J是根据发明一个实施例的形成衬底结构的一个阶段的截面图；
图10A和图10B分别示出了使用本发明的一个示例性微孔电极来检测不同的游离的标签分子的检测结果；
图11给出了在本发明的一个示例性微孔电极中通过模拟计算所得到的单个标签分子与电极的碰撞次数分布。
具体实施方式
现在将参照附图来详细描述本发明的各种示例性实施例。应理解，除非另外具体说明，否则在这些实施例中阐述的部件和步骤的相对布 置、数字表达式和数值不应被理解为对本发明范围的限制。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
此外，应当理解，为了便于描述，附图中所示出的各个部件的尺寸并不必然按照实际的比例关系绘制，例如某些层的厚度或宽度可以相对于其他层有所夸大。
以下对示例性实施例的描述仅仅是说明性的，在任何意义上都不作为对本发明及其应用或使用的任何限制。
对于相关领域普通技术人员已知的技术、方法和装置可能不作详细讨论，但在适用这些技术、方法和装置情况下，这些技术、方法和装置应当被视为本说明书的一部分。
应注意，相似的标号和字母在下面的附图中表示类似项，因此，一旦某一项在一个附图中被定义或说明，则在随后的附图的说明中将不需要对其进行进一步讨论。
图1A是根据本发明一个实施例的微孔电极的俯视图。图1B是沿着图1A所示的B-B’截取的截面图。图1C是沿着图1A所示的C-C’截取的截面图。
参考图1A、图1B和图1C，微孔电极100可以包括：
一个或多个第一电极301。不同的第一电极301之间可以被隔开，例如不同的第一电极301之间可以具有缝隙，或者该缝隙中可以填充有绝缘层。示例性地，第一电极301的厚度d1可以约为1-1000nm，例如，例如30nm、50nm、200nm、600nm、800nm等。在一个实施例中，第一电极301的材料可以包括：铂、金、银、氧化铟锡、碳基材料(例如金刚石、石墨、无定形碳、碳纳米管等)、硅或者前述材料的组合等。
微孔电极100还包括与每个第一电极301相对设置的第二电极303。每个第一电极301和与其相对的第二电极303之间具有通道601，通道601的至少一端连通有腔401。示例性地，第二电极303的厚度d2可以约为1-1000nm，例如，30nm、50nm、200nm、600nm、800nm等。 在一个实施例中，第二电极303的材料可以包括：铂、金、银、氧化铟锡、碳基材料(例如金刚石、石墨、无定形碳、碳纳米管等)、硅或者前述材料的组合等。在一个实施例中，腔401的底面与601的底面可以位于同一平面。在另一个实施例中，腔401的底面与601的底面可以位于不同的平面。
微孔电极100还包括位于腔401中的一个或多个引导电极501。在一个实施例中，引导电极501能够引导带电物质进入通道601。此外，引导电极501还能够控制带电物质在通道601中的运动。在向通道601两端的引导电极501施加不同的电压时，引导电极501之间产生的引导电场可以控制带电物质(例如氧化还原活性分子)进入通道601，并且，可以控制通道601中的带电物质(例如氧化还原活性分子)的运动，使得带电物质不会轻易运动到通道601之外。如果腔401中有一个引导电极501，则该引导电极501可以用于控制与腔401连通的一个或多个通道601中的带电物质的运动；如果腔401中有多个引导电极501，则每个引导电极501可以分别控制与其邻近的通道601中的带电物质的运动。示例性地，引导电极501的材料可以包括：硅、铂、金、氧化铟锡、碳基材料或前述材料的组合。
优选地，引导电极501可以与第一电极301或第二电极303基本上垂直(如图1A所示)。然而，应理解，引导电极501与第一电极301或第二电极303之间不一定必须垂直，也可以具有其他的角度。
本发明提供的微孔电极可以用于但不限于核酸测序，在用于核酸测序时，腔401可以是微流体腔。微孔电极的制造工艺可以与CMOS工艺兼容，易于大规模制造；另外，可以采用多种信号机制来进行核酸测序，并且读长更长。另外，由于微孔电极可以同时包括多个通道，这可以减少电极污染对测序结果造成的影响，使得测序结果更加准确。
图2A示出了根据本发明一个例子的微孔电极的工作原理图。如图2A所示，微孔电极包含一组检测电极(第一电极301和第二电极303)和一组引导电极501。第一电极301上施加的电压为V1，第二电极303上施加的电压为V2，两个引导电极之间的电压差为Vg。两个引导电极 501之间的引导电场可以将待检测分子(例如，具有氧化还原活性的标签分子)牵引至两个检测电极之间的通道中。标签分子在两个检测电极之间进行氧化还原循环反应，氧化还原循环反应会产生相应的电流信号，该电流信号可以被两个检测电极检测到。不同的标签分子对应不同的电流信号，因此，可以通过检测到的电流信号来确定标签分子的种类。
图2B示出了不同的碱基与第一电极和第二电极上施加的检测电压的对应关系的示意图。根据氧化还原反应的原理，只有当电压V1和V2位于待检测分子对应的氧化还原电位窗口时才可以检测到该待检测分子的电流信号。因此，在一个时间周期t0内，可以通过多次改变电压V1和V2的大小使得电压V1和V2位于待检测分子对应的氧化还原电位窗口，进而可以识别该待检测分子的种类。这样，可以在不同的电压V1和V2下检测到不同待检测分子的电流信号。如图2B所示，碱基A、T、C、G分别对应不同的电压V1和V2。具体地，碱基A对应的电压为V1A和V2A，碱基T对应的电压为V1T和V2T，碱基C对应的电压为V1C和V2C，碱基G对应的电压为V1G和V2G。以碱基A为例，当V1为V1A、V2为V2A时，如果通道中有碱基A对应的标签分子，则检测电极可以检测到碱基A对应的标签分子在氧化还原反应中产生的电流信号i。类似地，通过改变电压V1和V2的大小可以使得电压V1和V2位于碱基T、C或G对应的标签分子的氧化还原电位窗口，从而可以识别出标签分子的种类，进而识别出于标签分子对应的碱基类型。
在一个实施例中，参见图1B和图1C，微孔电极还可以包括衬底101和位于衬底101上的绝缘层102。第一电极301、第二电极303、以及引导电极501可以位于绝缘层102上。衬底101例如可以是硅衬底、III-V族半导体材料的衬底、绝缘体上硅衬底(SOI)，或者也可以是诸如ZnO、CdO、TiO2、Al2O3、SnO等的氧化物半导体衬底，或者也可以是诸如石英玻璃、苏打玻璃等绝缘材料的衬底。绝缘层102典型地可以为硅的氧化物(例如二氧化硅)、硅的氮化物(例如SiN)、硅的氮氧化物等等。
在实际应用中，上述第一电极301和第二电极303可以由支撑元件 来支撑。在一个实施例中，参见图1A，微孔电极还可以包括用于支撑第一电极301的第一支撑元件201。在一个实施例中，微孔电极可以包括多个第一电极301，所有的第一电极301可以均由第一支撑元件201来支撑。在另一个实施例中，微孔电极可以包括多个第一电极301，第一支撑元件201可以包括多个第一支撑元件201，每个第一电极301可以分别由对应的第一支撑元件201支撑。在又一个实施例中，微孔电极可以包括多个第二电极303，并且包括多个第二支撑元件304，每个第二电极303可以分别由对应的第二支撑元件304来支撑。
上述第一支撑元件201和第二支撑元件304可以为导电元件或非导电元件，优选为导电元件。在第一支撑元件201和第二支撑元件304为非导电元件的情况下，第一支撑元件201和第二支撑元件304主要起到支撑第一电极301和第二电极303的作用。在第一支撑元件201和第二支撑元件304为导电元件的情况下，可以通过向第一支撑元件201和第二支撑元件304施加电压来向第一电极301和第二电极303施加电压。在一个实施例中，上述导电元件的材料可以包括硅(例如多晶硅)、铂、金、银、氧化铟锡或碳基材料等；上述非导电元件的材料可以包括氧化硅、氮化硅、氮氧化硅、硼磷硅玻璃等。另外，第一支撑元件201平行于衬底101表面的方向的截面形状可以是椭圆形、圆形、多边形、方形或齿轮形。应理解，本发明并不限于上述示例性的形状。
在本发明微孔电极的另一个实施例中，上述第一电极301的材料和第二电极303的材料可以相同，也可以不同。在一个实施例中，第一电极301和第二电极303中的其中一个电极可以采用易于氧化的电极材料，另一个电极则可以采用易于还原的电极材料。
在本发明微孔电极的另一个实施例中，如图1B所示，微孔电极100还可以包括在第一电极301、第二电极303、第一支撑元件201(如果有的话)、第二支撑元件304(如果有的话)中的至少一个部件的表面上的钝化层701。在一个实施例中，钝化层701可以是硅的氮化物(例如SiN)、硅的氧化物(例如SiO2)等等。钝化层701可以起到保护微孔电极的作用，具体地，在利用微孔电极进行核酸测序时，只有未被钝化 层701覆盖的区域可以接触到反应液，这可以避免微孔电极的某些部件受损。另外，钝化层701还能够降低测序过程中产生的噪音。
在本发明微孔电极的再一个实施例中，微孔电极100还可以包括：能够固定酶或待检测化学物质802的纳米结构801(例如纳米点)。纳米结构801可以位于腔401的底部(如图1A和图1B所示)或侧壁，或者，位于通道601的底部(未示出)或侧壁，或者，位于引导电极211上。示例性地，纳米结构801的尺寸可以为1-100nm，例如8nm、20nm、60nm等。优选地，纳米结构801的材料可以包括金属氧化物，优选地，所述金属氧化物是诸如二氧化锆(ZrO2)或二氧化铪(HfO2)的过渡金属氧化物；或者，纳米结构801的材料也可以包括金属，优选地，所述金属是诸如金、铂的惰性金属；或者，纳米结构801的材料也可以是无机聚合物、有机聚合物或前述材料的组合。
在一些实现方案中，第一电极301和/或第二电极303可以包括间隔开的多段，例如两段或更多段，下面结合不同的实现方式进行说明。
在一个实现方式中，第一电极301包括间隔开的多段，第二电极303仅包括一段。微孔电极可以包括多个第一支撑元件201，每段第一电极301可以分别由对应的第一支撑元件201来支撑。在这种情况下，可以向第二电极303施加某个电压，向每段第一电极301施加与不同的待检测分子(例如氧化还原标签分子)对应的电压。在待检测分子通过每段第一电极301时，除了能得到待检测分子的电流信号信息外还能得到待检测分子从一段第一电极301运动到另一段第一电极301的时间信息，这有利于提升不同待检测分子的检测分辨率。
在另一个实现方式中，第一电极301包括间隔开的多段，第二电极303也包括间隔开的多段，每段第一电极301与每段第二电极303相对设置。每段第一电极301可以分别由对应的第一支撑元件201来支撑，每段第二电极303可以分别由对应的第二支撑元件304支撑。在这种情况下，可以向每段第一电极301和与该段第一电极301相对设置的一段第二电极303施加不同的电压，通过引导电场控制的待检测分子依次通过每段电极时，只有施加于该标签分子的氧化还原电位窗口对应的电压 的一段电极可以检测到电流信号。因此，通过不同段的第一电极301和对应的第二电极303可以检测到不同的待检测分子。
在又一个实现方式中，第一电极301可以包括一段，第二电极303可以包括间隔开的多段。在这种情况下，可以向第一电极301施加某个电压，向每段第二电极303施加与不同的待检测分子(例如氧化还原标签分子)的氧化还原窗口对应的电压。在待检测分子通过每段第二电极303时，除了能得到待检测分子的电流信号信息外还能得到待检测分子从一段第一电极303运动到另一段第一电极303的时间信息，这有利于提升不同待检测分子的检测分辨率。
图3示出了根据本发明一个实施例的微孔电极的第一电极和第二电极均包括四段的示意图。如图3所示，第一电极301包括第一段第一电极311、第二段第一电极321、第三段第一电极331和第四段第一电极341，第一段第一电极311至第四段第一电极341上施加的电压分别为V11、V21、V31和V41。第二电极303包括第一段第二电极313、第二段第二电极323、第三段第二电极333和第四段第二电极343，第一段第二电极313至第四段第二电极343上施加的电压分别为V12、V22、V32和V42。每段第一电极301和一段第二电极303相对设置，并且每段第一电极301和与其相对设置的一段第二电极303作为一组检测电极。例如，第一段第一电极311与第一段第二电极313相对设置，这两段电极可以作为一组检测电极。每组检测电极上施加的检测电压分别对应一种待检测标签分子。两个引导电极501上施加的电压差为Vg。通过引导电极501的引导电场控制的待检测标签分子依次通过四组检测电极时，只有施加的检测电压与该待检测标签分子的氧化还原窗口对应的的一组检测电极可以检测到电流信号。这样，在预先设定好检测电压后，即可以根据检测到电流信号的检测电极确定待检测标签分子的种类。例如，第一组检测电极上施加的检测电压与某个待检测标签分子的氧化还原窗口对应，则在第一组检测电极检测到电流信号后即可确定检测到了该待检测标签分子。
本发明还提供了一种微孔电极阵列，包括上述任意一个实施例所述 的一个或多个微孔电极。在一个实施例中，多个微孔电极可以共用一个引导电极。
图4A示出了由两个微孔电极组成的微孔电极阵列的示意图。如图4A所示，微孔电极包括第一微孔电极和第二微孔电极，第一微孔电极和第二微孔电极共用中间的引导电极501。第一微孔电极的第一电极301和第二电极303上施加的电压分别为V11和V12，第二微孔电极的第一电极301和第二电极303上施加的电压分别为V21和V22。第一微孔电极的两个引导电极501上施加的电压差为Vg1，第二微孔电极的两个引导电极501上施加的电压差为Vg2。带负电的标签分子被牵引至第一微孔电极的通道中，带正电的标签分子被牵引至第二微孔电极的通道中，从而实现了对标签分子的筛选。这样，由两个微孔电极组成的微孔电极阵列可以识别出标签分子的电荷极性，并且根据电流信号可以确定标签分子的种类。
图4B示出了由四个微孔电极组成的微孔电极阵列的示意图。如图4B所示，微孔电极阵列包括第一微孔电极、第二微孔电极、第三微孔电极和第四微孔电极。四个微孔电极共用中间的引导电极501。第一微孔电极的第一电极301和第二电极303上施加的电压分别为V11和V12，第二微孔电极的第一电极301和第二电极303上施加的电压分别为V21和V22，第三微孔电极的第一电极301和第二电极303上施加的电压分别为V31和V32，第四微孔电极的第一电极301和第二电极303上施加的电压分别为V41和V42。第一至第四微孔电极的引导电极501上施加的电压差分别为Vg1、Vg2、Vg3和Vg4。虚线框中示出了Vg1、Vg2、Vg3和Vg4随时间变化的示意图。利用周期性的引导电场，可以在一个时序周期内的不同时刻将不同的标签分子分别输送到四个微孔电极的通道中，这样可以有效增加每个标签分子在通道中的反应及检测时间，从而有效增强标签分子的检测信号、改善检测信噪比，并且可以避免相邻的标签分子间的信号干扰。
在微孔电极阵列中，多个微孔电极可以被布置为不同的形状。
图5A、5B和5C分别示出了微孔电极阵列中的多个微孔电极的三 种布置方式。在图5A中，多个微孔电极被布置为环形。在图5B中，多个微孔电极被布置为方形，例如矩形或正方形。在图5C中，多个微孔电极被布置为行列矩阵形。然而，本发明并不限于上述几种布置方式。例如，多个微孔电极还可以被布置为椭圆形、圆形、扇形、锯齿形、齿轮形等。另外，多个微孔电极的布置方式也不限于二维平面的布置方式，例如，多个微孔电极还可以被布置为上下叠层形的三维布置方式。
此外，微孔电极阵列中的多个微孔电极也可以是串联的，即，每个微孔电极的通道是串联的，如图5A或图5B所示。多个微孔电极也可以是相互独立的，如图5C所示，每个微孔电极可以用于分别检测不同的标签分子。或者，多个微孔电极还可以是并联的，即，每个微孔电极的通道是并联的，如图5D所示，多个(例如4个)微孔电极的引导电极上施加的电压差均为Vg，多个微孔电极可以在各自的检测电压(即，第一电极和第二电极上施加的电压)下并行对标签分子进行检测。
本发明还提供了一种传感器芯片，包括上述实施例所述的微孔电极阵列。在传感器芯片的一个实施例中，可以包括微孔电极阵列，还可以包括向电极(例如第一电极、第二电极或引导电极)施加电压的电路、放大装置、电流感应电路、电压感应电路等等。
本发明还提供了一种测序系统，包括上述实施例所述的传感器芯片。
图6示出了根据本发明的一个实施例测序系统的示意图。如图6所示，测序系统包括流通池(flow cell)、传感器芯片、印制电路板PCB、现场可编程门阵列(FPGA)卡和计算机/移动设备。流通池可以向传感器芯片供应微流体，封装后的传感器芯片可以通过PCB板上的插座固定在PCB板上。PCB板上可以包含电流和/或电压感应电路芯片、电流和/或电压放大电路芯片、数据存储芯片、系统控制芯片等等。FPGA卡可以与PCB板集成，并可以通过接口与计算机/移动设备相连，从而可以在计算机或移动设备上开发应用控制软件，实现对测序系统的控制。
在实际应用中，可以将测序系统设计成不同的样式，例如手掌式、便携式、桌面式或大型机式。微孔电极阵列可以整合到测序系统的传感器芯片中，并且可以通过FPGA卡进行软件编程，从而实现软硬件的整 合。
本发明提出的微孔电极能够与CMOS工艺兼容，下面将介绍微孔电极的一种示例性的制造方法。
图7是根据本发明一个实施例的微孔电极的制造方法的简化流程示意图。图8A-图8K示出了根据本发明一个实施例的微孔电极的制造方法的各个阶段。下面参考图7以及图8A-图8K对微孔电极的制造方法进行详细说明。
首先，在步骤702中，提供衬底结构。
图8A示出了根据本发明一个实施例的衬底结构的截面图。图8B示出了图8A所示衬底结构的俯视图。如图8A和图8B所示，衬底结构包括表面具有绝缘层102的衬底101、以及位于绝缘层102上的第一支撑元件材料层201，并且，第一支撑元件材料层201的侧壁(例如，第一支撑元件材料层201的一侧或两侧的侧壁)上依次具有第一电极材料层301、牺牲材料层302、第二电极材料层303和第二支撑元件材料层304。注意，图8A和8B所示的衬底结构中，第一支撑元件材料层201一侧的侧壁上具有第一电极材料层301、牺牲材料层302、第二电极材料层303和第二支撑元件材料层304。在一些实施例中，第一支撑元件材料层201的另一侧的侧壁上也可以具有第一电极材料层301、牺牲材料层302、第二电极材料层303和第二支撑元件材料层304。
作为一个非限制性示例，第一电极材料层301的厚度可以约为1-1000nm，例如30nm、50nm、200nm、600nm、800nm等。牺牲材料层302的厚度可以约为0.5-100nm，例如5nm、10nm、20nm、40nm、80nm等。第二电极材料层303的厚度可以约为1-1000nm，例如30nm、50nm、200nm、600nm、800nm等。
第一电极材料层301和第二电极材料层303的组成材料可以相同，也可以不同。另外，牺牲材料层302的材料可以根据第一电极材料层301和第二电极材料层303来选择。示例性地，第一电极材料层301和第二电极材料层303的材料可以包括：硅、铂、金、银、氧化铟锡、碳基材料(例如金刚石、石墨、无定形碳等)或者前述材料的组合；牺牲材料 层302的材料可以包括：铬、钨、铝、铝的氧化物、硅、硅的氧化物、硅的氮化物或者前述材料的组合。在一个具体实施例中，第一电极材料层301和第二电极材料层303的材料可以是铂，牺牲材料层302的材料可以是铬或氧化硅。在另一个具体实施例中，第一电极材料层301和第二电极材料层303的材料可以是金，牺牲材料层302的材料可以是钨。
接下来，在步骤704中，图案化第一支撑元件材料层201、第一电极材料层301、牺牲材料层302、第二电极材料层303和第二支撑元件材料层304，从而形成一个或多个腔401，并形成第一支撑元件201、以及依次位于第一支撑元件201的侧壁上的第一电极301、牺牲层302、第二电极303和第二支撑元件304，如图8C所示。这里，第一支撑元件201、第一电极301、牺牲层302、第二电极303和第二支撑元件304分别由前述的第一支撑元件材料层、第一电极材料层、牺牲材料层、第二电极材料层和第二支撑元件材料层形成而来。所形成的腔401与之后形成的通道601连通。另外，该步骤中，还可以将第一电极材料层301和/或第二电极材料层303分成间隔开的多段，从而使得形成的第一电极301和/或第二电极303包括间隔开的多段。
之后，在步骤706中，在腔401中形成一个或多个引导电极501，如图8D所示。例如，可以通过光刻、沉积、剥离等工艺形成引导电极501，也可以通过沉积、平坦化、刻蚀等工艺形成引导电极501。优选地，引导电极501可以与第一电极301基本垂直；或者引导电极501也可以与第二电极303基本垂直。也即，对引导电极501、第一电极301、以及第二电极302分别施加电压后，引导电极501之间的电场的方向与第一电极301和第二电极303之间的电场的方向可以基本垂直。
之后，在步骤708中，去除第一支撑元件201的侧壁上的牺牲层302，以在第一电极301和第二电极303之间形成通道601，其中，通道601的至少一端连通有腔401，如图8E所示。
在一个实施例中，在去除第一支撑元件201的侧壁上的牺牲层302之前，可以在第一支撑元件201、第二支撑元件304、第一电极301和第二电极303中的至少一个部件的表面上形成钝化层701，如图8F所 示。示例性地，钝化层701的材料可以是硅的氮化物、硅的氧化物等等。例如，可以在第一支撑元件201、第一电极301、第二电极303和第二支撑元件304的顶部沉积钝化材料，然后对沉积的钝化材料进行图案化，以在第一支撑元件201、第二支撑元件304、第一电极301和第二电极303中的至少一个部件的表面上形成钝化层701。之后，可以通过选择性刻蚀工艺去除牺牲层302，从而在第一电极301和第二电极303之间形成通道601，如图8G所示。
在另一个实施例中，在去除第一支撑元件201的侧壁上的牺牲层302之前，可以先去除第一支撑元件201和第二支撑元件304的顶部的一部分，以露出第一电极301、牺牲层302和第二电极303的一部分，如图8H所示。然后，在剩余的第一支撑元件201和第二支撑元件304上、以及露出的第一电极301、牺牲层302和第二电极303上沉积钝化层701，如图8I所示。之后，对沉积的钝化层701进行平坦化，以在剩余的第一支撑元件201和第二支撑元件304上形成钝化层701并露出牺牲层302，如图8J所示。在一个实现方式中，平坦化工艺可以停止在第一电极301、牺牲层302、以及第二电极303的上表面。替代地，平坦化工艺也可以去除第一电极301、牺牲层302、以及第二电极303的一部分，只要在剩余的第一支撑元件201和第二支撑元件304上保留一部分钝化层701即可。之后，可以通过选择性刻蚀工艺去除露出的牺牲层302，以在第一电极301和第二电极303之间形成通道601，如图8K所示。
作为示例，参见图1A和图1B，所形成的通道601的宽度W可以约为0.5-100nm(例如1nm、2nm、10nm、50nm、80nm等)，通道601的长度L可以约为50nm-100μm(例如100nm、500nm、5μm、10μm、30μm等)，通道601的深度H可以约为0-10μm(例如100nm、300nm、1μm、2μm、8μm等)。这里，通道601的宽度为第一电极301和第二电极303之间的距离，通道601的长度为围绕第一支撑元件201延伸的长度，通道601的深度为第一电极301和第二电极303的上表面与绝缘层102之间的距离。
通过如上方法，形成了图1A、图1B和图1C所示的微孔电极。然而，应明白，本发明不限于采用上述方法来形成微孔电极。在实际应用中，可以根据微孔电极的具体结构采用相应的制造方法，例如，可以在上述方法的基础上增加某些步骤或调整上述方法中的某些步骤。例如，在第一支撑元件的侧壁上形成第一电极材料层时，可以根据所需的第一电极的数量来确定是第一支撑元件的一侧还是两侧的侧壁上形成第一电极材料层。
在形成微孔电极后，还可以在腔401的底部或侧壁，或者通道601的底部或侧壁，或者引导电极501上形成能够固定酶或待检测化学物质802的纳米结构801。图1C示出了纳米结构位于腔401的底部的情况。在一个实施例中，纳米结构801可以是纳米点。示例性地，纳米结构801的尺寸可以为1-100nm，例如8nm、20nm、50nm、80nm等。在一个实施例中，纳米结构802的材料可以包括诸如二氧化锆(ZrO2)或二氧化铪(HfO2)的过渡金属氧化物、惰性金属、无机聚合物、有机聚合物或前述材料的组合。
图8A和图8B所示的衬底结构可以通过不同的方式来实现，下面结合图9A-图9J介绍形成衬底结构的一种具体实现方式。
首先，如图9A所示，提供表面具有绝缘层102的衬底101。衬底101例如可以是硅衬底、III-V族半导体材料的衬底、绝缘体上硅衬底(SOI)，或者也可以是诸如ZnO、CdO、TiO2、Al2O3、SnO等的氧化物半导体衬底，或者也可以是诸如石英玻璃、苏打玻璃等绝缘材料的衬底。可以通过热氧化的方式在衬底101上形成绝缘层102，也可以通过沉积(例如物理气相沉积PVD、化学气相沉积CVD等)的方式在衬底上形成绝缘层102。典型地，绝缘层102可以是二氧化硅。
接下来，如图9B所示，在绝缘层102的一部分上形成第一支撑元件材料层201。第一支撑元件材料层201的材料可以为导电材料，也可以为非导电材料，优选为导电材料。例如，可以通过沉积(例如PVD或CVD)的方式在绝缘层102上沉积导电材料，然后对导电材料进行图案化，从而形成第一支撑元件材料层201。作为一个非限制性示例， 第一支撑元件材料层201在平行于衬底101表面方向的截面为椭圆形、方形、圆形或多边形。然而，应理解，本发明不限于此，第一支撑元件材料层201也可以是其他合适的形状。
然后，如图9C所示，沉积第一电极材料层301，以覆盖第一支撑元件材料层201的上表面和侧壁。这里，第一电极材料层301也可以覆盖暴露的绝缘层102的一部分或全部。
然后，如图9D所示，可以通过各向异性干法刻蚀，例如反应离子刻蚀(RIE)、离子束刻蚀(IBE)等去除第一支撑元件材料层201的上表面上的第一电极材料层301，仅保留第一支撑元件材料层201的侧壁上的第一电极材料层301。在一个实施例中，如果绝缘层102上也沉积有第一电极材料层301，则将绝缘层102上的第一电极材料层301去除。
接下来，如图9E所示，沉积牺牲材料层302，以覆盖第一支撑元件材料层201的上表面、剩余的第一电极材料层301(也即，第一支撑元件材料层201的侧壁上的第一电极材料层301)的上表面和侧壁。这里，牺牲材料层302也可以覆盖暴露的绝缘层102的一部分或全部。
接下来，如图9F所示，去除第一支撑元件材料层201的上表面、剩余的第一电极材料层301的上表面上的牺牲材料层302，仅保留剩余的第一电极材料层301的侧壁上的牺牲材料层302。在一个实施例中，如果绝缘层102上也沉积有牺牲材料层302，则将绝缘层102上的牺牲材料层302去除。
然后，如图9G所示，沉积第二电极材料层303，以覆盖第一支撑元件材料层201的上表面、剩余的第一电极材料层301的上表面、以及剩余的牺牲材料层302的上表面和侧壁。这里，第二电极材料层303也可以覆盖暴露的绝缘层102的一部分或全部。
之后，如图9H所示，去除第一支撑元件材料层201的上表面、剩余的第一电极材料层301的上表面、剩余的牺牲材料层302的上表面上的第二电极材料层303，保留剩余的牺牲材料层302的侧壁上的第二电极材料层303。在一个实施例中，如果绝缘层102上也沉积有第二电极 材料层303，则将绝缘层102上的第二电极材料层303去除。
之后，如图9I所示，沉积第二支撑元件材料层304，以覆盖第一支撑元件材料层201、第一支撑元件材料层201的侧壁上的第一电极材料层301、牺牲材料层302、第二电极材料层303、以及暴露的绝缘层102。
之后，如图9J所示，对沉积的第二支撑元件材料层304进行平坦化，以暴露第一支撑元件材料层201的侧壁上的牺牲层302，从而形成衬底结构。在一个实现方式中，平坦化工艺可以使得第一支撑元件材料层201及其侧壁上的第一电极材料层301、牺牲层302、以及第二电极材料层303的上表面和第二支撑元件材料层304的上表面基本齐平。
按照图9A至图9J形成衬底结构后，可以按照上面给出的方式执行后续步骤704-步骤708，在此不再赘述。
在一个实施例中，用于氧化还原循环反应的标签分子例如可选自：
Figure PCTCN2016086846-appb-000001
在一个实施例中，所述标签分子修饰的dNTP或其类似物例如可以通过如下方法合成：
Figure PCTCN2016086846-appb-000002
在本发明中，可以设计不同的标签分子来修饰四种不同的dNTP、NTP分子或其类似物，使游离的标签分子具有不同的氧化还原电位，进而能够区分不同的dNTP、NTP分子或其类似物，在一个实施例中，所述不同的标签分子可如下所示：
Figure PCTCN2016086846-appb-000003
当游离的标签分子携带的电荷数量不同时，其在引导电极的作用 下具有不同的移动速度，在一个实施例中，不同的标签分子携带的电荷如下所示：
Figure PCTCN2016086846-appb-000004
在一个实施例中，不带电荷或带负电荷的标签分子修饰的dNTP在DNA聚合酶和碱性磷酸酶的作用下成为带正电荷的氧化还原活性物质，如下所示：
Figure PCTCN2016086846-appb-000005
在一个实施例中，第一电极和第二电极发生氧化还原循环反应的原理如下所示：
Figure PCTCN2016086846-appb-000006
图10A和图10B分别示出了使用本发明的一个示例性微孔电极来检测不同的游离的标签分子的检测结果。其中，图10A为使用本发明的微孔电极检测到的六氰合铁分子的氧化还原电流曲线，图10B为使用本发明的微孔电极检测到的二茂铁分子氧化还原电流曲线。图10A和图10B的结果显示，六氰合铁分子和二茂铁分子的氧化还原电位窗口和曲线形状存在显著差异。这一结果表明，可利用本发明的微孔电极所检测到的电流信号来区分和辨别各种游离的标签分子。当反应液中的各种基本单位(例如dNTP、NTP或其类似物)各自用不同的标签分子进行修饰时，本发明的微孔电极即可通过所检测到的独特的电信号来区分和辨别标签分子的种类，并进而区分和辨别在反应液中参与反应的基本单位(例如dNTP、NTP或其类似物)的种类，最终实现对待测化学物质(例如核酸)的分析。
图11给出了在本发明的一个示例性微孔电极中通过模拟计算所得到的单个标签分子与电极的碰撞次数分布。忽略分子吸附等非理想条件，如果该电化学活性分子每次与电极碰撞交换一个电子，那么单位时间内的碰撞次数即可以直接转化为产生的最大理论电流值。因此，通过进一步缩小通道最小尺寸，改变标签分子化学结构，增加每次碰撞电子交换数目，对电极表面进行处理以降低分子吸附等方式，可以实现电流信号的进一步放大，从而提高电信号检测的准确度。
至此，已经详细描述了根据本发明实施例的微孔电极及其制造方法，以及基于该微孔电极的核酸分析方法。为了避免遮蔽本发明的构思，没有描述本领域所公知的一些细节，本领域技术人员根据上面的描述，完全可以明白如何实施这里公开的技术方案。另外，本说明书公开所教导的各实施例可以自由组合。本领域的技术人员应该理解，可以对上面说明的实施例进行多种修改而不脱离如所附权利要求限定的本发明的精神和范围。

Claims (59)

  1. 一种微孔电极，其特征在于，包括：
    一个或多个第一电极；
    与每个第一电极相对设置的第二电极，每个第一电极和与其相对的第二电极之间具有通道，通道的至少一端连通有腔；
    位于所述腔中的一个或多个引导电极。
  2. 根据权利要求1所述的微孔电极，其特征在于，还包括：
    用于支撑所述第一电极的第一支撑元件。
  3. 根据权利要求2所述的微孔电极，其特征在于，所述微孔电极包括多个第一电极，所述第一支撑元件包括多个第一支撑元件，每个第一电极分别由对应的第一支撑元件支撑。
  4. 根据权利要求1所述的微孔电极，其特征在于，所述微孔电极包括多个第二电极，所述微孔电极还包括：
    多个第二支撑元件，每个第二电极分别由对应的第二支撑元件支撑。
  5. 根据权利要求1所述的微孔电极，其特征在于，
    所述第一电极包括间隔开的多段。
  6. 根据权利要求1或5所述的微孔电极，其特征在于，
    所述第二电极包括间隔开的多段。
  7. 根据权利要求5所述的微孔电极，其特征在于，还包括：
    多个第一支撑元件，每段第一电极分别由对应的第一支撑元件支撑。
  8. 根据权利要求6所述的微孔电极，其特征在于，还包括：
    多个第二支撑元件，每段第二电极分别由对应的第二支撑元件支撑。
  9. 根据权利要求3或7所述的微孔电极，其特征在于，
    所述第一支撑元件为导电元件。
  10. 根据权利要求4或8所述的微孔电极，其特征在于，
    所述第二支撑元件为导电元件。
  11. 根据权利要求1所述的微孔电极，其特征在于，还包括：
    能够固定酶或待检测化学物质的纳米结构，所述纳米结构位于所述腔的底部或侧壁，或者位于所述通道的底部或侧壁，或者位于所述引导电极上。
  12. 根据权利要求11所述的微孔电极，其特征在于，
    所述纳米结构的材料包括金属、金属氧化物、无机聚合物、有机聚合物或前述材料的组合。
  13. 根据权利要求1所述的微孔电极，其特征在于，
    所述通道的宽度为0.5-100nm；和/或
    所述通道的长度为50nm-100μm；和/或
    所述通道的深度为0-10μm。
  14. 根据权利要求1所述的微孔电极，其特征在于，
    所述第一电极的厚度为1-1000nm；和/或
    所述第二电极的厚度为1-1000nm。
  15. 根据权利要求1所述的微孔电极，其特征在于，
    所述第一电极的材料与所述第二电极的材料相同。
  16. 根据权利要求1所述的微孔电极，其特征在于，
    所述第一电极的材料与所述第二电极的材料不同。
  17. 根据权利要求1所述的微孔电极，其特征在于，
    所述第一电极的材料包括：硅、铂、金、银、氧化铟锡或碳基材料；和/或
    所述第二电极的材料包括：硅、铂、金、银、氧化铟锡或碳基材料；和/或
    所述引导电极的材料包括：硅、铂、金、银、氧化铟锡或碳基材料。
  18. 根据权利要求9所述的微孔电极，其特征在于，
    所述导电元件的材料包括：硅、铂、金、银、氧化铟锡或碳基材料。
  19. 根据权利要求10所述的微孔电极，其特征在于，
    所述导电元件的材料包括：硅、铂、金、银、氧化铟锡或碳基材料。
  20. 根据权利要求2所述的微孔电极，其特征在于，
    所述第一支撑元件沿着与衬底表面平行的方向的截面为椭圆形、圆形、多边形或齿轮形。
  21. 根据权利要求1所述的微孔电极，其特征在于，
    所述腔的底面与所述通道的底面位于同一平面或不同平面。
  22. 根据权利要求1所述的微孔电极，其特征在于，还包括：
    衬底和位于衬底上的绝缘层；
    所述第一电极、所述第二电极、以及所述引导电极位于所述绝缘层上。
  23. 根据权利要求1所述的微孔电极，其特征在于，还包括：
    位于所述第一电极和/或所述第二电极的表面上的钝化层。
  24. 一种微孔电极阵列，其特征在于，包括：权利要求1-23任意一项所述的微孔电极。
  25. 根据权利要求24所述的微孔电极阵列，其特征在于，所述微孔电极阵列包括多个所述微孔电极；
    多个所述微孔电极被布置为椭圆形、圆形、环形、扇形、矩形、正方形、锯齿形、齿轮形、行列矩阵形或上下叠层形。
  26. 根据权利要求25所述的微孔电极阵列，其特征在于，
    多个所述微孔电极是相互独立的或串联的或并联的。
  27. 根据权利要求25所述的微孔电极阵列，其特征在于，
    多个所述微孔电极共用一个引导电极。
  28. 一种传感器芯片，其特征在于，包括：权利要求24-27任意一项所述的微孔电极阵列。
  29. 一种测序系统，其特征在于，包括：权利要求28所述的传感器芯片。
  30. 一种微孔电极的制造方法，其特征在于，包括：
    提供衬底结构，所述衬底结构包括表面具有绝缘层的衬底、位于绝缘层上的第一支撑元件材料层，所述第一支撑元件材料层的侧壁上依次具有第一电极材料层、牺牲材料层、第二电极材料层和第二支撑元件材料层；
    图案化所述第一支撑元件材料层、所述第一电极材料层、所述牺牲材料层、所述第二电极材料层和所述第二支撑元件材料层，从而形成一 个或多个腔，并形成第一支撑元件、以及依次位于所述第一支撑元件的侧壁上的第一电极、牺牲层、第二电极和第二支撑元件；
    在所述腔中形成一个或多个引导电极；
    去除所述第一支撑元件的侧壁上的牺牲层，以在所述第一电极和所述第二电极之间形成通道；
    其中，所述通道的至少一端连通有所述腔。
  31. 根据权利要求30所述的方法，其特征在于，所述提供衬底结构的步骤包括：
    提供表面具有绝缘层的衬底；
    在所述绝缘层的一部分上形成第一支撑元件材料层；
    沉积第一电极材料层，以覆盖所述第一支撑元件材料层的上表面和侧壁；
    去除所述第一支撑元件材料层的上表面上的第一电极材料层；
    沉积牺牲材料层，以覆盖所述第一支撑元件材料层的上表面、剩余的第一电极材料层的上表面和侧壁；
    去除所述第一支撑元件材料层的上表面和剩余的第一电极材料层的上表面上的牺牲材料层；
    沉积第二电极材料层，以覆盖所述第一支撑元件材料层的上表面、剩余的第一电极材料层的上表面、以及剩余的牺牲材料层的上表面和侧壁；
    去除所述第一支撑元件材料层的上表面、剩余的第一电极材料层的上表面和剩余的牺牲材料层的上表面上的第二电极材料层；
    沉积第二支撑元件材料层，以覆盖所述第一支撑元件材料层、所述第一支撑元件材料层的侧壁上的第一电极材料层、牺牲材料层和第二电极材料层、以及暴露的绝缘层；
    对沉积的第二支撑元件材料层进行平坦化，以暴露所述第一支撑元件材料层的侧壁上的牺牲材料层。
  32. 根据权利要求30所述的方法，其特征在于，在去除所述第一支撑元件的侧壁上的牺牲层之前，还包括：
    在所述第一支撑元件、所述第二支撑元件、所述第一电极和所述第二电极中的至少一个部件的表面上形成钝化层。
  33. 根据权利要求30所述的方法，其特征在于，在去除所述第一支撑元件的侧壁上的牺牲层之前，还包括：
    去除所述第一支撑元件和所述第二支撑元件的顶部的一部分，以露出所述第一电极、所述牺牲层和所述第二电极的一部分；
    在剩余的第一支撑元件和第二支撑元件上、以及露出的所述第一电极、所述牺牲层和所述第二电极上沉积钝化层；
    对沉积的钝化层进行平坦化，以在剩余的第一支撑元件和第二支撑元件上形成钝化层并露出所述牺牲层。
  34. 根据权利要求30所述的方法，其特征在于，所述图案化所述第一支撑元件材料层、所述第一电极材料层、所述牺牲材料层、所述第二电极材料层和所述第二支撑元件材料层的步骤包括：
    将所述第一电极材料层和/或所述第二电极材料层分成间隔开的多段，从而使得形成的第一电极和/或第二电极包括间隔开的多段。
  35. 根据权利要求30所述的方法，其特征在于，所述方法还包括：
    在所述腔的底部或侧壁，或者所述通道的底部或侧壁，或者所述引导电极上形成能够固定酶或待检测化学物质的纳米结构。
  36. 根据权利要求35所述的方法，其特征在于，
    所述纳米结构的材料包括金属、金属氧化物、无机聚合物、有机聚合物或前述材料的组合。
  37. 根据权利要求30所述的方法，其特征在于，
    所述通道的宽度为0.5-100nm；和/或
    所述通道的长度为50nm-100μm；和/或
    所述通道的深度为0-10μm。
  38. 根据权利要求30所述的方法，其特征在于，
    所述第一电极的厚度为1-1000nm；和/或
    所述牺牲层的厚度为0.5-100nm；和/或
    所述第二电极的厚度为1-1000nm。
  39. 根据权利要求30所述的方法，其特征在于，
    所述第一电极的材料和所述第二电极的材料相同。
  40. 根据权利要求30所述的方法，其特征在于，
    所述第一电极的材料和所述第二电极的材料不同。
  41. 根据权利要求30所述的方法，其特征在于，
    所述第一电极的材料包括：硅、铂、金、银、氧化铟锡或碳基材料；和/或
    所述牺牲层的材料包括：硅、铬、钨、铝、铝的氧化物、硅的氧化物或硅的氮化物；和/或
    所述第二电极的材料包括：铂、金、银、氧化铟锡或碳基材料；和/或
    所述引导电极的材料包括：硅、铂、金、银、氧化铟锡或碳基材料。
  42. 根据权利要求30所述的方法，其特征在于，
    所述第一支撑元件和/或所述第二支撑元件包括导电元件。
  43. 根据权利要求42所述的方法，其特征在于，
    所述导电元件的材料包括：硅、铂、金、银、氧化铟锡或碳基材料。
  44. 根据权利要求30-43任意一项所述的方法，其特征在于，
    所述第一支撑元件和/或所述第二支撑元件沿着与衬底表面平行的方向的截面为椭圆形、圆形、矩形、正方形或齿轮形。
  45. 一种分析化学物质的方法，所述方法包括以下步骤：
    (1)提供权利要求1-23任意一项的微孔电极或权利要求24-27任意一项所述的微孔电极阵列；
    (2)将含有待测化学物质的反应液加入微孔电极或微孔电极阵列中，并使所述反应液发生反应，以生成带电分子；
    (3)使带电分子在引导电极和/或流体力学的作用下进入通道，或者在引导电极的作用下聚集在通道中；和
    (4)利用第一电极、第二电极和/或引导电极确定带电分子的种类，进而获得待测化学物质的信息。
  46. 根据权利要求45所述的方法，其中步骤(4)中，通过选自下列的一种或多种方法，利用第一电极、第二电极和/或引导电极来确定带电分子的种类：氧化还原效应、电阻效应、电容效应、场效应、隧穿效应。
  47. 根据权利要求45所述的方法，其用于分析化学物质的组成、序列、电荷、大小或浓度等。
  48. 一种分析核酸分子的方法，所述方法包括以下步骤：
    (1)提供权利要求1-23任意一项的微孔电极或权利要求24-27任意一项所述的微孔电极阵列；
    (2)将聚合酶(例如DNA聚合酶或RNA聚合酶)固定于微孔电极或微孔电极阵列的腔或通道中或引导电极上；
    (3)在所述微孔电极或微孔电极阵列中加入反应液，所述反应液包含待测核酸分子，引物，以及至少一种(例如一种、两种、三种、 四种)脱氧核糖核苷三磷酸(dNTP)分子、核糖核苷三磷酸(NTP)分子或它们的类似物，其中，所述引物能够与所述待测核酸分子的部分序列杂交或退火，并且所述至少一种dNTP、NTP分子或它们的类似物中的每一种各自用标签分子修饰；随后，在适当的条件下，使所述待测核酸分子与所述引物杂交形成复合物；
    (4)在聚合酶的催化作用下，所述用标签分子修饰的dNTP、NTP分子或它们的类似物中的一种被掺入(或并入)所述引物，形成与待测核酸分子互补的延伸产物，并且掺入所述引物的dNTP、NTP分子或它们的类似物所携带的标签分子被去除而成为游离的标签分子，所述游离的标签分子带有电荷；
    (5)使所述游离的标签分子在引导电极和/或流体力学的作用下进入通道，或者在引导电极的作用下聚集在通道中；优选地，利用电荷极性或反应和释放顺序来控制标签分子进入或聚集在不同的微孔电极通道中；
    (6)利用第一电极和第二电极，确定标签分子的种类；进而，通过标签分子与dNTP、NTP分子或它们的类似物之间的对应性，确定掺入所述引物的dNTP、NTP分子或它们的类似物的种类；进而，通过碱基互补配对原则，确定待测核酸分子相应位置的碱基；
    (7)重复进行步骤(4)、(5)和(6)，直至所述复合物的延伸反应结束。
  49. 根据权利要求48所述的方法，其中游离的标签分子为可发生氧化还原循环反应的氧化还原活性物质，或者可以转换为可发生氧化还原循环反应的氧化还原活性物质；优选地，所述氧化还原活性物质在第一电极和第二电极之间发生氧化还原循环反应，并产生可检测的电流。
  50. 根据权利要求48所述的方法，所述反应液中还含有磷酸酶。
  51. 根据权利要求48所述的方法，其中步骤(4)中所述游离的标签分子在磷酸酶的作用下进一步脱去磷酸基团。
  52. 根据权利要求48所述的方法，所述游离的标签分子带正电荷或负电荷。
  53. 根据权利要求48所述的方法，其中所述的标签分子连接于dNTP、NTP分子或其类似物的磷酸基团、碱基或糖基团。
  54. 根据权利要求48所述的方法，通过选择标签分子来调整游离的标签分子携带的电荷数量，进而调整游离的标签分子在引导电极作用下的移动速度。
  55. 根据权利要求48所述的方法，其中步骤(1)中所述的聚合酶固定于腔或通道的底部绝缘层上，或者固定于引导电极上；优选地，所述聚合酶固定于腔的底部靠近通道入口处的部位。
  56. 根据权利要求55所述的方法，其中所述绝缘层的材料选自二氧化硅、氮氧化硅、氮化硅或其他绝缘材料。
  57. 根据权利要求55所述的方法，其中在所述绝缘层与聚合酶之间还包括可功能化区和/或分子结合区；
    优选地，所述可功能化区包含二氧化硅、氧化铪、氧化铝、氧化钽、和/或氧化锆，更优选地，所述可功能化材料使用选自以下的链接分子进行功能化：硅烷(例如氨丙基三乙氧基硅烷)、硫醇(-SH)、二硫化物(-S-S-)、异硫氰酸盐、烯烃和炔烃；
    优选地，所述分子结合区包括探针分子；优选地，所述探针分子例如选自生物素、亲和素、抗体、抗原、受体、配体、DNA序列、RNA序列、蛋白及其配体。
  58. 根据权利要求48所述的方法，其中步骤(6)中，通过氧化还原效应、电阻效应、电容效应、场效应、隧穿效应中的一种或多种来确定标签分子的种类。
  59. 根据权利要求48所述的方法，所述方法用于分析核酸分子的序列、组成、电荷、大小或浓度。
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