Gene sequencing method based on silicon nanowire / graphene monomolecular device

Abstract

The invention provides a gene sequencing method based on a silicon nanowire / graphene single-molecule device, and belongs to the technical field of single-molecule gene sequencing. Two materials of a one-dimensional silicon nano material and a molecular heterojunction are used as electrical detection elements, source and drain currents are output, and a single functional DNA polymerase is used as an element grid synchronously, so that the single-molecule gene sequencing is realized. Preparing a single DNA polymerase modified single molecule field effect transistor device; a bionic liquid phase test module is designed and constructed by utilizing a micro-nano processing and microfluidic technology, polymerase test conditions are optimized, real-time sequencing of non-amplification genes is realized, and the purposes of single-base resolution, high fidelity and low-cost gene sequencing can be achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of single-molecule gene sequencing, and in particular relates to a gene sequencing method based on silicon nanowire / graphene single-molecule devices. Background Art

[0002] DNA sequences contain rich genetic information in the genes of organisms. Accurately achieving high-fidelity reading of DNA sequences is of great value for the discovery and treatment of potential genetic diseases and related diseases such as early cancer. Since Sanger first proposed the concept of DNA sequencing in 1977, the development of DNA sequencing technology has undergone continuous iterations and updates. At present, the DNA sequencing market scale has been established, which is mainly based on the second-generation sequencing technology (Illumina's HiSeq series, Thermo Fisher's Ion Torrent series) and the third-generation technology (PacBio's SMRT technology, ONT's Nanopore technology). The second-generation sequencing technology has broken through the technical difficulties of scale and parallelization, and adopted the concept of sequencing while synthesis, so that DNA samples can be analyzed simultaneously, improving the sequencing throughput. However, short read length (within 1000bp) is still the shortcoming of this generation of sequencing technology, and a large number of PCR amplification links have greatly increased the sequencing cost. The third-generation sequencing technology achieves the goal of high-precision, high-throughput, and long-read sequence information reading by capturing the fluorescent signal of the single-base polymerization event of a single polymerase and real-time recording of the ion current when a single DNA chain passes through a pore, and greatly reduces the workload and cost of sequencing. Therefore, the sequencing technology based on single molecules has forward-looking research value and a strong market blue ocean. However, the current single-molecule sequencing method still has certain limitations, such as background interference of optical signals and single-base accuracy problems in electrical sequencing, and has the shortcoming of low sequencing accuracy. Summary of the invention

[0003] The purpose of the present invention is to develop a high-precision single-molecule electrical sequencing method with DNA single-base recognition, using one-dimensional silicon nanomaterials and molecular heterojunction materials as electrical detection elements, outputting source-drain current, and synchronously using a single functional DNA polymerase as the element gate to prepare a single-molecule field effect transistor device modified by a single DNA polymerase; using micro-nano processing and microfluidics technology, designing and constructing a bionic liquid phase test module, optimizing the polymerase test conditions, and realizing real-time recording of the polymerase single-base polymerization process; introducing modified deoxynucleotide substrate molecules (sulfated deoxynucleotides and dideoxynucleotides), distinguishing the relaxation time and synthesis continuity of different deoxynucleotide single-base polymerization characteristics, realizing reliable distinction of the electrical characteristics of four bases, and combining the test analysis of the gene NPM-415 related to the expression of cancer inhibitors to verify the sequencing accuracy of the technical method.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] The present invention provides the first single-molecule sequencing sensor based on a silicon nanowire field effect transistor device, which includes silicon nanowires arranged on the surface of a silicon substrate, and a metal array coated on the silicon substrate, a layer of SiO2 is attached to the surface of the metal array, a groove is longitudinally punched on the surface of the silicon wire, and the surface of the silicon wire in the groove is modified with a molecular bridge having a maleimide end for connecting to the cysteine ​​site of the polymerase.

[0006] Furthermore, the polymerase is KF polymerase having an amino acid sequence shown in SEQ ID NO.1, and a prefabricated window with a width of 10 nm is longitudinally punched on the surface of the silicon wire, and after development, a channel suitable only for the limited connection of a single KF polymerase is obtained.

[0007] The present invention provides a second single-molecule sequencing sensor based on a graphene-based molecular heterojunction transistor device. The preparation of the device includes processing of graphene strips, coating of metal arrays around the strips, exposure treatment of dotted line prefabricated windows between electrodes, oxygen plasma etching and point burning to gradually process graphene electrode pairs with nanogaps. The ends of the electrode pairs have been functionalized with carboxyl groups, and maleimide molecular bridges are connected between the graphene nanogap electrode pairs for connecting to the cysteine ​​sites of polymerases.

[0008] Furthermore, the graphene-based single-molecule transistor device is a high-density arrayed graphene-based transistor device obtained by evaporating Cr and Au onto graphene strips, and the polymerase is a Poly (β) enzyme with a single cysteine ​​thiol site, having an amino acid sequence shown in SEQ ID NO.4.

[0009] The present invention provides a first method for preparing a single-molecule sequencing sensor based on a silicon nanowire-based field effect transistor device, comprising the following steps:

[0010] 1) Fabrication of silicon nanowire-based field effect transistor devices

[0011] (1) Use CVD thermal growth method to prepare high-quality silicon nanowires, transfer silicon wires to the surface of silicon substrate, rely on template lithography to protect the silicon wires between the electrodes to be etched, and remove excess silicon wires.

[0012] (2) After deprotection, a metal array is coated on the silicon substrate again by template lithography and thermal evaporation, and a layer of SiO2 is attached to the surface of the metal array;

[0013] (3) Using template lithography, a layer of positive resist SU-8 is thermally cured on the device surface, and the window process only exposes the silicon wires between the electrodes and the source and drain electrodes;

[0014] 2) Single-molecule modification of silicon nanowire-based field-effect transistor devices

[0015] (1) Spin-coat a layer of PMMA on the device surface. After curing, a prefabricated window is punched longitudinally on the surface of the silicon wire between the electrodes. After development, a channel suitable for confined connection of a single KF polymerase is obtained;

[0016] (2) The surface of the silicon wire in the channel is etched with HF wet etching to obtain an active surface with Si-H dangling bonds. The PMMA coating is then removed and a molecular bridge with a maleimide end is modified on the active surface of the silicon wire through Si-H addition reaction, esterification reaction and substitution reaction in sequence, which is used to connect the cysteine ​​site of KF polymerase;

[0017] (3) KF polymerase is dissolved in physiological buffer solution and circulated on the surface of the silicon wire of the device by microfluidics. By observing the change in the conductivity of the silicon wire during the electrical test, the purpose of visualizing the successful connection of KF polymerase is achieved.

[0018] Furthermore, the physiological buffer solution in step 2) includes 200 nM KF, 10 mM Tris, 50 mM NaCl, 10 mM MgCl2, 100 μM DTT, and the pH value is 7.8.

[0019] The present invention provides a second method for preparing a single molecule sequencing sensor based on a graphene-based molecular heterojunction transistor device, comprising the following steps:

[0020] 1) Preparation of graphene-based transistor devices

[0021] (1) Using low-pressure chemical vapor deposition to grow a single layer of graphene on copper foil, and using ferric chloride etching and wet transfer technology to transfer the graphene on the copper foil to the silicon wafer;

[0022] (2) Graphene strips are obtained by processing, and Cr and Au are evaporated onto the graphene strips by photolithography and thermal evaporation to prepare high-density arrayed graphene-based transistor devices;

[0023] 2) Micro-nano processing of graphene-based nanogap electrode pairs

[0024] (1) Spin-coat a layer of PMMA on the surface of the transistor array and solidify it. Use an electron beam exposure system to make a dotted line prefabricated window in the middle of the electrode pair. After development, the graphene surface inside the dotted line is exposed;

[0025] (2) First, the graphene inside the dotted line is etched away by oxygen plasma etching, and then the oxygen plasma etching and electrical burning techniques are alternately used to gradually form a graphene-based nanogap electrode pair with carboxyl groups at the ends;

[0026] 3) Single-molecule enzyme modification of graphene-based nanogap electrode pairs

[0027] (1) Dissolve an appropriate amount of maleimide molecular bridge, EDCI and HOBT in anhydrous pyridine protected by argon, and then place the device in the anhydrous pyridine for a certain period of time. After taking out the device, clean the device with acetone and deionized water, and use a semiconductor parameter analyzer to screen each pair of electrodes to obtain a graphene-based single-molecule transistor device in which maleimide is successfully connected between graphene nanogap electrode pairs;

[0028] (2) Drop an enzyme buffer solution with only a single cysteine ​​thiol group on the surface onto the graphene-based single-molecule transistor device. After rinsing and drying, use a semiconductor parameter meter to test the electrode pair. The decrease in conductivity indicates the successful connection of the enzyme.

[0029] Furthermore, the poly(β) polymerase buffer having a single cysteine ​​thiol group in step 3) includes 200 nM polymerase Poly(β), 50 mM Tris–HCl, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, and a pH value of 8.0.

[0030] The present invention provides a first gene sequencing method based on a single molecule sequencing sensor of a silicon nanowire-based field effect transistor device, comprising the following steps:

[0031] (1) Attach a PDMS tank with a cylindrical cavity in the middle to the device surface, and inject the KF polymerase physiological buffer solution into the internal cavity;

[0032] (2) Add adenine deoxynucleotide, thiothymine deoxynucleotide, thiocytosine dideoxynucleotide, thiocytosine deoxynucleotide, guanine dideoxynucleotide, guanine deoxynucleotide, and test the DNA chain polymerization process of the cell sequence template chain;

[0033] (3) Relying on the difference in conductivity values, the number of events and data sites of base repulsion steps are classified, and the electrical data between repulsion steps with regular polymerization lengths are screened and sorted out, and the corresponding esterification reaction time of each single base polymerization is obtained by fitting;

[0034] (4) By setting a threshold for the esterification reaction, the electrical data fragments are converted into binary arrays of short esterification polymerization and long esterification polymerization. These binary arrays are then sorted from short to long to infer the termination site information of each array. By associating the results from the binary arrays with the length of the identified chain termination site, the base information of the sequence site is obtained.

[0035] The present invention provides a second gene sequencing method of a single molecule sequencing sensor based on a graphene-based molecular heterojunction transistor device, comprising the following steps:

[0036] (1) Attach the PDMS tank to the device surface and inject the polymerase Poly(β) physiological buffer solution into the internal cavity;

[0037] (2) Each time, one of the substrates was used as a thionucleotide substrate, and four sets of repeated tests were performed, and the electrical data were recorded in real time;

[0038] (3) Screen out the two-state jump current data consistent with the template length, and use the difference in conductance values ​​to fit the corresponding esterification reaction time of each single base polymerization;

[0039] (4) By establishing a threshold value for the esterification reaction, the electrical data fragments are converted into binary arrays of short esterification polymerization and long esterification polymerization. Combined with the type of thionucleotide substrate added each time, the site information of the corresponding base is read, and the four sets of results are combined to obtain the base information of the sequence site.

[0040] The technical effect of the present invention is as follows: the present invention establishes an electrical single-molecule gene sequencing technology based on silicon nanowires and graphene molecular heterojunctions, achieving single-base precision and high-fidelity reading of DNA sequences. Using a single polymerase chain reaction as the sequence information feedback source, the intrinsic physiological functions of biological molecules are utilized to achieve high-precision reading of sequences, and an effective signal analysis method is constructed to improve the effectiveness of information reading and improve the accuracy and efficiency of single-molecule sequencing technology. At the same time, the present invention systematizes the device preparation process, optimizes the preparation process and performance parameters, and provides a preliminary foundation for realizing the universal preparation of devices in the single-molecule sequencing market and the broad-spectrum application of sequencing platforms. In addition, the technical solution is also applicable to sequencing applications of other functional biological enzymes combined with single-molecule devices, such as other types of DNA polymerases, RNA, DNA degrading enzymes and polypeptide degrading enzymes, which can achieve the universal single-molecule sequencing goals of RNA, DNA and polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Single-molecule polymerase modification process for silicon nanowire-based field-effect transistor devices.

[0042] Figure 2 Single-molecule polymerase modification process for graphene-based molecular heterojunction transistor devices.

[0043] Figure 3 Real-time electrical testing of the device before polymerase ligation and after ligation with the addition of NPM-415 sequence (a) and time distribution of esterification reaction during the complete chain polymerization of NPM-415 sequence (b).

[0044] Figure 4 Data analysis statistical methods (a), NPM-415 sequencing accuracy (b), and four base recognition accuracies (c).

[0045] Figure 5 Real-time electrical testing of the device before polymerase connection (top) and after polymerase modification (bottom). DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0047] (I) Silicon nanowire-based single-molecule electrical sequencing method

[0048] 1) Fabrication of silicon nanowire-based field effect transistor devices

[0049] (1) High-quality silicon nanowires were prepared by CVD thermal growth method (475°C, 2.2 sccm disilane, 0.24 sccm diborane, 7.2 sccm hydrogen, and 25 min growth time). The silicon wires were transferred to the surface of a 1.5×1.7 cm silicon substrate. The silicon wires between the electrodes to be etched were protected by template lithography (lithography for 20 s and development for 8 s), and the excess silicon wires were removed by ultrasound.

[0050] (2) After deprotection, use template lithography (lithography 20s, development 8s) and thermal evaporation ( chromium, A metal array is coated on a silicon substrate by using a gold coating method, and a layer of SiO2 is attached to the surface of the metal array using an electron beam coating device. Prevent the occurrence of liquid phase leakage between arrays.

[0051] (3) Finally, a layer of positive resist SU-8 was thermally cured (95°C) on the device surface by template photolithography (photolithography 40s, development 17s) to achieve large-area coverage protection of the device surface. Window processing (8μm) was performed to expose only the silicon wires between the electrodes and the source and drain electrodes to prevent other impurity molecules from interfering with the test signal in the liquid phase test.

[0052] 2) Single-molecule modification of silicon nanowire-based field-effect transistor devices

[0053] (1) A layer of PMMA was spin-coated on the device surface and baked for curing (180°C, 2 min). Then, a prefabricated window with a width of about 10 nm was longitudinally punched on the surface of the silicon wire between the electrodes using a high-precision electron beam exposure system. After development (15 s), a channel suitable for confined connection of a single Klenow Fragment (KF) polymerase (~8.5 nm) was obtained.

[0054] (2) The surface of the silicon wire in the channel was wet-etched with HF (7s) to obtain an active surface with Si–H dangling bonds. The PMMA coating was then removed and the active surface of the silicon wire was modified with a maleimide-terminated molecular bridge through Si–H addition reaction (reaction with undecynoic acid for 9h, 95°C), esterification reaction (reaction with N-hydroxysuccinimide for 4h, 25°C) and substitution reaction (reaction with N-2-aminoethylmaleimide hydrochloride for 2h, 25°C) to connect the cysteine ​​site of KF polymerase.

[0055] (3) KF polymerase was dissolved in a physiological buffer solution (200nM KF, 10mM Tris, 50mM NaCl, 10mM MgCl2, 100μM DTT, pH 7.8) and circulated on the surface of the silicon wire of the device using microfluidics. By observing the changes in the conductivity of the silicon wire during the electrical test, the purpose of visualizing the successful connection of KF polymerase was achieved. Figure 1 )

[0056] 3) Electrical real-time testing of single-molecule modified silicon nanowire-based field-effect transistor devices

[0057] (1). A PDMS liquid tank with a cylindrical cavity of ~2 mm in diameter in the middle was attached to the device surface, and the internal cavity was injected with KF polymerase physiological buffer solution (10 mM Tris, 50 mM NaCl, 10 mM MgCl2, 100 μM DTT, pH 7.8). Liquid phase circulation was achieved under the drive of a microfluidic liquid pump. The device was placed on a temperature-controlled platform (INSTEC), and a phase-locked amplifier (HF2LI) combined with a current amplifier (DL1211) was used to record the source and drain current of the device in real time, with a sampling frequency of 57.6 kHz.

[0058] (2) Adenine deoxynucleotide (2.5 μM), thiothymine deoxynucleotide (2.5 μM), thiocytosine dideoxynucleotide (1.25 μM), thiocytosine deoxynucleotide (1.25 μM), guanine dideoxynucleotide (1.25 μM), and guanine deoxynucleotide (1.25 μM) were added to test the DNA chain polymerization process of the cell sequence template chain (1 μM primer-template composite chain) to verify the feasibility of sequencing.

[0059] 4) Analysis of electrical sequencing results

[0060] (1). The number of events and data points of the base repulsion step are classified based on the difference in conductivity values. The electrical data between the repulsion steps with regular polymerization lengths are screened and sorted out, and the corresponding esterification reaction time of each single base polymerization is obtained by fitting.

[0061] (2) By setting the threshold of the esterification reaction (1ms), the electrical data fragments are converted into binary arrays (divided into short esterification polymerization and long esterification polymerization). These binary arrays are then sorted from short to long to infer the termination site information (template chain synthesis length) of each array. By correlating the results from the binary array with the length of the identified chain termination site, the base information of the sequence site is obtained.

[0062] (II) Single-molecule sequencing method of graphene-based molecular heterojunction transistor devices

[0063] 1) Preparation of graphene-based transistor devices

[0064] (1) Low-pressure chemical vapor deposition (CVD) was used to grow a single-layer graphene on a 25 μm thick copper foil (1050°C, 8 cm 3 / min hydrogen, 1.6cm 3 / min methane, 25min growth time). The graphene on the copper foil was transferred to a 1.5×1.5cm silicon wafer (the thickness of SiO2 is )superior.

[0065] (2) Through photolithography and oxygen plasma selective etching technology, graphene strips with a length of 250μm and a width of 40μm were processed. Then, 8nm of Cr and 80nm of Au were evaporated onto the graphene strips using photolithography and thermal evaporation methods again to prepare high-density arrayed graphene-based transistor devices.

[0066] 2) Micro-nano processing of graphene-based nanogap electrode pairs (dashed line etching method)

[0067] (1) After the graphene-based transistor device is prepared, a layer of PMMA950 is spin-coated on the surface of the transistor array and baked at 180°C for 2 minutes to solidify.

[0068] (2) Using an ultra-high resolution electron beam exposure system, a dotted window (with a period of 150 nm real part and 50 nm imaginary part) was processed on the PMMA layer. The window pattern was drawn using Design CAD software, and the design size was 5 nm wide.

[0069] (3) Develop in a mixed solution of isopropanol:methyl isobutyl ketone = 3:1 volume ratio for 20 seconds, rinse with isopropanol for fixing and blow dry with N2 to reveal the dotted window of the pattern design.

[0070] (4) Adjust the time of oxygen plasma (RIE, ME-3A) etching and the power of RIE to oxidize and etch the graphene in the dotted window, while the graphene protected by PMMA is oxidized and extended. The etching speed is slower, and slow etching can be achieved to finally obtain a graphene electrode pair with a nano-gap (pA-fA current at 50mV bias). The whole process is as follows: Figure 2 As shown. Moreover, the ends of the graphene electrode pairs obtained by oxygen plasma etching have been functionalized with carboxyl groups, laying the foundation for the covalent connection of the molecule-electrode interface of this type of device.

[0071] 3) Single-molecule enzyme modification of graphene-based nanogap electrode pairs

[0072] (1) Dissolve an appropriate amount of maleimide molecular bridge, EDCI and HOBT in anhydrous pyridine protected by argon, and then place the device in it for 1 to 2 days. After taking out the device, clean the device with acetone and deionized water, and use a semiconductor parameter analyzer to screen each pair of electrodes to obtain a graphene-based single-molecule transistor device with maleimide successfully connected between graphene nanogap electrode pairs.

[0073] (2). Drop an enzyme buffer (200nM polymerase Poly(β), 50mMTris–HCl, pH 8.0, 300mM NaCl, 1mM DTT, 1mM EDTA) with only a single cysteine ​​thiol group on the surface onto the graphene-based single-molecule transistor device and place it at 4°C for 24h. Take out the device, rinse the surface with the buffer, blow dry it, and test the electrode pair using a semiconductor parameter meter. The decrease in conductivity indicates the successful connection of the enzyme. ( Figure 2 )

[0074] 4) Electrical real-time testing of graphene-based single-molecule transistor devices

[0075] (1). A PDMS liquid tank with a diameter of ~2 mm in the middle was attached to the device surface, and the internal cavity was injected with a polymerase Poly(β) physiological buffer solution (50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 1 mM DTT, 1 mM EDTA). The liquid phase circulation was achieved under the drive of a microfluidic liquid pump. The device was placed on a temperature-controlled platform (INSTEC), and a phase-locked amplifier (HF2LI) combined with a current amplifier (DL1211) was used to record the source and drain current of the device in real time, with a sampling frequency of 57.6 kHz.

[0076] (2) Each time, one of the substrates is controlled as a thionucleotide, and four sets of repeated tests are performed. When the thionucleotide substrate is incorporated into the end of the primer chain, the esterification reaction time of this site will be prolonged, and the electrical data will be recorded in real time to verify the feasibility of sequencing.

[0077] 5) Analysis of electrical sequencing results

[0078] (1) The binary transition current data consistent with the template length were selected, and the difference in conductance values ​​was used to fit the corresponding esterification reaction time of each single base polymerization.

[0079] (2) By establishing the threshold value of the esterification reaction (1ms), the electrical data fragments are converted into binary arrays (divided into short esterification polymerization and long esterification polymerization). Combined with the type of thionucleotide substrate added each time, the site information of the corresponding base is read. The four groups of results are combined to obtain the base information of the sequence site.

[0080] Example

[0081] (I) DNA sequencing example using single-molecule modified silicon nanowire-based field-effect transistor devices

[0082] 1) DNA polymerase and test sample (NPM-415) sequence information

[0083] (1) The enzyme used in the example is a single thiol mutant KF polymerase, and the amino acid sequence of the enzyme is as follows (SEQ ID NO.1):

[0084] Protein molecular weight = 614MW = 69229.9

[0085] Isoelectric point = 6.14

[0086] 001MVQIPQNPLILVDGSSYLYRAYHAFPPLTN SAGEPTGAMYGVLNMLRSLI MQYKPTHAAV

[0087] 061VFDAKGKTFRDELFEHYKSH RPMPDDLRAQIEPLHAMVKAMGLPLLAVS GVEADDVIGT

[0088] 121LAREAEKAGR PVLISTGDKD MAQLVTPNIT LINTMTNTIL GPEEVVNKYG VPPELIIDFL

[0089] 181ALMGDSSDNIPGVPGVGEKTAQALLQGLGGLDTLYAEPEKIAGLSFRGAKTMAAKLEQNK

[0090] 241EVAYLSYQLATIKTDVELELTCEQLEVQQPAAEELLGLFKKYEFKRWTADVEAGKWLQAK

[0091] 301GAKPAAKPQE TSVADEAPEV TATVISYDNYVTILDEETLKAWIAKLEKAP VFAFDTETDS

[0092] 361LDNISANLVG LSFAIEPGVAAYIPVAHDYL DAPDQISRERALELLKPLLE DEKALKVGQN

[0093] 421LKYDRGILANYGIELRGIAF DTMLESYILN SVAGRHDMDS LAERWLKHKT ITFEEIAGKG

[0094] 481KNQLTFNQIALEEAGRYAAE DADVTLQLHLKMWPDLQKHK GPLNVFENIE MPLVPVLSRI

[0095] 541ERNGVKIDPK VLHNHSEELT LRLAELEKKAHEIAGEEFNL SSTKQLQTIL FEKQGIKPLK

[0096] 601KTPGGAPSTS EEVLEELALD YPLPKVILEYRGLAKLKSTY TDKLPLMINP KTGRVHTSYH

[0097] 661QAVTATGRLS STDPNLQNIP VRNEEGRRIR QAFIAPEDYV IVSADYSQIE LRIMAHLSRD

[0098] 721KGLLTAFAEG KDIHRATAAE VFGLPLETVT SEQRRSAKAI NFGLIYGMSAFGLARQLNIP

[0099] 781RKEAQKYMDLYFERYPGVLQYMERTRAQAKEQGYVETLDGRRLYLPDIKS SNGARRAAAE

[0100] 841RAAINAPMQG TAADIIKRAM IAVDAWLQAE QPRVRMIMQVHDELVFEVHKDDVDAVAKQI

[0101] 901LMENCTRLDVPLLVEVGS GENWDQAH

[0102] (2) The NPM-415 gene sequence used in the example is specifically:

[0103] Template (SEQ ID NO.2):

[0104] CCAGAGACAAGAAAGACAACTAAAGGAATTCATTTATTACGATTTGATAA

[0105] TTTATTAATAATCATATAACAAGTGTAAAAATACTGACTAATTTCAAAAC

[0106] CTTAATTTAATGTAGACTCATATTTAAAAGAACCTCAGTATAGAAATAGAT

[0107] CTCAATTGAGAGACCACCATCTTACTTTTTATCTACAACTTGATACGTTTC

[0108] TCTGTAATTAAATAACTACAGATACTTCACAACACCAAGGAATTGGTGT

[0109] AAAGAAAAAAAAAAAAAGGTCCGATAAGTTCTAGAGACCGTCACCTCCTT

[0110] CAGAGAAATTCTTTTATCAAATTTGTTAAACAATTTTTTAAAAGGCAGAAT

[0111] AAAGTAAAGACATTGTCAACTATAGACCGACAGGAAAAATATTACGTCTC

[0112] ACTCTTGAAAGGGATGGCACAAACTATTTACAACAGG

[0113] Primer (SEQ ID NO.3):

[0114] GGTCTCTGTTCTTTCTGTTGATTTCC

[0115] 2) Blank experiment and real-time sequencing experiment

[0116] (1) Add physiological buffer solution (37°C, 10mM Tris, 50mM NaCl, 10mM MgCl2, 100μM DTT, pH 7.8) to the PDMS liquid pool and record the current signal in real time ( Figure 3a, up).

[0117] (2) After polymerase modification, the device was tested in real time in a physiological buffer solution (37°C, 1 μM primer-template complex chain, 2.5 μM dATP, 2.5 μM dTTPSα, 1.25 μM ddCTPSα, 1.25 μM dCTPSα, 1.25 μM ddGTP, 1.25 μM dGTP, 10 mM Tris, 50 mM NaCl, 10 mM MgCl2, 100 μM DTT, pH 7.8). Figure 3 a, below).

[0118] (3) It can be observed that KF polymerase completes the chain polymerization of the 415bp site of the NPM-415 sequence ( Figure 3 b), and the three-stage polymerization process (open conformation, base checking, esterification polymerization) in the polymerization of a single base ( Figure 3 a, below).

[0119] 3) Analysis of electrical sequencing results

[0120] (1). The binary array of the chain polymerization process (long esterification polymerization and short esterification polymerization) is arranged in order by length, the terminal site information is derived, and finally the site information in the sequence is derived (terminal site: ddCTPSα and ddGTP, long esterification site: dTTPSα and dCTPSα, short esterification site: dATP and dGTP) Figure 4 a).

[0121] (2) Compare the sequence analysis results with the sample information ( Figure 4 b and 4c), the sequencing accuracy (~99.5%) and base recognition accuracy (A: 83.5%, T: 69.5%, C: 85.1%, G: 68.7%) were obtained.

[0122] (II) DNA sequencing example of graphene-based molecular heterojunction single-molecule transistor device

[0123] 1) DNA polymerase and test sample (NPM-415) sequence information

[0124] (1) The enzyme used in the example is a single thiol mutant polymerase Poly (β), the amino acid sequence of the enzyme is as follows (SEQ ID NO.4):

[0125] Protein molecular weight = 348MW = 39485

[0126] Isoelectric point = 8.88

[0127] 001MHHHHHHLGG GGSMSKRKAPQETLNGGITD MLTELANFEKNVSQAIHKYNAYRKAASVIA

[0128] 061KYPHKIKSGA EAKKLPGVGT KIAEKIDEFLATGKLRKLEK IRQDDTSSSI NFLTRVSGIG

[0129] 121PSAARKFVDE GIKTLEDLRKNEDKLNHHQR IGLKYFGDFE KRIPREEMLQ MQDIVLNEVK

[0130] 181KVDSEYIATV CGSFRRGAES SGDMDVLLTH PSFTSESTKQ PKLLHQVVEQ LQKVHFITDT241LSKGETKFMGVCQLPSKNDE KEYPHRRIDI RLIPKDQYYC GVLYFTGSDI FNKNMRAHAL

[0131] 301EKGFTINEYTIRPLGVTGVAGEPLPVDSEKDIFDYIQCKYREPKDRSE

[0132] (2). The NPM-415 gene sequence used in the example is specifically as follows:

[0133] Template (SEQ ID NO.5):

[0134] CCAGAGACAAGAAAGACAACTAAAGGAATTCATTTATTACGATTTGATAA

[0135] TTTATTAATAATCATATAACAAGTGTAAAAATACTGACTAATTTCACAAAC

[0136] CTTAATTTAATGTAGACTCATATTTAAAAGAACCTCAGTATAGAAATAGAT

[0137] CTCAATTGAGAGACCACCATCTTACTTTTTATCTACAACTTGATACGTTTC

[0138] TCTGTAAATTAAATAACTACAGATACTTCACAACACCAAGGAATTGGTGT

[0139] AAAGAAAAAAAAAAAAAGGTCCGATAAGTTCTAGAGACCGTCACCTCCTT

[0140] CAGAGAAATTCTTTTATCAAATTTGTTAAACAATTTTTTAAAAGGCAGAAT

[0141] AAAGTAAAGACATTGTCAACTATAGACCGACAGGAAAAATATTACGTCTC

[0142] ACTCTTGAAAGGGATGGCACAAACTATTTACAACAGG

[0143] Primer (SEQ ID NO.6):

[0144] GGTCTCTGTTCTTTCTGTTGATTTCC

[0145] 2) Blank experiment and real-time sequencing experiment

[0146] (1) Add physiological buffer solution (37°C, 50mM Tris-HCl, pH 8.0, 300mMNaCl, 1mM DTT, 1mM EDTA) to the PDMS liquid pool and record the current signal in real time ( Figure 5 ,superior).

[0147] (2) After polymerase modification, the device was tested in real time in a physiological buffer solution (37°C, 1 μM primer-template complex chain, 2.5 μM dNTP, 2.5 μM dNTPSα, 50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 1 mM DTT, 1 mM EDTA). Figure 5 ,Down).

[0148] (3) The complete chain polymerization of the polymerase Poly(β) at the 415bp site of the NPM-415 sequence and the bidirectional conversion process of the enzyme conformation during single base polymerization can be observed.

[0149] 3) Analysis of electrical sequencing results

[0150] The position information of each nucleotide substrate in the sequence can be tested by using the single-base polymerization event resolution and the method of extending the polymerization time of a single type of nucleotide.

[0151] Base A T C G Accuracy (%) 68.1 72.6 69.8 77.5

[0152] Based on the embodiments of the present invention, the present invention can realize real-time sequencing of non-amplified genes through electrical single-molecule gene sequencing technology based on silicon nanowires and molecular heterojunctions, and can achieve the purpose of single-base resolution, high-fidelity and low-cost gene sequencing.

[0153] It should be further explained that the above implementation modes are only used for understanding the technical solutions of the present invention, and are not used to limit the protection scope of the present invention. Any obvious changes and adjustments made to the technical solutions of the present invention within the technical concept of the present invention should also fall within the protection scope of the present invention.

Claims

1. A single-molecule sequencing sensor based on a silicon nanowire-based field effect transistor device, characterized by: It includes silicon nanowires arranged on the surface of a silicon substrate and a metal array coated on the silicon substrate. A layer of SiO2 is attached to the surface of the metal array. A groove is longitudinally punched on the surface of the silicon wire, and the polymerase is connected in the groove. The surface of the silicon wire in the groove has a molecular bridge with a maleimide end, which is used to connect to the cysteine ​​site of the polymerase.

2. The single-molecule sequencing sensor based on silicon nanowire-based field effect transistor device according to claim 1, characterized in that: The polymerase is KF polymerase, having the amino acid sequence shown in SEQ ID NO.

1. A prefabricated window with a width of 10 nm is longitudinally punched on the surface of the silicon wire, and a channel suitable for limited connection of a single KF polymerase is obtained after development.

3. Single molecule sequencing sensor based on graphene-based molecular heterojunction transistor device, characterized by: The invention comprises a graphene-based single-molecule transistor device, wherein the device has a graphene electrode pair with a nanogap, the ends of the electrode pair have been functionalized with carboxyl groups, and a maleimide molecular bridge is connected between the graphene nanogap electrode pairs for connecting to the cysteine ​​site of the polymerase.

4. The single molecule sequencing sensor based on the graphene-based molecular heterojunction transistor device according to claim 3, characterized in that: The graphene-based single-molecule transistor device is a high-density arrayed graphene-based transistor device obtained by evaporating Cr and Au onto graphene strips. The polymerase is a poly(β) polymerase having a single cysteine ​​thiol group and has an amino acid sequence shown in SEQ ID NO.

4.

5. The method for preparing a single-molecule sequencing sensor based on a silicon nanowire-based field effect transistor device according to claim 1 or 2, characterized in that The following steps are involved: 1) Fabrication of silicon nanowire-based field effect transistor devices (1) Prepare high-quality silicon nanowires using the CVD thermal growth method, transfer the silicon wires to the surface of the silicon substrate, rely on template photolithography to directional protect the silicon wires between the electrodes to be etched, and remove excess silicon wires; (2) After deprotection, a metal array is coated on the silicon substrate again by template lithography and thermal evaporation, and a layer of SiO2 is attached to the surface of the metal array; (3) Using template lithography, a layer of positive resist SU-8 is thermally cured on the device surface, and the window process only exposes the silicon wires between the electrodes and the source and drain electrodes; 2) Single-molecule modification of silicon nanowire-based field-effect transistor devices (1) Spin-coat a layer of PMMA on the device surface. After curing, a prefabricated window is punched longitudinally on the surface of the silicon wire between the electrodes. After development, a channel suitable for confined connection of a single KF polymerase is obtained; (2) The surface of the silicon wire in the channel is etched with HF wet etching to obtain an active surface with Si-H dangling bonds. The PMMA coating is then removed and a molecular bridge with a maleimide end is modified on the active surface of the silicon wire through Si-H addition reaction, esterification reaction and substitution reaction in sequence, which is used to connect the cysteine ​​site of KF polymerase; (3) KF polymerase is dissolved in physiological buffer solution and circulated on the surface of the silicon wire of the device by microfluidics. By observing the change in the conductivity of the silicon wire during the electrical test, the purpose of visualizing the successful connection of KF polymerase is achieved.

6. The method for preparing a single-molecule sequencing sensor based on a silicon nanowire-based field effect transistor device according to claim 5, characterized in that: The physiological buffer solution in step 2) includes 200 nM KF, 10 mM Tris, 50 mM NaCl, 10 mM MgCl2, 100 μM DTT, and has a pH value of 7.

8.

7. The method for preparing a single molecule sequencing sensor based on a graphene-based molecular heterojunction transistor device according to claim 3 or 4, characterized in that The following steps are involved: 1) Preparation of graphene-based transistor devices (1) Using low-pressure chemical vapor deposition to grow a single layer of graphene on copper foil, and using ferric chloride etching and wet transfer technology to transfer the graphene on the copper foil to the silicon wafer; (2) Graphene strips are obtained by processing, and Cr and Au are evaporated onto the graphene strips by photolithography and thermal evaporation to prepare high-density arrayed graphene-based transistor devices; 2) Micro-nano processing of graphene-based nanogap electrode pairs (1) Spin-coat a layer of PMMA on the surface of the transistor array and solidify it. Use an electron beam exposure system to make a dotted line prefabricated window in the middle of the electrode pair. After development, the graphene surface inside the dotted line is exposed; (2) First, the graphene inside the dotted line is etched away by oxygen plasma etching, and then the oxygen plasma etching and electrical burning techniques are alternately used to gradually form a graphene-based nanogap electrode pair with carboxyl groups at the ends; 3) Single-molecule enzyme modification of graphene-based nanogap electrode pairs (1) Dissolve an appropriate amount of maleimide molecular bridge, EDCI and HOBT in anhydrous pyridine protected by argon, and then place the device in the anhydrous pyridine for a certain period of time. After taking out the device, clean the device with acetone and deionized water, and use a semiconductor parameter analyzer to screen each pair of electrodes to obtain a graphene-based single-molecule transistor device in which maleimide is successfully connected between graphene nanogap electrode pairs; (2) Drop an enzyme buffer solution with only a single cysteine ​​thiol group on the surface onto the graphene-based single-molecule transistor device. After rinsing and drying, use a semiconductor parameter meter to test the electrode pair. The decrease in conductivity indicates the successful connection of the enzyme.

8. The method for preparing a single molecule sequencing sensor based on a graphene-based molecular heterojunction transistor device according to claim 7, characterized in that: In step 3), the Poly(β) polymerase buffer includes 200 nM polymerase Poly(β), 50 mM Tris–HCl, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, and has a pH value of 8.

0.

9. The gene sequencing method of the single molecule sequencing sensor based on silicon nanowire-based field effect transistor device according to claim 1 or 2, characterized in that The following steps are involved: (1) Attach a PDMS tank with a cylindrical cavity in the middle to the device surface, and inject KF polymerase physiological buffer solution into the internal cavity; (2) Add adenine deoxynucleotide, thiothymine deoxynucleotide, thiocytosine dideoxynucleotide, thiocytosine deoxynucleotide, guanine dideoxynucleotide, guanine deoxynucleotide, and test the DNA chain polymerization process of the cell sequence template chain; (3) Relying on the difference in conductivity values, the number of events and data sites of base repulsion steps are classified, and the electrical data between repulsion steps with regular polymerization lengths are screened and sorted out, and the corresponding esterification reaction time of each single base polymerization is obtained by fitting; (4) By setting a threshold for the esterification reaction, the electrical data fragments are converted into binary arrays of short esterification polymerization and long esterification polymerization. These binary arrays are then sorted from short to long to infer the termination site information of each array. By associating the results from the binary arrays with the length of the identified chain termination site, the base information of the sequence site is obtained.

10. The gene sequencing method of the single molecule sequencing sensor based on the graphene-based molecular heterojunction transistor device according to claim 3 or 4, characterized in that The following steps are involved: (1) Attach the PDMS tank to the device surface and inject the polymerase Poly(β) physiological buffer solution into the internal cavity; (2) Each time, one of the substrates was used as a thionucleotide substrate, and four sets of repeated tests were performed, and the electrical data were recorded in real time; (3) Screen out the two-state jump current data consistent with the template length, and use the difference in conductance values ​​to fit the corresponding esterification reaction time of each single base polymerization; (4) By establishing a threshold value for the esterification reaction, the electrical data fragments are converted into binary arrays of short esterification polymerization and long esterification polymerization. Combined with the type of thionucleotide substrate added each time, the site information of the corresponding base is read, and the four sets of results are combined to obtain the base information of the sequence site.

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