WO2024021686A1 - 一种超灵敏纳米孔结构及芯片及分析装置及氨基酸和蛋白质检测方法及应用 - Google Patents

一种超灵敏纳米孔结构及芯片及分析装置及氨基酸和蛋白质检测方法及应用 Download PDF

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WO2024021686A1
WO2024021686A1 PCT/CN2023/088193 CN2023088193W WO2024021686A1 WO 2024021686 A1 WO2024021686 A1 WO 2024021686A1 CN 2023088193 W CN2023088193 W CN 2023088193W WO 2024021686 A1 WO2024021686 A1 WO 2024021686A1
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nanopore
ultra
sensitive
disulfide
helicase
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French (fr)
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冯建东
王馥实
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浙江大学
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • 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
    • 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
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to the field of single molecule detection, in particular to an ultra-sensitive nanopore structure, chip and analysis device, as well as amino acid and protein detection methods and applications.
  • Protein is an extremely important biological substance.
  • the development of protein sequencing technology can not only provide key information for the study of protein structure and function, but is also expected to bring revolutionary progress to proteomics research and medical research.
  • Edman degradation method and mass spectrometry have shortcomings in detection speed, read length, or in achieving routine, complete proteome quantification at low abundance.
  • Nanopore technology is of great significance in fields such as gene sequencing and pathogen sequencing due to its extremely high resolution.
  • nanopore technology has made remarkable progress in DNA sequencing, but its development in protein sequencing lags behind, mainly due to the very complex structure of proteins.
  • Proteins are composed of 20 kinds of amino acids, while DNA is composed of only 4 kinds of bases.
  • the detection of 20 distinguishable signals is a big problem.
  • the multi-level structure and uneven charging of proteins also bring many challenges to their sequencing. .
  • the length of the sensing region of the nanopore is crucial for improving the spatial resolution of the nanopore.
  • the length of the sensing region of currently widely used biological nanopores is several amino acids.
  • the length of the sensing region of MspA biological nanopore is 8 amino acids (H. Brinkerhoff et al., Science, 374(6574), 1509–1513.). This limits further improvements in spatial resolution.
  • the present invention proposes an ultra-sensitive nanometer Pore structure, that is, a nanopore structure with a sensing region length smaller than the shortest sensing region length (8 amino acids) that traditional biological nanopores can provide.
  • the ultra-sensitive nanopore structure provides a basis for the identification of single amino acid molecules and de novo protein sequencing because the length of the sensing area is smaller than the shortest sensing area length that traditional biological nanopores can provide.
  • the present invention also proposes A strategy for integrating ultra-sensitive nanopore structures with biological enzymes to achieve protein sequencing was developed.
  • the present invention improves the shortcomings of the existing technology and provides an ultra-sensitive nanopore structure and chip and analysis device as well as amino acid and protein detection methods and applications.
  • the present invention is implemented by adopting the following technical solutions:
  • An ultra-sensitive nanopore structure characterized in that the ultra-sensitive nanopore structure is a nanopore, the pore diameter of the nanopore is in the range of 0.35-5nm, and the length of the sensing area of the nanopore is in the range of 0.3-2nm, which is consistent with a single amino acid molecule Similar sizes.
  • the nanopore of the present invention has a pore diameter in the range of 0.35-2nm, which can realize the detection and identification of single amino acid molecules.
  • the nanopore of the present invention has a pore diameter in the range of 1-5 nm, which can realize the detection and identification of post-translational chemically modified amino acid molecules, the detection of polypeptides, the differentiation of heteropolypeptide amino acid sequences, and protein sequencing.
  • the nanopores of the present invention are artificial nanopores or biological nanopores.
  • the length of the material sensing area of the nanopore of the present invention is less than 2 nm and has an atomic level thickness, including molybdenum disulfide, molybdenum diselenide, molybdenum diselenide, tungsten disulfide, tungsten diselenide, Tungsten telluride, titanium disulfide, di Titanium selenide, titanium ditelluride, zirconium disulfide, zirconium diselenide, zirconium ditelluride, hafnium disulfide, hafnium diselenide, hafnium disulfide, vanadium disulfide, vanadium diselenide, vanadium ditelluride , niobium disulfide, niobium diselenide, niobium diselenide, tantalum disulfide, tantalum diselenide, tantalum disulfide, technetium disulfide, technetium disulfide, tech
  • the invention discloses an ultra-sensitive nanopore chip including an ultra-sensitive nanopore structure.
  • the chip includes a lower substrate and an upper nano-film containing the ultra-sensitive nanopore structure.
  • the substrate of the present invention is provided with support holes for supporting ultra-sensitive nanopores, and the diameter of the support holes is in the range of 5-500 nm.
  • the substrate of the present invention includes a silicon layer, and the resistance of the silicon layer is higher than 2700 Ohmcm.
  • the substrate of the present invention includes five layers of silicon nitride, silicon oxide, silicon, silicon oxide and silicon nitride from top to bottom.
  • the thickness of the silicon layer is 300-500 ⁇ m.
  • the surface layer is an insulating layer and supports holes. Opened on the surface silicon nitride layer.
  • the chip of the present invention can achieve a background noise lower than 20pA.
  • the dielectric noise is reduced and a signal-to-noise ratio sufficient to resolve individual amino acid molecules can be provided.
  • the invention discloses an ultra-sensitive nanopore analysis device of an ultra-sensitive nanopore chip.
  • the device includes a fluid tank filled with ionic solution, and an ultra-sensitive nanopore that divides the fluid tank into a cis side chamber and a trans side chamber.
  • Chip electrode connected to fluid tank solution or chip, current amplifier and power supply connected in series with the electrode, digital-to-analog conversion equipment and computer connected to the current amplifier.
  • the invention discloses a method for preparing an ultra-sensitive nanopore structure by an ultra-sensitive nanopore analysis device.
  • a salt solution is injected into chambers on both sides of a fluid tank, and a voltage in the range of 0-10V is directly applied to the nanofilm through a power supply and a current amplifier.
  • Nanopores are obtained by etching, and the size of the pores can be controlled by controlling the electrochemical reaction by controlling the size and duration of the applied voltage; real-time data of the transmembrane conductance of the nanopores are obtained.
  • the conductance value reaches the value corresponding to the target pore size, the application of voltage is stopped. Complete the nanopore preparation process.
  • the invention discloses a method for rapid amino acid detection in nanopores using an ultra-sensitive nanopore analysis device.
  • the pore size of the nanopores is in the range of 0.35-2nm, and includes the following steps:
  • the invention discloses a method for rapid protein sequencing of nanopores using an ultra-sensitive nanopore analysis device.
  • the pore size of the nanopores is in the range of 1-5nm, and includes the following steps:
  • the biological enzymes of the present invention include phi29 DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Pfu HS DNA polymerase, T4 DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, Taq II DNA polymerase, Taq plus DNA polymerase, LA Taq DNA polymerase, VentR DNA polymerase, Phusion DNA polymerase, KOD DNA polymerase, Iproof DNA polymerase, Hel308 helicase, T4 helicase, DnaB helicase Enzyme, Rep protein helicase, PcrA helicase, Tte UvrD helicase, Tth UvrD helicase, T4 Dda helicase, T4 gp41 helicase, RecQ helicase, NS3 HCV helicase, eLF4A helicase Gyrase, WRN helicase, TRCF helicase, LTag helicase, E1 helicase, T7 g
  • the invention discloses an ultra-sensitive nanopore chip applied to nanopores in amino acid detection.
  • the pore diameter of the nanopores is in the range of 0.35-2nm, realizing the detection and differentiation of 20 kinds of natural amino acids.
  • the invention discloses an ultra-sensitive nanopore chip that is used in the detection of post-translationally modified amino acids by nanopores.
  • the pore diameter of the nanopore is in the range of 1-5 nm, realizing the detection and differentiation of post-translationally modified amino acids.
  • the invention discloses an ultra-sensitive nanopore chip used in polypeptide detection.
  • the pore diameter of the nanopore is in the range of 1-5 nm, realizing the detection of polypeptides and the differentiation of heteropolypeptide amino acid sequences.
  • the invention discloses an ultra-sensitive nanopore chip used in protein sequencing.
  • the pore diameter of the nanopore is in the range of 1-5 nm to realize protein sequencing.
  • the invention designs an ultra-sensitive nanopore structure, an ultra-sensitive nanopore chip and an ultra-sensitive nanopore analysis device based on the ultra-sensitive nanopore structure.
  • the sensitive length of this ultra-sensitive nanopore structure is smaller than the shortest sensing area length that traditional biological nanopores can provide, and the pore size can be controlled at 0.35-5nm.
  • This ultra-sensitive nanopore chip can provide background noise of less than 20pA.
  • Using this ultra-sensitive nanopore analysis device we achieved a single-molecule detection limit of less than 1 Dalton, realized the detection of amino acid molecules at the single-molecule level, and completed the direct detection of 20 natural amino acid molecules and post-translational chemically modified amino acids. , proposed a strategy to achieve protein sequencing by integrating technology with ultra-sensitive nanopore structures and biological enzymes.
  • the present invention provides a direct solution for improving the spatial resolution of nanopore detection.
  • Electrochemical etching method is used to prepare ultra-sensitive nanopores.
  • the pore diameter is accurately controllable in the range of 0.35-5nm and is easy to operate.
  • the electrochemical etching drilling method is used Not only can nanopores smaller than 2nm be prepared, but the operation is simple and low-cost.
  • the nanopore can be accurately designed and prepared according to the size of the sample to be tested, which increases the amplitude of the generated current signal, making the nanopore capable of identifying different types of amino acids.
  • nanopores with atomic-level thickness have been proven to be able to resolve single nucleotide molecules.
  • the present invention further reduces the nanopore diameter and achieves a size in the pore diameter range of 0.35-2nm. Detection and resolution of smaller amino acids.
  • this invention improves the performance of nanopores from the direct detection of 4 kinds of nucleotide molecules to the direct detection of 20 kinds of amino acid molecules.
  • it can realize the identification of molecules that are isomers of each other and the identification of differences in individual groups in molecules, improving the resolution ability of ultra-sensitive nanopore structures to less than 1 Dalton.
  • the ultra-sensitive nanopore structure proposed by the present invention with a sensing area length of 0.3-2 nm can provide sufficient spatial resolution for amino acid detection and protein sequencing.
  • the base structure of the ultra-sensitive nanopore chip used in the present invention is shown in Figure 2, which is a sandwich structure composed of silicon nitride, silicon oxide and silicon.
  • the thickness of the silicon layer is 380 ⁇ 25 ⁇ m, with high resistance (greater than 2700Ohmcm); the thickness of the silicon oxide layer is 60 ⁇ 6nm, prepared by dry thermal oxidation; the thickness of the silicon nitride layer is 20 ⁇ 4nm, and uses a low-stress Silicon nitride facilitates the subsequent preparation of nanoscale support holes.
  • the design of the sandwich structure greatly reduces the dielectric noise.
  • the ultra-sensitive nanopore analysis device using the sandwich structure can achieve a background noise lower than 20pA, compared with previous literature reports (Feng, J. et al., (2015) ).Nature nanotechnology,10(12),1070–1076.) has lower noise.
  • the support hole size on the ultrasensitive nanopore chip substrate of the present invention is controllable in the range of 5-500nm. Using a support hole size less than 60nm can improve the mechanical noise caused by the vibration of the upper nanofilm during the detection process.
  • the ultra-sensitive nanopore analysis device of the present invention can realize the direct detection of 20 natural amino acids without the need for modification and pretreatment of the molecules to be measured, and is simple to operate. And the analysis device has a single-molecule detection limit of less than 1 Dalton, providing a basis for nanopore protein sequencing.
  • the ultra-sensitive nanopore analysis device of the present invention can identify post-translational chemically modified amino acids within the nanopore diameter range of 1-5 nm. Since post-translational modification of proteins is closely related to diseases, the device is beneficial to medical research and disease diagnosis. development of.
  • the ultra-sensitive nanopore analysis device of the present invention can detect polypeptides and differentiate amino acid sequences of heteropolypeptides in the range of nanopore diameters of 1-5 nm.
  • the ultra-sensitive nanopore analysis device of the present invention can realize protein detection in the nanopore diameter range of 1-5nm. Sequencing. Design and synthesize the connection complex between the target peptide chain and the nucleotide chain, introduce biological enzymes (such as phi29 DNA polymerase, Hel308 helicase), so that the target chain contacts the nanopore and biological enzymes, and the enzyme controls the target chain to pass through the nanometer By measuring and analyzing the current pattern generated during the pore movement, the sequence of the target peptide chain can be obtained.
  • biological enzymes such as phi29 DNA polymerase, Hel308 helicase
  • the ultra-sensitive nanopore structure of the present invention has high stability and can be stored for several weeks and detect tens of thousands of perforation events.
  • Figure 1 is a schematic structural diagram of the analysis device of the present invention.
  • 1 is the power supply
  • 2 is the current amplifier
  • 3 is the digital-to-analog conversion equipment and computer
  • 4 is the electrode
  • 5 is the substrate
  • 6 is the support hole
  • 7 is the nanofilm
  • 8 is the nanopore
  • 9 is the fluid tank
  • 10 is the sample to be tested
  • 11 is the cis side chamber
  • 12 is the trans side chamber.
  • Figure 2 is a schematic diagram of the substrate
  • Figure 3 is a schematic diagram of the fluid tank
  • the fluid tank structure used in this embodiment is shown in Figure 3.
  • the fluid tank is mainly composed of two parts. The only difference between the two parts is that the part shown in Figure 16 contains internal threads, while the part shown in Figure 15 has a smooth inner wall. Designed for easy assembly of fluid body with screws.
  • Figure 13 is for the silver/silver chloride electrode to be inserted,
  • Figures 11 and 12 are the chambers, and the annular groove in Figure 14 fits the O-ring.
  • Figure 4 is a characterization diagram of a single layer of molybdenum disulfide
  • Figure 5 is a schematic diagram of the detection of single amino acids in molybdenum disulfide nanopores
  • a Schematic diagram of the experimental device (not to scale); b. Current trace collected during glutamate perforation; c. Current trace collected during alanine perforation. The right side shows the normalized current histogram. Fitting curve; heat map of residence time and blocking current of signals generated by dG, GG, and GGG in the same device; e. Flow chart of using deep learning to identify amino acids; f. Glycine (G) and alanine (A) in Histogram of ⁇ I/I 0 caused in the same device; g. For the data in f, the score plots of precision, recall and F1 obtained by deep learning;
  • Figure 6 shows the effect of voltage on the detection of amino acids through nanopores.
  • a Diagram of perforation frequency of serine at different voltages
  • bc Diagram of blocking current values ⁇ I and ⁇ I/I 0 caused by perforation of glycine at different voltages.
  • Figure 7 shows the detection results of all 20 natural amino acids
  • the picture shows the ⁇ I/I 0 histogram obtained from nanopore experiments on 20 kinds of amino acids, in order of charged amino acids (ab), hydrophobic non-aromatic amino acids (cd), polar uncharged amino acids (ef), hydrophobic aromatic amino acids Amino acids (g) and special types of amino acids (h) Display.
  • the corresponding confusion matrix plot is attached below each histogram.
  • Figure 8 shows the results of identifying chemical groups by molybdenum disulfide nanopores
  • ac Aspartic acid (D) and asparagine (N), glutamic acid (E) and glutamine (Q), asparagine (N) and leucine (L) in nanopore experiments Histogram of ⁇ I/I 0 values for perforation events caused by; df. Serine (S) and threonine (T), phenylalanine (F) and tyrosine (Y), leucine ( Heat map of L) and isoleucine (I). The width of the box in the figure df represents the standard deviation.
  • Figure 9 is a diagram showing the identification results of phosphorylated amino acids
  • Figure 10 shows the single molecule detection results of the peptide chain
  • the figure shows the typical current signal diagram of the peptide chains EEEGEEE and EEEWEEE during the experiment.
  • Figure 11 is a schematic diagram of introducing enzymes to achieve protein sequencing.
  • the invention discloses an ultra-sensitive nanopore 8 structure.
  • the ultra-sensitive nanopore 8 structure is a nanopore 8.
  • the aperture of the nanopore 8 is in the range of 0-50nm, and the length of the sensing area of the nanopore 8 is in the range of 0.3-2nm. , similar to the size of a single amino acid molecule.
  • the pore diameter of the nanopore 8 is further limited to the range of 0.35-2nm, which can realize the detection and identification of single amino acid molecules.
  • the nanopore 8 is an artificial nanopore 8 or a biological nanopore 8 .
  • the length of the sensing area of the material of the nanopore 8 is less than 2 nm and has an atomic level thickness, including molybdenum disulfide, molybdenum diselenide, molybdenum diselenide, tungsten disulfide, tungsten diselenide, Tungsten telluride, titanium disulfide, titanium diselenide, titanium diselenide, titanium disulfide, zirconium disulfide, zirconium diselenide, zirconium disulfide, hafnium disulfide, hafnium diselenide, hafnium disulfide, vanadium disulfide, Vanadium disulfide, vanadium diselenide, niobium disulfide, niobium diselenide, niobium disulfide, tantalum disulfide, tantalum diselenide, tantalum disulfide, technetium disulfide, technetium diselec
  • the invention discloses an ultra-sensitive nanopore 8 chip.
  • the chip includes a lower substrate 5 and an upper nano-film 7 containing an ultra-sensitive nanopore 8 structure.
  • the substrate 5 is provided with a support hole 6 for supporting the ultra-sensitive nanopore 8.
  • the support hole 6 The pore diameter is between 5-500nm Within the range, the substrate 5 includes a silicon layer, the resistance value of the silicon layer is higher than 2700 Ohmcm, the substrate 5 includes five layers of silicon nitride, silicon oxide, silicon, silicon oxide and silicon nitride from top to bottom, and the thickness of the silicon layer is 300- 500 ⁇ m, the surface layer is an insulating layer, and the support hole 6 is opened on the surface silicon nitride layer.
  • the chip can achieve a background noise lower than 20pA. The dielectric noise is reduced and a signal-to-noise ratio sufficient to resolve individual amino acid molecules can be provided.
  • the invention discloses an ultra-sensitive nanopore 8 analysis device.
  • the device includes a fluid tank 9 filled with ionic solution, and an ultra-sensitive nanopore 8 that divides the fluid tank 9 into a cis side chamber 11 and a trans side chamber 12
  • the invention discloses a method for preparing an ultra-sensitive nanopore 8 structure by an ultra-sensitive nanopore 8 analysis device.
  • a salt solution is injected into the chambers on both sides of the fluid tank 9, and 0- is directly applied to the nano-film 7 through the power supply 1 and the current amplifier 2.
  • the voltage in the 10V range is etched to obtain nanopore 8.
  • the size of the pore size can be controlled; real-time data of the transmembrane conductance of nanopore 8 is obtained.
  • the conductance value reaches the target pore size
  • the value reaches the corresponding value, stop applying the voltage and complete the preparation process of nanopore 8.
  • the invention discloses a method for rapid amino acid detection of nanopore 8 using an ultra-sensitive nanopore 8 analysis device, which includes the following steps:
  • the invention discloses a method for nanopore 8 rapid protein sequencing using an ultra-sensitive nanopore 8 analysis device, which includes the following steps:
  • the biological enzymes include phi29 DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Pfu HS DNA polymerase, T4 DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, Taq II DNA polymerase, Taq plus DNA polymerase Enzymes, LA Taq DNA Polymerase, VentR DNA Polymerase, Phusion DNA Polymerase, KOD DNA Polymerase, Iproof DNA polymerase, Hel308 helicase, T4 helicase, DnaB helicase, Rep protein helicase, PcrA helicase, Tte UvrD helicase, Tth UvrD helicase, T4 Dda helicase, T4 gp41 Helicase, RecQ helicase, NS3 HCV helicase, eLF4A helicase, WRN helicase, TRCF helicase, LTag helicase, E1 helicase, T7 gp4 helicase, Rho helicase
  • the invention discloses an ultra-sensitive nanopore 8 chip that is applied to the nanopore 8 in amino acid detection to realize the detection and differentiation of 20 kinds of natural amino acids.
  • the invention discloses an ultra-sensitive nanopore 8 chip that is used in the detection of post-translation chemically modified amino acids in the nanopore 8 to realize the detection and differentiation of post-translationally modified amino acids.
  • the invention discloses an ultra-sensitive nanopore 8 chip used in polypeptide detection to realize the detection of polypeptides and the differentiation of heteropolypeptide amino acid sequences.
  • the invention discloses an ultra-sensitive nanopore 8 chip used in protein sequencing to realize protein sequencing.
  • Example 1 Detection of 20 natural amino acids using molybdenum disulfide nanopore 8.
  • Figure 1 is a schematic structural diagram of the analysis device of the present invention; including a fluid tank 9 filled with ionic solution, an ultra-sensitive nanopore 8 chip that divides the fluid tank 9 into a cis side chamber 11 and a trans side chamber 12, and access Fluid tank 9 solution or electrode 4 of the chip, current amplifier 2 and power supply 1 connected in series with the electrode 4, digital-to-analog conversion equipment and computer 3 connected to the current amplifier 2.
  • the substrate 5 is provided with a support hole 6, and the nanofilm 7 is provided with an ultra-sensitive nanopore 8.
  • the nanofilm 7 used in this embodiment is a single layer of molybdenum disulfide with a thickness of about 0.65 nanometers, and the ultra-sensitive nanopore 8 has a diameter of 0- It is controllable in the range of 50nm, and the substrate 5 is a sandwich structure composed of silicon nitride, silicon oxide and silicon.
  • a wafer with a sandwich structure is used to prepare the substrate 5.
  • the structure of the substrate 5 is shown in Figure 2.
  • the sandwich structure is composed of silicon nitride, silicon oxide and silicon.
  • the silicon layer thickness is 380 ⁇ 25 ⁇ m and has a high resistance value (greater than 2700Ohmcm); the silicon oxide layer thickness is 60 ⁇ 6nm and is prepared by dry heat oxidation; nitriding
  • the thickness of the silicon layer is 20 ⁇ 4nm, and the use of silicon nitride with low stress is beneficial to the subsequent preparation of nanoscale support holes 6.
  • a single layer of molybdenum disulfide was prepared using chemical vapor deposition (CVD).
  • Molybdenum trioxide powder is used as the molybdenum source
  • sulfur powder is used as the sulfur source
  • high-purity argon gas is used as the protective gas and carrier gas
  • a single-sided polished silicon dioxide wafer is used as the substrate for depositing the sample 5.
  • the quartz boat filled with molybdenum trioxide and substrate 5 is placed in the central heating area of the quartz tube of the tube furnace, and the quartz boat filled with sulfur powder is placed on the edge of the quartz tube of the tube furnace.
  • Check the sealing of the tube furnace use a mechanical pump to extract the air in the tube, and then introduce argon gas as a protective gas.
  • the argon flow rate is 100 sccm
  • the temperature is increased to 700°C at a constant speed, and the heating rate is about 15°C/minute.
  • the heating is continued at a constant temperature for 10 minutes, then the heating is stopped, and the heating is naturally cooled to room temperature.
  • Figure 4 shows the characterization results of a single layer of molybdenum disulfide.
  • Picture a is an optical image of a single layer of molybdenum disulfide grown on a SiO 2 /Si surface.
  • Picture b is an image of a single layer of molybdenum disulfide grown on a high-resolution sphere. Images under a difference-corrected transmission electron microscope (AC-TEM).
  • Picture c is the electron diffraction pattern of a single-layer single crystal molybdenum disulfide obtained under a transmission electron microscope (200kV).
  • Picture d is a sample of molybdenum disulfide transferred to a sapphire sheet. Image under an atomic force microscope. The thickness of the sample is approximately 0.78nm.
  • a KCl solution with a concentration of 1mol/L is injected into the chambers on both sides of the fluid tank 9, and a voltage in the range of 0-10V is directly applied to the nanofilm 7 through the power supply 1 and the current amplifier 2, and the nanofilm 7 is etched.
  • Nanopore 8 is obtained by etching, and the electrochemical reaction is controlled by controlling the size and duration of the applied voltage.
  • the conductance value reaches the value corresponding to the target pore diameter, the application of voltage is stopped and the pore diameter is controlled within the range of 0.35-2nm.
  • the fluid tank 9 is made of PMMA material.
  • the structure of the fluid tank 9 is shown in Figure 3.
  • the fluid tank 9 is mainly composed of two parts. The only difference between the two parts is that the part shown in Figure 16 contains internal threads, while the part shown in Figure 15 is smooth.
  • the inner wall is designed to facilitate the assembly of the fluid tank 9 with screws.
  • Figure 13 is for the silver/silver chloride electrode 4 to be inserted, Figures 11 and 12 are chambers, and the annular groove in Figure 14 fits the O-ring. Use an O-ring to seal the chip between the two parts of the fluid tank 9 and fix it with 4 screws device.
  • the sample concentration is 2 ⁇ mol/L.
  • a patch clamp (Axopatch 200B amplifier) low-noise system was used for testing, and an NI PXI-1042Q acquisition card was used for data acquisition. Take two pieces of silver wire about 3cm in length and polish them with sandpaper to remove the oxide layer on the surface. Dip three-quarters of the silver wire length and platinum electrode 4 (as anode and cathode respectively) in 1M KCl solution. A voltage of 2V was applied for a period of time to prepare Ag/AgCl electrode 4. Then connect the two Ag/AgCl electrodes 4 to the probes of the patch clamp as the positive electrode and ground wire.
  • the patch clamp needs to be calibrated with a model cell before use to eliminate errors.
  • a current-voltage field sweep was performed in a KCl solution with a concentration of 1 mol/L to determine the pore size of the device.
  • the corresponding buffer solution was injected into the cis-side chamber 11 and the trans-side chamber 12 of the fluid tank 9, and a bias voltage of 200mV was applied to the trans-side chamber 12.
  • the positive and negative voltages depended on different The relative size of the isoelectric point of the amino acid and the pH value of the buffer solution.
  • the isoelectric point of the amino acid to be tested is less than 7.8. At this time, the amino acid is negatively charged in the buffer solution environment, and the applied bias voltage is positive.
  • a stable ion current with an average value of I0 can be detected through the nanopore 8.
  • a brief block in the ionic current can be detected.
  • Each current block corresponds to the perforation behavior of a single amino acid molecule in the nanopore 8, and its current characteristics are the relative current drop value ⁇ I/I 0 and the residence time ⁇ t.
  • the experimental results of each group are normalized.
  • the amino acid molecules pass through the molybdenum disulfide nanopore 8 driven by electrophoretic force.
  • the molecules to be measured pass through the molybdenum disulfide nanopore 8
  • they will briefly block the pore channel, and the current in the pore channel will change, which is By analyzing the amplitude and duration of the current change and the current waveform, the corresponding molecular information can be obtained.
  • the molybdenum disulfide nanopore 8 with a diameter in the range of 0.35-2nm can detect the current signals generated by 20 natural amino acids when passing through the pores and distinguish different amino acid molecules.
  • Figure 5b shows a section Typical current signals, and the types of amino acids can be distinguished by performing histogram distribution statistics and fitting on the relative blocking current value ⁇ I/I 0 of the signal ( Figure 5f).
  • the selection of the size of the molybdenum disulfide nanopore 8 is very important. When the size of the nanopore 8 is close to the size of the amino acid molecule to be measured, the ion current can be reduced. is 0nA ( Figure 5c). When the size of the nanopore 8 is larger than 2 nm, the detection sensitivity of amino acid molecules decreases.
  • the detection sensitive area of the molybdenum disulfide nanopore 8 is equivalent to the size of a single amino acid.
  • short chains G, GG, and GGG of different lengths composed of the same amino acid were selected for experiments.
  • the blocking current values produced by the three are very close ( Figure 5d), which is completely different from the results produced in the previously reported biological nanopore 8.
  • a deep learning model was introduced during the data analysis process, and the neural network diagram is shown in Figure 5e.
  • the deep learning model learns both the eigenvalues and waveforms of the current signal, and each set of experimental results is displayed in the form of a confusion matrix.
  • the ⁇ I/I 0 values of lysine and arginine are positively correlated with the relative mass of the molecule (as shown in Figure 7a), and the discrimination accuracy after deep learning analysis reached 82.18%.
  • the relative current blocking values of glutamate and aspartic acid are similar ( Figure 7b), and the ⁇ I/I 0 value distribution of histidine molecules is obviously different from that of glutamic acid and aspartic acid ( Figure 7b).
  • methionine (M, 149.21 Da) has a greater probability of blocking the pore channels and causing the interruption of the experiment. This may be due to its sulfur atom, which is different from molybdenum disulfide. Easy to interact.
  • isomeric amino acids leucine (L) and isoleucine (I) with the same molecular mass produced different relative blocking currents (Figure 7d).
  • ⁇ I/I 0 values were higher than L in 5 devices, but the opposite result was found in the other 2 devices.
  • nanopores 8 with a diameter of approximately 0.6 nm. Attributing this change to the fact that the size of nanopore 8 may affect the pore entry direction of amino acids, the resolution accuracy of these two isomeric amino acids reached 87.25% through deep learning.
  • proline For special types of amino acids, glycine (G, 75.07Da), cysteine (C, 121.16Da) and proline (P, 115.13Da), among which proline produced the largest relative current blocking value in the experiment (such as Figure 7h), this is because the molecular structure of proline contains a five-membered ring, which makes it have the largest molecular volume among the special family of amino acids. Because cysteine contains sulfur atoms, it may also block nanopore 8 during the experiment. Through the above experiments, overall, the relative blocking current value generated by the amino acid molecules when passing through the molybdenum disulfide nanopore 8 increases as its volume increases.
  • FraC Biological Nanopore 8 can distinguish polypeptides that differ by only one amino acid residue, with a resolution of 44Da (Huang, G. et al., (2019). Nature communications, 10(1), 835.). Aerolysin nanopore 8 shows high sensitivity for detecting peptide chains formed by connecting polycationic polymers to amino acids (Ouldali, H. et al., (2020). Nature biotechnology, 38(2), 176–181 .). However, these measurements distinguish individual amino acids in an indirect way, measuring differences in current flow by replacing amino acid residues.
  • the present invention further studies the ultimate resolution of the current molybdenum disulfide nanopore 8 system.
  • the relative molecular mass difference between the two is only 0.99Da, and except for the terminal chemical groups, the molecular skeleton is the same, among which the charged amino acid (day The molecular structure end of (partic acid, glutamic acid) is -OH (17.01Da), and the molecular structure end of polar uncharged amino acids (asparagine, glutamine) is -NH 2 (16.02Da).
  • Example 2 Using molybdenum disulfide nanopore 8 to detect post-translational modifications of amino acids.
  • the present invention can be applied to detect post-translational modifications (PTMs) of amino acids.
  • PTMs post-translational modifications
  • the recognition of post-translational modifications of amino acids is of great significance in the study of diseases.
  • tyrosine (Y) phosphorylation plays a key regulatory role in cell activity, and abnormal tyrosine phosphorylation is closely related to carcinogenesis. .
  • This example uses molybdenum disulfide nanopore 8 to detect and identify tyrosine (Y) and phosphorylated tyrosine (p-Y).
  • Figure 9a shows the molecules of tyrosine (Y) and phosphorylated tyrosine (p-Y). Structure diagram. Repeat the experimental operations (1), (2), (3), (4), (5), (6), (7), (8), (9) described in Example 1, wherein operation (5) ) are tyrosine solution and phosphorylated tyrosine solution.
  • Example 3 Detection of short peptide chains using molybdenum disulfide nanopore 8.
  • the present invention can be applied to the detection of short peptide chains.
  • the current waveform shown in the box in Figure 10 was observed during the experiment.
  • the waveform shown at the bottom of Figure 10 is the ideal waveform of the actual measured current signal without being affected by any noise.
  • the larger W produces larger current blockage, while the smaller G produces smaller current blockage, which is consistent with expectations.
  • a nanopore 8 resolution lower than 1 Da and a detection sensitive area equivalent to the size of a single amino acid molecule can theoretically provide sufficient spatial resolution for protein sequencing. It is proposed that enzymes are introduced into the molybdenum disulfide nanopore 8 system to precisely control the rate of peptide chains passing through the pores, which is expected to provide sufficient time resolution to enable de novo protein sequencing.
  • enzymes are introduced into biological nanopores to control peptide movement have been widely used, and many literatures have also reported the compatibility between DNA polymerase and solid nanopore systems, so this strategy is feasible.
  • Example 4 Protein sequencing using enzymes and molybdenum disulfide nanopore 8.
  • the present invention can be applied to protein sequencing.
  • the detection principle is shown in Figure 11.
  • the complex designed and synthesized connecting the peptide chain and the nucleotide chain is HOOC-GEPGEPDDGDD-5'-X-AGAACTTTAGAACTTTTCAGATCTCACTATCGCATTCTCATGCAGGTCGTAGCC-3'
  • the primer chain is 5'-GCGTACGCCTACGGTTTTCCGTAGGCGTACGCGGCTACGACCTGCATGAGAATGC-3'
  • the blocking chain 5'- GATAGTGAGATCTGAXXXXXXZ-3'.
  • the X in the sequence represents the abasic spacer
  • the Z represents the 3' inter-arm.
  • a buffer solution containing phi29 DNA polymerase at a concentration of 5 nmol/L, deoxyribonucleoside triphosphates (dNTPs) at a concentration of 1 mmol/L and the sample 10 to be tested at a concentration of 10 nmol/L into the cis-side chamber 11, and reverse Add blank buffer solution to the formula side.
  • a voltage in the range of +200-300mV is applied, and after the sample comes into contact with phi29 DNA polymerase and nanopore 8, it passes through nanopore 8 under the action of the electric field.
  • the current perforation pattern is recorded through the current amplifier 2, and the sequence of the target protein is obtained by analyzing the current perforation pattern.

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Abstract

一种超灵敏纳米孔结构及芯片及分析装置及氨基酸和蛋白质检测方法及应用，超灵敏纳米孔结构为纳米孔（8），纳米孔（8）的孔径在0.35-5nm的范围内，纳米孔（8）的感应区域长度在0.3-2nm范围内，与单个氨基酸分子尺寸相近。超灵敏纳米孔芯片孔径为5-500nm范围内的基底（5），用于支撑超灵敏纳米孔（8），利用该超灵敏纳米孔分析装置，实现了小于1道尔顿的单分子检测极限，实现了氨基酸分子在单分子水平上的检测，完成了20种天然氨基酸分子和翻译后化学修饰氨基酸的直接检测，提出了将超灵敏纳米孔结构与生物酶进行技术融合实现蛋白质测序的策略，为提高纳米孔检测的空间分辨率提供了直接的解决方案。

Description

一种超灵敏纳米孔结构及芯片及分析装置及氨基酸和蛋白质检测方法及应用 技术领域
本发明涉及单分子检测领域，特别是涉及一种超灵敏纳米孔结构及芯片及分析装置及氨基酸和蛋白质检测方法及应用。
背景技术
蛋白质是一种极为重要的生命物质，蛋白质测序技术的发展不仅能为蛋白质结构和功能的研究提供关键信息，还有望为蛋白质组学研究和医学研究带来变革性的进步。Edman降解法和质谱法作为目前蛋白质测序的主流方法，在检测速度、读取长度或在低丰度下实现常规、完整的蛋白质组定量方面存在不足。
纳米孔技术由于具有极高的分辨率，在基因测序，病原体测序等领域中具有重要意义。目前，纳米孔技术在DNA测序上已经取得了瞩目进展，但在蛋白质测序方面的发展较为滞后，这主要是由于蛋白质的结构非常复杂。蛋白质由20种氨基酸组成，而DNA只由4种碱基构成，20个可分辨信号的检测是一大难题，此外，蛋白质的多级结构和带电不均一等特性也给其测序带来诸多挑战。
目前利用纳米孔技术已经实现了蛋白质和短肽链的检测，但距离实现蛋白质从头测序仍然面临两个至关重要的挑战：一、时间分辨率，控制肽链穿过纳米孔的速度可以提高有效数据点的数量，通过采用引入酶拉拽肽链通过纳米孔等方法，已经可以为纳米孔蛋白测序提供足够的时间分辨率；二、空间分辨率，近年来诸多文献报道了在纳米孔中提高蛋白质检测灵敏度的方法，已经实现了对载体聚合物中氨基酸取代、不同大小的均带电短同肽和翻译后修饰的区分，然而，对于直接解析单个氨基酸残基而言，空间分辨率仍然是制约纳米孔蛋白测序发展的主要瓶颈。要分辨20种天然氨基酸之间的微小差异所产生的信号是极其困难的。
纳米孔的感应区域长度，即线性穿过多肽链时，纳米孔同时读取氨基酸的数量，这对于纳米孔空间分辨率的提高至关重要。目前广泛使用的生物纳米孔的感应区域长度均为数个氨基酸，例如MspA生物纳米孔的感应区域长度为8个氨基酸(H.Brinkerhoff et al.,Science,374(6574),1509–1513.)，这限制了其进一步提高空间分辨率。本发明提出一种超灵敏纳米 孔结构，即具有感应区域长度小于传统生物纳米孔可以提供的最短传感区域长度(8个氨基酸)的纳米孔结构。基于这种超灵敏纳米孔结构设计超灵敏纳米孔芯片和超灵敏纳米孔分析装置。超灵敏纳米孔结构由于感应区域长度小于传统生物纳米孔可以提供的最短传感区域长度，为单个氨基酸分子的识别和蛋白质从头测序提供了基础。此外，基于现有文献报道的利用生物酶控制肽链穿过纳米孔以提高时间分辨率的方法(H.Brinkerhoff et al.,Science,374(6574),1509–1513.)，本发明还提出了将超灵敏纳米孔结构与生物酶进行技术融合实现蛋白质测序的策略。
目前，对于实现纳米孔蛋白质测序的挑战在于：
1、空间分辨率的提升。鉴于目前生物纳米孔面临的感应区域长度过大的问题，开发一种感应区域长度和单个氨基酸分子尺寸相当的超灵敏纳米孔至关重要。
2、纳米孔孔径的控制。专利US10648965B2中提及的采用电子束打孔法，可实现3-20纳米范围的纳米孔直径，但这一尺寸对于单个氨基酸的检测而言仍然偏大。将纳米孔尺寸控制在亚纳米级至2纳米范围内将对氨基酸检测和蛋白质测序带来新的契机。
3、氨基酸识别信噪比的控制。由于不同氨基酸的结构差异较小，在单分子实验过程中保证高信噪比是必需条件。传统人工纳米孔面临着噪声过大的问题，噪声的降低对人工纳米孔的发展是一大挑战。
发明内容
本发明针对现有技术的不足之处作出了改进，提供了一种超灵敏纳米孔结构及芯片及分析装置及氨基酸和蛋白质检测方法及应用，本发明是采用以下技术方案来实现的：
一种超灵敏纳米孔结构，其特征在于，超灵敏纳米孔结构为纳米孔，纳米孔的孔径在0.35-5nm的范围内，纳米孔的感应区域长度在0.3-2nm范围内，与单个氨基酸分子尺寸相近。
作为进一步地改进，本发明所述的纳米孔的孔径在0.35-2nm的范围内，可实现单个氨基酸分子的检测和识别。
作为进一步地改进，本发明所述的纳米孔的孔径在1-5nm的范围内，可实现翻译后化学修饰氨基酸分子的检测和识别、多肽的检测和杂多肽氨基酸序列的区分以及蛋白质测序。
作为进一步地改进，本发明所述的纳米孔为人工纳米孔或生物纳米孔。
作为进一步地改进，本发明所述的纳米孔的材料感应区域长度小于2nm，具有原子级厚度，包括二硫化钼、二硒化钼、二碲化钼、二硫化钨、二硒化钨、二碲化钨、二硫化钛、二 硒化钛、二碲化钛、二硫化锆、二硒化锆、二碲化锆、二硫化铪、二硒化铪、二碲化铪、二硫化钒、二硒化钒、二碲化钒、二硫化铌、二硒化铌、二碲化铌、二硫化钽、二硒化钽、二碲化钽、二硫化锝、二硒化锝、二碲化锝、二硫化铑、二硒化铑、二碲化铑、二硫化铼、二硒化铼、二碲化铼、二硫化铂、二硒化铂、二碲化铂、二硫化钯、二硒化钯、二碲化钯、石墨烯、石墨烯氮化碳、氮化硼、黑磷、层状金属氧化物、层状金属碳化物、层状金属氮化物或金属氮氧化物复合二维材料、金属有机骨架材料、共价有机骨架材料、钙钛矿材料。
本发明公开了一种包括超灵敏纳米孔结构的超灵敏纳米孔芯片，芯片包括下层基底和上层含超灵敏纳米孔结构的纳米薄膜。
作为进一步地改进，本发明所述的基底开设有支撑孔，用于支撑超灵敏纳米孔，支撑孔的孔径在5-500nm范围内。
作为进一步地改进，本发明所述的基底包括硅层，硅层的阻值高于2700Ohmcm。
作为进一步地改进，本发明所述的基底包括从上到下的氮化硅、氧化硅、硅、氧化硅和氮化硅五层，硅层厚度为300-500μm，表层为绝缘层，支撑孔开设于表层氮化硅层上。
作为进一步地改进，本发明芯片可实现低于20pA的背景噪声。降低了介电噪声，可以提供足够分辨单个氨基酸分子的信噪比。
本发明公开了一种超灵敏纳米孔芯片的超灵敏纳米孔分析装置，装置包括注满离子溶液的流体槽、将流体槽分为顺式侧腔室和反式侧腔室的超灵敏纳米孔芯片、接入流体槽溶液或芯片的电极、与电极串联的电流放大器和电源、与电流放大器相连的数模转换设备和计算机。
本发明公开了一种超灵敏纳米孔分析装置制备超灵敏纳米孔结构的方法，在流体槽两侧腔室内注入盐溶液，通过电源和电流放大器对纳米薄膜直接施加0-10V范围内的电压，刻蚀得到纳米孔，通过控制施加电压的大小和时长控制电化学反应，即可控制孔径的尺寸；获得纳米孔跨膜电导的实时数据，当电导值达到目标孔径对应值时，停止施加电压，完成纳米孔制备过程。
本发明公开了一种利用超灵敏纳米孔分析装置用于纳米孔快速氨基酸检测的方法，所述的纳米孔的孔径在0.35-2nm的范围内，包括如下步骤：
1)、将氨基酸样品溶于缓冲液；
2)、在流体槽溶液中加入氨基酸样品，样品在电场作用下通过纳米孔；
3)、通过电流放大器记录电流穿孔图谱；
4)、通过分析电流穿孔图谱和/或由电流穿孔图谱经过统计学分析得到的相对电流阻塞电导降和/或穿孔时间和/或捕获速率来分析检测氨基酸。
本发明公开了一种利用超灵敏纳米孔分析装置用于纳米孔快速蛋白质测序的方法，所述的纳米孔的孔径在1-5nm的范围内，包括如下步骤：
1)、设计并合成肽链与核苷酸链相连的复合物，引物链以及阻断链，在每次实验前将这三种物质按所需比例混合并退火，即得待测样品；
2)、在顺式侧腔室中加入生物酶，脱氧核糖核苷三磷酸(dNTPs)，以及待测样品；
3)、施加电压，样品在电场作用下通过纳米孔，通过电流放大器记录电流穿孔图谱；
4)、通过分析电流穿孔图谱解析蛋白质序列。
作为进一步地改进，本发明所述的生物酶包括phi29 DNA聚合酶、Tth DNA聚合酶、Pfu DNA聚合酶、Pfu HS DNA聚合酶、T4 DNA聚合酶、Bst DNA聚合酶、Taq DNA聚合酶、Taq Ⅱ DNA聚合酶、Taq plus DNA聚合酶、LA Taq DNA聚合酶、VentR DNA聚合酶、Phusion DNA聚合酶、KOD DNA聚合酶、Iproof DNA聚合酶、Hel308解旋酶、T4解旋酶、DnaB解旋酶、Rep蛋白解旋酶、PcrA解旋酶、Tte UvrD解旋酶、Tth UvrD解旋酶、T4 Dda解旋酶、T4 gp41解旋酶、RecQ解旋酶、NS3 HCV解旋酶、elF4A解旋酶、WRN解旋酶、TRCF解旋酶、LTag解旋酶、E1解旋酶、T7 gp4解旋酶、Rho解旋酶、ClpX蛋白酶。
本发明公开了一种超灵敏纳米孔芯片应用于纳米孔在氨基酸检测，纳米孔的孔径在0.35-2nm的范围内，实现20种天然氨基酸的检测和区分。
本发明公开了一种超灵敏纳米孔芯片应用于纳米孔在在翻译后化学修饰氨基酸检测，纳米孔的孔径在1-5nm的范围内，实现翻译后修饰氨基酸的检测和区分。
本发明公开了一种超灵敏纳米孔芯片应用于多肽检测，纳米孔的孔径在1-5nm的范围内，实现多肽的检测和杂多肽氨基酸序列的区分。
本发明公开了一种超灵敏纳米孔芯片应用于蛋白质测序，纳米孔的孔径在1-5nm的范围内，实现蛋白质测序。
本发明设计了一种超灵敏纳米孔结构，以及基于超灵敏纳米孔结构的超灵敏纳米孔芯片和超灵敏纳米孔分析装置。该超灵敏纳米孔结构的感应灵敏长度小于传统生物纳米孔可以提供的最短传感区域长度，孔径尺寸可控制在0.35-5nm。该超灵敏纳米孔芯片，可以提供小于20pA的背景噪声。利用该超灵敏纳米孔分析装置，实现了小于1道尔顿的单分子检测极限，实现了氨基酸分子在单分子水平上的检测，完成了20种天然氨基酸分子和翻译后化学修饰氨基酸的直接检测，提出了将超灵敏纳米孔结构与生物酶进行技术融合实现蛋白质测序的策略。本发明为提高纳米孔检测的空间分辨率提供了直接的解决方案。
进一步地，本发明的有益效果如下：
(1)采用电化学刻蚀法制备超灵敏纳米孔，孔径在0.35-5nm范围内精确可控且操作简单。相比于使用扫描电子显微镜(TEM)进行电子束打孔法所能达到的最小孔径2nm(Storm,A.et al.Nature Mater 2,537–540(2003).)，采用电化学刻蚀打孔法不仅可以制备出小于2nm的纳米孔，而且操作简单、陈本低廉。可以根据待测样品的尺寸精确设计并制备纳米孔，提高了所产生电流信号的幅度，使得该种纳米孔具备鉴别不同种类氨基酸的能力。
(2)此前，具有原子级厚度的纳米孔已被证明可用于分辨单个核苷酸分子，本发明相较于这一工作进一步缩小了纳米孔孔径，在0.35-2nm范围的孔径中实现了尺寸更小的氨基酸的检测与分辨。本发明相较于专利US10648965B2，将纳米孔的性能从4种核苷酸分子的直接检测提升至20种氨基酸分子的直接检测。此外，可以实现互为同分异构体的分子的辨别，分子中单个基团差异的辨别，将超灵敏纳米孔结构的分辨能力提升至小于1道尔顿。
(3)本发明提出的具有0.3-2nm感应区域长度的超灵敏纳米孔结构，可以为氨基酸检测和蛋白质测序提供足够的空间分辨率。
(4)本发明中所采用的超灵敏纳米孔芯片中的基底结构如图2所示，由氮化硅，氧化硅和硅构成夹心结构。其中硅层厚度为380±25μm，具有较高阻值(大于2700Ohmcm)；氧化硅层厚度为60±6nm，采用干热氧化制备；氮化硅层厚度为20±4nm，并使用具有低应力的氮化硅有利于后续制备纳米级支撑孔。该夹心结构的设计大大降低了介电噪声，采用该夹心结构的超灵敏纳米孔分析装置可以实现低于20pA的背景噪声，相较于先前的文献报道(Feng,J.et al.,(2015).Nature nanotechnology,10(12),1070–1076.)具有更低的噪声。
(5)本发明中超灵敏纳米孔芯片基底上的支撑孔尺寸在5-500nm范围内可控，使用小于60nm的支撑孔尺寸可以改善检测过程中由于上层纳米薄膜振动导致的机械噪声。
(6)本发明的超灵敏纳米孔分析装置由于具有高空间分辨率和高信噪比，可以实现20种天然氨基酸的直接检测，无需对待测分子进行修饰和预处理，操作简单。并且该分析装置具有小于1道尔顿的单分子检测极限，为纳米孔蛋白质测序提供基础。
(7)本发明的超灵敏纳米孔分析装置在纳米孔孔径为1-5nm范围内可以识别翻译后化学修饰氨基酸，由于蛋白质的翻译后修饰与疾病息息相关，因此该装置有利于医学研究和疾病诊断的发展。
(8)本发明的超灵敏纳米孔分析装置在纳米孔孔径为1-5nm范围内可以实现多肽的检测和杂多肽氨基酸序列的区分。
(9)本发明的超灵敏纳米孔分析装置在纳米孔孔径为1-5nm范围内可以实现蛋白质 测序。设计并合成目标肽链与核苷酸链的连接复合物，引入生物酶(例如phi29DNA聚合酶、Hel308解旋酶)，使目标链与纳米孔和生物酶相接触，酶控制目标链穿过纳米孔运动，通过测定并分析穿孔过程中产生的电流图谱，即可得到目标肽链的序列。
(10)本发明的超灵敏纳米孔结构稳定性高，可以贮存数周并完成上万次穿孔事件的检测。
附图说明
图1为本发明分析装置的结构示意图；
图中，1是电源，2是电流放大器，3是数模转换设备和计算机，4是电极，5是基底，6是支撑孔，7是纳米薄膜，8是纳米孔，9是流体槽，10是待测样品，11为顺式侧腔室，12为反式侧腔室。
图2为基底的示意图；
图3为流体槽的示意图；
本实施例中使用的流体槽结构如图3所示，流体槽主要由两部分组成，该两部分的唯一差别在于图中16所示的部分含内螺纹，而图示15为光滑内壁，该设计便于用螺丝组装流体槽。图示13供银/氯化银电极插入，图示11和12为腔室，图示14中的环形凹槽适配O型密封圈。
图4为单层二硫化钼的表征图；
图5为二硫化钼纳米孔中单个氨基酸的检测示意图；
a.实验装置示意图(非按比例)；b.谷氨酸穿孔过程中采集到的电流轨迹；c.丙氨酸穿孔过程中采集到的电流轨迹，右侧展示了归一化电流直方图的拟合曲线；d.G、GG、GGG在同一器件中产生信号的停留时间和阻塞电流的热图；e.利用深度学习识别氨基酸的流程图；f.甘氨酸(G)和丙氨酸(A)在同一器件中引起的ΔI/I0的直方图；g.对于f中的数据，利用深度学习得到的精确度、召回率和F1的得分图；
图6为电压对纳米孔检测氨基酸的影响。
a.丝氨酸在不同电压下的穿孔频率图；b-c.甘氨酸在不同电压下穿孔引起的阻塞电流值ΔI和ΔI/I0图。
图7为对所有20种天然氨基酸的检测结果图；
图中为对20种氨基酸进行纳米孔实验所得到ΔI/I0直方图，依次按带电类氨基酸(a-b)、疏水非芳香族氨基酸(c-d)、极性不带电氨基酸(e-f)、疏水芳香族氨基酸(g)和特殊类氨基酸 (h)展示。相应的混淆矩阵图附在每个直方图下方。
图8为二硫化钼纳米孔识别化学基团的结果图；
a-c.分别是天冬氨酸(D)和天冬酰胺(N)，谷氨酸(E)和谷氨酰胺(Q)，天冬酰胺(N)和亮氨酸(L)在纳米孔实验中引起的穿孔事件的ΔI/I0值的直方图；d-f.分别是丝氨酸(S)和苏氨酸(T)，苯丙氨酸(F)和酪氨酸(Y)，亮氨酸(L)和异亮氨酸(I)的热图。图d-f中框的宽度表示标准差。
图9为对磷酸化氨基酸的识别结果图；
a.酪氨酸(Y)和磷酸化酪氨酸(p-Y)的化学结构。b.酪氨酸和磷酸化酪氨酸在同一器件中的穿孔事件的ΔI/I0直方图。c.对b中的数据进行深度学习后得到的精确度、召回率和F1的得分图。
图10为肽链的单分子检测结果图；
图中展示了肽链EEEGEEE和EEEWEEE在实验过程中的典型电流信号图。
图11为引入酶实现蛋白质测序的示意图。
具体实施方式
本发明公开了一种超灵敏纳米孔8结构，超灵敏纳米孔8结构为纳米孔8，纳米孔8的孔径在0-50nm的范围内，纳米孔8的感应区域长度在0.3-2nm范围内，与单个氨基酸分子尺寸相近。纳米孔8的孔径进一步限定在0.35-2nm的范围内，可实现单个氨基酸分子的检测和识别，纳米孔8为人工纳米孔8或生物纳米孔8。当为人工纳米孔8时，纳米孔8的材料的感应区域长度小于2nm，具有原子级厚度，包括二硫化钼、二硒化钼、二碲化钼、二硫化钨、二硒化钨、二碲化钨、二硫化钛、二硒化钛、二碲化钛、二硫化锆、二硒化锆、二碲化锆、二硫化铪、二硒化铪、二碲化铪、二硫化钒、二硒化钒、二碲化钒、二硫化铌、二硒化铌、二碲化铌、二硫化钽、二硒化钽、二碲化钽、二硫化锝、二硒化锝、二碲化锝、二硫化铑、二硒化铑、二碲化铑、二硫化铼、二硒化铼、二碲化铼、二硫化铂、二硒化铂、二碲化铂、二硫化钯、二硒化钯、二碲化钯、石墨烯、石墨烯氮化碳、氮化硼、黑磷、层状金属氧化物、层状金属碳化物、层状金属氮化物或金属氮氧化物复合二维材料、金属有机骨架材料、共价有机骨架材料、钙钛矿材料。
本发明公开了超灵敏纳米孔8芯片，芯片包括下层基底5和上层含超灵敏纳米孔8结构的纳米薄膜7，基底5开设有支撑孔6，用于支撑超灵敏纳米孔8，支撑孔6的孔径在5-500nm 范围内，基底5包括硅层，硅层的阻值高于2700Ohmcm，基底5包括从上到下的氮化硅、氧化硅、硅、氧化硅和氮化硅五层，硅层厚度为300-500μm，表层为绝缘层，支撑孔6开设于表层氮化硅层上，该芯片可实现低于20pA的背景噪声。降低了介电噪声，可以提供足够分辨单个氨基酸分子的信噪比。
本发明公开了一种超灵敏纳米孔8分析装置，装置包括注满离子溶液的流体槽9、将流体槽9分为顺式侧腔室11和反式侧腔室12的超灵敏纳米孔8芯片、接入流体槽9溶液或芯片的电极4、与电极4串联的电流放大器2和电源1、与电流放大器2相连的数模转换设备和计算机3。
本发明公开了一种超灵敏纳米孔8分析装置制备超灵敏纳米孔8结构的方法，在流体槽9两侧腔室内注入盐溶液，通过电源1和电流放大器2对纳米薄膜7直接施加0-10V范围内的电压，刻蚀得到纳米孔8，通过控制施加电压的大小和时长控制电化学反应，即可控制孔径的尺寸；获得纳米孔8跨膜电导的实时数据，当电导值达到目标孔径对应值时，停止施加电压，完成纳米孔8制备过程。
本发明公开了一种利用超灵敏纳米孔8分析装置用于纳米孔8快速氨基酸检测的方法，包括如下步骤：
1)、将氨基酸样品溶于缓冲液；
2)、在流体槽9溶液中加入氨基酸样品，样品在电场作用下通过纳米孔8；
3)、通过电流放大器2记录电流穿孔图谱；
4)、通过分析电流穿孔图谱和/或由电流穿孔图谱经过统计学分析得到的相对电流阻塞电导降和/或穿孔时间和/或捕获速率来分析检测氨基酸。
本发明公开了一种利用超灵敏纳米孔8分析装置用于纳米孔8快速蛋白质测序的方法，包括如下步骤：
1)、设计并合成肽链与核苷酸链相连的复合物，引物链以及阻断链，在每次实验前将这三种物质按所需比例混合并退火，即得待测样品10；
2)、在顺式侧腔室11中加入生物酶，脱氧核糖核苷三磷酸(dNTPs)，以及待测样品10；
3)、施加电压，样品在电场作用下通过纳米孔8，通过电流放大器2记录电流穿孔图谱；
4)、通过分析电流穿孔图谱解析蛋白质序列。
其中生物酶包括phi29 DNA聚合酶、Tth DNA聚合酶、Pfu DNA聚合酶、Pfu HS DNA聚合酶、T4 DNA聚合酶、Bst DNA聚合酶、Taq DNA聚合酶、Taq Ⅱ DNA聚合酶、Taq plus DNA聚合酶、LA Taq DNA聚合酶、VentR DNA聚合酶、Phusion DNA聚合酶、KOD DNA聚合酶、Iproof  DNA聚合酶、Hel308解旋酶、T4解旋酶、DnaB解旋酶、Rep蛋白解旋酶、PcrA解旋酶、Tte UvrD解旋酶、Tth UvrD解旋酶、T4 Dda解旋酶、T4 gp41解旋酶、RecQ解旋酶、NS3 HCV解旋酶、elF4A解旋酶、WRN解旋酶、TRCF解旋酶、LTag解旋酶、E1解旋酶、T7 gp4解旋酶、Rho解旋酶、ClpX蛋白酶。
本发明公开了一种超灵敏纳米孔8芯片应用于纳米孔8在氨基酸检测，实现20种天然氨基酸的检测和区分。
本发明公开了一种超灵敏纳米孔8芯片应用于纳米孔8在在翻译后化学修饰氨基酸检测，实现翻译后修饰氨基酸的检测和区分。
本发明公开了一种超灵敏纳米孔8芯片应用于多肽检测，实现多肽的检测和杂多肽氨基酸序列的区分。
本发明公开了一种超灵敏纳米孔8芯片应用于蛋白质测序，实现蛋白质测序。
下面结合说明书附图，通过具体实施例对本发明的技术方案作进一步地说明。
实施例1：采用二硫化钼纳米孔8检测20种天然氨基酸。
图1为本发明分析装置的结构示意图；包括注满离子溶液的流体槽9、将流体槽9分为顺式侧腔室11和反式侧腔室12的超灵敏纳米孔8芯片、接入流体槽9溶液或芯片的电极4、与电极4串联的电流放大器2和电源1、与电流放大器2相连的数模转换设备和计算机3。基底5上开有支撑孔6，纳米薄膜7上开有超灵敏纳米孔8，本实施例采用的纳米薄膜7为单层二硫化钼，厚度约0.65纳米，超灵敏纳米孔8孔径在0-50nm范围内可控，基底5为氮化硅，氧化硅和硅组成的夹心结构。
本实施例的具体实验操作如下：
(1)器件支撑基底5的加工
为了得到具有较高信噪比的器件，采用具有夹心结构的晶圆制备基底5，基底5结构如图2所示。该夹心结构由氮化硅、氧化硅和硅构成，其中硅层厚度为380±25μm，具有较高阻值(大于2700Ohmcm)；氧化硅层厚度为60±6nm，采用干热氧化制备；氮化硅层厚度为20±4nm，并使用具有低应力的氮化硅有利于后续制备纳米级支撑孔6。
先利用激光直写在4寸晶圆上写出0.5cm×0.5cm的正方形图案，然后进行反应离子刻蚀和湿法刻蚀，将部分氮化硅、氧化硅和硅刻蚀掉，得到图2所示的氮化硅悬空结构，最后利用聚焦离子束(FIB)在氮化硅支撑窗口上制备支撑孔6。其中支撑窗口尺寸为15-20μm，并控制支撑孔6尺寸为60nm左右。采用该夹心结构的超灵敏纳米孔8分析装置的背景噪声约为18pA。
(2)二硫化钼的制备
采用化学气相沉积法(CVD)制备单层二硫化钼。使用三氧化钼粉末作为钼源，硫粉作为硫源，高纯氩气作为保护气和载气，单面抛光的二氧化硅片作为沉积样品的基底5。取2mg三氧化钼粉末和0.15g硫粉分别装于两只石英舟内，基底5按抛光面朝下的方向置于装有三氧化钼粉末的石英舟上。装有三氧化钼和基底5的石英舟置于管式炉石英管中央加热区，装有硫粉的石英舟置于管式炉石英管的边缘。检查管式炉密封性，使用机械泵将管内空气抽出，后通入氩气作为保护气。在氩气流量为100sccm的条件下匀速升温至700℃，升温速率约为15℃/分钟，升至700℃后恒温继续加热10分钟后停止加热，自然冷却至室温，此时基底5氧化层上已沉积有单层二硫化钼薄膜。图4展示了单层二硫化钼的各项表征结果，其中a图为在SiO2/Si表面生长得到的单层二硫化钼的光学图像，b图为单层二硫化钼在高分辨率球差校正透射电子显微镜(AC-TEM)下的图像，c图为透射电镜(200kV)下获得的单层单晶二硫化钼的电子衍射图，d图为转移到蓝宝石片上的二硫化钼样品在原子力显微镜下的图像，该样品厚度约为0.78nm。
(3)二硫化钼的转移
取一小片生长所得的结晶度良好的单层二硫化钼样品，使用匀胶机在2500r/s的转速均匀旋涂PMMA胶，180℃条件下烘烤15min。然后将样品分割为2mm×2mm的尺寸，在30％的KOH溶液中80℃湿法刻蚀直至PMMA膜浮起。将刻蚀得到的PMMA薄膜在纯水中清洗2-3次，借助显微操作系统转移至含有60nm支撑孔6的氮化硅片上，待水蒸发后在180℃条件下烘烤20min。采用丙酮溶液在60℃条件下去除氮化硅表面的PMMA胶，随后在200℃条件下氩气氛围中褪火4h。
(4)超灵敏纳米孔8制备
采用电化学刻蚀打孔的方式，在流体槽9两侧腔室内注入浓度为1mol/L的KCl溶液，通过电源1和电流放大器2对纳米薄膜7直接施加0-10V范围内的电压，刻蚀得到纳米孔8，通过控制施加电压的大小和时长控制电化学反应，当电导值达到目标孔径对应值时，停止施加电压，控制孔径在0.35-2nm范围内。
(5)流体槽9组装
流体槽9采用PMMA材料，流体槽9结构如图3所示，流体槽9主要由两部分组成，该两部分的唯一差别在于图中16所示的部分含内螺纹，而图示15为光滑内壁，该设计便于用螺丝组装流体槽9。图示13供银/氯化银电极4插入，图示11和12为腔室，图示14中的环形凹槽适配O型密封圈。利用O型圈将芯片密封在两部分流体槽9之间，通过4个螺丝钉固定 器件。
(6)溶液配制
配制pH＝7.8的Tris-EDTA-KCl缓冲溶液，其中Tris浓度为10mmol/L,EDTA浓度为1mmol/L，KCl的浓度为1mol/L。将氨基酸固体粉末溶解于缓冲溶液配制相应的氨基酸溶液，样品浓度为2μmol/L。
(7)实验装置的搭建
采用膜片钳(Axopatch 200B放大器)低噪音系统进行测试，采用NI PXI-1042Q采集卡进行数据采集。取两段长度约为3cm的银丝，使用砂纸抛光以除去表面的氧化层。在1M KCl溶液中浸入四分之三的银丝长度和铂电极4(分别作为阳极和阴极)。施加2V电压一段时间以制备Ag/AgCl电极4。然后将两根Ag/AgCl电极4分别连接在膜片钳的探头上，作为正极和地线。膜片钳在使用前需用model cell进行校准，消除误差。
(8)测量
在每次实验前，在浓度为1mol/L的KCl溶液中进行电流-电压扫场，确定器件的孔径。在正式实验中，在流体槽9顺式侧腔室11和反式侧腔室12内注入相应的缓冲溶液，反式侧腔室12上施加200mV的偏置电压，电压的正负取决于不同氨基酸的等电点与缓冲溶液pH值的相对大小，例如待测氨基酸的等电点小于7.8，此时该氨基酸在缓冲溶液环境中带负电，则施加的偏置电压为正。在未加入氨基酸分子的空白实验中，可以检测到平均值为I0的稳定离子电流通过纳米孔8。在顺式侧腔室11注入含氨基酸的缓冲溶液后，可以检测到离子电流的短暂阻断。每个电流阻断对应于纳米孔8中单个氨基酸分子的穿孔行为，其电流特征是相对电流下降值ΔI/I0和停留时间Δt。对每一组实验结果均作归一化。
(9)数据分析
采用Matlab程序对数据进行处理和分析，并整合数据呈现实验结果。后续结合深度学习进一步分析实验数据。
如图5a所示，氨基酸分子在电泳力驱动下穿过二硫化钼纳米孔8，当待测分子通过二硫化钼纳米孔8时会短暂地堵塞孔道，从而孔道中的电流会产生变化，对该电流变化的幅度和持续时间以及电流波形进行分析，即可获取相应的分子信息。本实施例中，在直径为0.35-2nm范围内的二硫化钼纳米孔8可以检测到20种天然氨基酸在过孔时产生的电流信号并对不同的氨基酸分子加以区分，图5b中展示了一段典型的电流信号，且可以通过对信号的相对阻塞电流值ΔI/I0进行直方图分布统计并拟合来区分氨基酸的种类(图5f)。二硫化钼纳米孔8的尺寸的选择非常重要，当纳米孔8的尺寸与待测氨基酸分子的尺寸接近时，离子电流可降 为0nA(图5c)。当纳米孔8的尺寸大于2nm时，对氨基酸分子的检测灵敏度下降。
本实施例中为了确定所得到的信号是由氨基酸穿孔引起的，进行了一组不同电压下的穿孔实验。结果表明随着电压的增大，阻塞电流ΔI的值增大，相对阻塞电流ΔI/I0的值基本不变(图6b，c)，且穿孔事件发生的频率随着电压的增大而先迅速增大后趋于饱和(图6a)。
本实施例中二硫化钼纳米孔8的检测灵敏区域与单个氨基酸尺寸相当，为了证明这一点，选用由同一种氨基酸构成的不同长度的短链G，GG，GGG进行实验，在同一纳米孔8中三者产生的阻塞电流值非常接近(图5d)，这与先前报道的生物纳米孔8中所产生的结果截然不同。
在具体实验过程中，由于二硫化钼纳米孔8基线电流存在一定的不稳定性，所以为了确保实验数据不受基线电流变化的影响，本实施例中将20种氨基酸按照其所属类别进行分组讨论，分别是带电类、极性不带电类、疏水芳香类、疏水非芳香类和特殊类氨基酸。
在数据的分析过程中引入了深度学习模型，神经网络图如图5e所示。该深度学习模型对电流信号的特征值和波形均进行了学习，每一组实验结果均以混淆矩阵的形式进行展示。
对于带电类氨基酸，考虑到实验中采用的Tris-EDTA缓冲溶液pH＝7.8，在这一条件下，由于谷氨酸(E,147.13Da)、天冬氨酸(D,133.11Da)和组氨酸(H,155.15Da)的pI值小于溶液pH值，在溶液中带负电，而赖氨酸(K,146.19Da)和精氨酸(R,174.20Da)的情况恰恰相反，在溶液中带正电，因此按实际带电情况分两组进行实验。赖氨酸和精氨酸的ΔI/I0值与分子的相对质量成正相关(如图7a)，且深度学习分析后的辨别准确率达到了82.18％。谷氨酸和天冬氨酸的相对电流阻塞值相近(如图7b)，组氨酸分子的ΔI/I0值分布与谷氨酸、天冬氨酸有明显区分(如图7b)，所得数据在经过深度学习后，谷氨酸、天冬氨酸和组氨酸三者的辨别准确率达到了82.08％。
疏水非芳香族包含的五种氨基酸中，甲硫氨酸(M,149.21Da)在实验中有较大的概率会堵塞孔道导致实验的中断，这可能是由于其含硫原子，与二硫化钼易发生相互作用。这组实验中还发现具有相同分子质量的异构氨基酸亮氨酸(L)和异亮氨酸(I)产生了不同的相对阻塞电流(如图7d)。在7个二硫化钼纳米孔8器件(孔径为0.6-1.4nm)中，观察到在5个器件中显示的ΔI/I0值高于L，但在另外2个器件中结果相反。后一种情况发生在直径约0.6nm的纳米孔8中。将这一变化归因于纳米孔8的大小可能会影响氨基酸的孔隙进入方向，通过深度学习，这两种同分异构氨基酸的分辨准确率达到了87.25％。
对于极性不带电类氨基酸，丝氨酸(S,105.09Da)、苏氨酸(T,119.10Da)、天冬酰胺(N,132.12Da)和谷氨酰胺(Q,146.15Da)在孔道中产生的ΔI/I0值随着分子质量的增大而变大(如图7e，f)。丝氨酸和苏氨酸，天冬酰胺和谷氨酰胺的分子结构均只相差一个 基团，该组氨基酸的检测结果表明，二硫化钼纳米孔8可以识别分子量仅为14.01Da的单个化学基团的差异。
对于疏水芳香族氨基酸，苯丙氨酸(F,165.20Da)、酪氨酸(Y,181.19Da)和色氨酸(W,204.20Da)的相对电流阻塞值与相对分子质量也呈现出正相关(如图7g)，这里值得注意的是，苯丙氨酸和酪氨酸之间仅有一个羟基之差，但两者的电流分布可以被区分，这砸一次证明了二硫化钼纳米孔8对化学基团的检测具有很高的灵敏度。
对于特殊类氨基酸，甘氨酸(G,75.07Da)，半胱氨酸(C,121.16Da)和脯氨酸(P,115.13Da)，其中脯氨酸在实验中产生的相对电流阻塞值最大(如图7h)，这是因为脯氨酸分子结构中含五元环，使其在特殊族氨基酸中的分子体积最大。半胱氨酸由于含有硫原子，在实验过程中也存在堵塞纳米孔8的情况。通过上述实验，整体而言，氨基酸分子在通过二硫化钼纳米孔8时产生的相对阻塞电流值随着其体积的增加而增加。
本实施例为了进一步证明二硫化钼纳米孔8对氨基酸的高灵敏度，对相对分子质量差仅为1Da的天冬氨酸(D)和天冬酰胺(N)进行了单独的实验，结果表明通过电流分析可以区分二者(如图8a)，证明所制备的二硫化钼纳米孔8达到了1Da的分辨率，这也是目前纳米孔8领域的最高分辨率。
近年来，用于检测多肽的纳米孔8的最终分辨率不断更新。FraC生物纳米孔8可以区分仅有一个氨基酸残基不同的多肽，其分辨率达到了44Da(Huang,G.et al.,(2019).Nature communications,10(1),835.)。气溶素纳米孔8对检测聚阳离子聚合物与氨基酸相连形成的肽链显示出了较高的灵敏度(Ouldali,H.et al.,(2020).Nature biotechnology,38(2),176–181.)。然而，这些测量均是以间接的方式区分单个氨基酸，通过替换氨基酸残基来测量电流的差异。本发明基于可以区分单个氨基酸的实验基础，进一步研究目前二硫化钼纳米孔8系统的极限分辨率。对于天冬氨酸和天冬酰胺，或谷氨酸和谷氨酰胺，两两之间的相对分子质量差仅为0.99Da，且除了末端化学基团外，分子骨架相同，其中带电氨基酸(天冬氨酸，谷氨酸)的分子结构末端为-OH(17.01Da)，极性不带电氨基酸(天冬酰胺，谷氨酰胺)的分子结构末端为-NH2(16.02Da)。虽然每一对氨基酸的分子量和体积都非常接近，但官能团的不同导致其分子构型和带电量的不同，从而导致穿过二硫化钼纳米孔8时产生不同的电流阻塞(图8a，b)。相对分子质量差为0.94Da的亮氨酸和天冬酰胺，由于其结构差异较大，穿孔过程中亦可产生可区分的相对电流阻塞(图8c)。据所知，这是实验报道的纳米孔8的最高分辨率(区分小于1Da的相对分子质量差)。
实施例2：采用二硫化钼纳米孔8检测氨基酸的翻译后修饰。
本发明可以应用于检测氨基酸的翻译后修饰(PTMs)。对氨基酸翻译后修饰的识别在疾病的研究中具有非常重要的意义，例如酪氨酸(Y)的磷酸化对细胞活性起着关键的调控作用，而酪氨酸的异常磷酸化与癌变密切相关。
本实施例使用二硫化钼纳米孔8对酪氨酸(Y)和磷酸化酪氨酸(p-Y)进行检测识别，图9a为酪氨酸(Y)和磷酸化酪氨酸(p-Y)的分子结构图。重复实施例1中所述的实验操作(1)、(2)、(3)、(4)、(5)、(6)、(7)、(8)、(9)，其中操作(5)中配制的样品溶液为酪氨酸溶液和磷酸化酪氨酸溶液。
如图9b所示，酪氨酸和磷酸化酪氨酸在实验中引起的ΔI/I0的分布可被区分，识别准确率为80.67％。这一结果表明二硫化钼纳米孔8具有识别翻译后修饰的氨基酸的潜力。
实施例3：采用二硫化钼纳米孔8检测短肽链。
本发明可以应用于短肽链的检测。
本实施例中设计了两条序列分别为EEEGEEE和EEEWEEE的肽链，重复实施例1中所述的实验操作(1)、(2)、(3)、(4)、(5)、(6)、(7)、(8)、(9)，其中操作(6)中配制的样品溶液为EEEGEEE和EEEWEEE这两种肽链分子溶液，操作(8)中施加电压为300mV。
在实验中观察到如图10中方框内所示的电流波形。图10底部所展示的波形为实际测得电流信号在不受任何噪声影响下的理想波形。体积较大的W产生较大的电流阻塞，而体积较小的G则产生了较小的电流阻塞，该结果与预期相符。
基于本发明目前的研究进展，低于1Da的纳米孔8分辨率以及与单个氨基酸分子尺寸相当的检测灵敏区域，在理论上已经可以为蛋白质测序提供足够的空间分辨率。提出，将酶引入二硫化钼纳米孔8体系来精确控制肽链通过孔道的速率，这有望提供足够的时间分辨率，进而实现蛋白质的从头测序。目前将酶引入生物纳米孔8来控制多肽运动的实验已被广泛应用，诸多文献也报道了DNA聚合酶与固体纳米孔8系统之间的相容性，因此这一策略具有可行性。
实施例4：利用酶和二硫化钼纳米孔8进行蛋白质测序。
本发明可以应用于蛋白质测序。检测原理如图11所示。
本实施例中设计并合成肽链与核苷酸链相连的复合物为HOOC-GEPGEPDDGDD-5'-X-AGAACTTTAGAACTTTTCAGATCTCACTATCGCATTCTCATGCAGGTCGTAGCC-3'，引物链为5'-GCGTACGCCTACGGTTTTCCGTAGGCGTACGCGGCTACGACCTGCATGAGAATGC-3'以及阻断链5'-GATAGTGAGATCTGAXXXXXXXZ-3'。序列中的X代表无碱基间隔物，Z代表3'间臂。在每次实验前将这三种物质按1：1：2比例混合并在室温下退火20分钟，即得待测样品10。配制pH＝7.5 的HEPES缓冲溶液，其中KCl的浓度为1mol/L，HEPES浓度为10mmol/L，MgCl2浓度为10mmol/L，(NH4)2SO4的浓度为10mmol/L，DTT的浓度为4mmol/L。重复实施例1中所述的实验操作(1)、(2)、(3)、(4)、(5)、(7)。在顺式侧腔室11中加入含浓度为5nmol/L的phi29DNA聚合酶，浓度为1mmol/L的脱氧核糖核苷三磷酸(dNTPs)和浓度为10nmol/L待测样品10的缓冲溶液，反式侧加入空白缓冲溶液。施加+200-300mV范围内电压，样品与phi29DNA聚合酶和纳米孔8接触后，在电场作用下通过纳米孔8。通过电流放大器2记录电流穿孔图谱，通过分析电流穿孔图谱解析得到目标蛋白质的序列。
显然，上述并非对实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可做出不同形式的变化，这些经变化后的实施方式仍处于本发明创造的保护范围中。

Claims (19)

  1. 一种超灵敏纳米孔(8)结构，其特征在于，所述的超灵敏纳米孔(8)结构为纳米孔(8)，所述的纳米孔(8)的孔径在0.35-5nm的范围内，所述的纳米孔(8)的感应区域长度在0.3-2nm范围内，与单个氨基酸分子尺寸相近。
  2. 根据权利要求1所述的超灵敏纳米孔(8)结构，其特征在于，所述的纳米孔(8)的孔径在0.35-2nm的范围内。
  3. 根据权利要求1所述的超灵敏纳米孔(8)结构，其特征在于，所述的纳米孔(8)的孔径在1-5nm的范围内。
  4. 根据权利要求1所述的超灵敏纳米孔(8)结构，其特征在于，所述的纳米孔(8)为人工纳米孔(8)或生物纳米孔(8)。
  5. 根据权利要求1所述的超灵敏纳米孔(8)结构，其特征在于，所述的纳米孔(8)的材料感应区域长度小于2nm，具有原子级厚度，包括二硫化钼、二硒化钼、二碲化钼、二硫化钨、二硒化钨、二碲化钨、二硫化钛、二硒化钛、二碲化钛、二硫化锆、二硒化锆、二碲化锆、二硫化铪、二硒化铪、二碲化铪、二硫化钒、二硒化钒、二碲化钒、二硫化铌、二硒化铌、二碲化铌、二硫化钽、二硒化钽、二碲化钽、二硫化锝、二硒化锝、二碲化锝、二硫化铑、二硒化铑、二碲化铑、二硫化铼、二硒化铼、二碲化铼、二硫化铂、二硒化铂、二碲化铂、二硫化钯、二硒化钯、二碲化钯、石墨烯、石墨烯氮化碳、氮化硼、黑磷、层状金属氧化物、层状金属碳化物、层状金属氮化物或金属氮氧化物复合二维材料、金属有机骨架材料、共价有机骨架材料、钙钛矿材料。
  6. 一种包括权利要求1至5任意一项的超灵敏纳米孔(8)结构的超灵敏纳米孔(8)芯片，其特征在于，所述的芯片包括下层基底(5)和上层含超灵敏纳米孔(8)结构的纳米薄膜(7)。
  7. 根据权利要求6所述的超灵敏纳米孔(8)芯片，其特征在于，所述的基底(5)开设有支撑孔(6)，用于支撑超灵敏纳米孔(8)，所述的支撑孔(6)的孔径在5-500nm范围内。
  8. 根据权利要求6所述的超灵敏纳米孔(8)芯片，其特征在于，所述的基底(5)包括硅层，所述的硅层的阻值高于2700 Ohmcm。
  9. 根据权利要求6所述的超灵敏纳米孔(8)芯片，其特征在于，所述的基底(5)包括从上到下的氮化硅、氧化硅、硅、氧化硅和氮化硅五层，所述的硅层厚度为300-500μm，所述的表层为绝缘层，所述的支撑孔(6)开设于表层氮化硅层上。
  10. 根据权利要求7或8或9所述的超灵敏纳米孔(8)芯片，其特征在于，所述的芯片可 实现低于20pA的背景噪声。
  11. 一种包括权利要求6-9任意一项的超灵敏纳米孔(8)芯片的超灵敏纳米孔(8)分析装置，其特征在于，所述的装置包括注满离子溶液的流体槽(9)、将流体槽(9)分为顺式侧腔室(11)和反式侧腔室(12)的超灵敏纳米孔(8)芯片、接入流体槽(9)溶液或芯片的电极(4)、与电极(4)串联的电流放大器(2)和电源(1)、与电流放大器(2)相连的数模转换设备和计算机(3)。
  12. 一种利用权利要求11所述的超灵敏纳米孔(8)分析装置制备超灵敏纳米孔(8)结构的方法，其特征在于，采用电化学刻蚀法制备纳米孔，在流体槽(9)两侧腔室内注入盐溶液，通过电源(1)和电流放大器(2)对纳米薄膜(7)直接施加0-10V范围内的电压，刻蚀得到纳米孔(8)，通过控制施加电压的大小和时长控制电化学反应，即可控制孔径的尺寸；获得纳米孔(8)跨膜电导的实时数据，当电导值达到目标孔径对应值时，停止施加电压，完成纳米孔(8)制备过程。
  13. 一种利用权利要求11所述的超灵敏纳米孔(8)分析装置用于纳米孔(8)快速氨基酸检测的方法，其特征在于，所述的纳米孔(8)的孔径在0.35-2nm的范围内，包括如下步骤：
    1)、将氨基酸样品溶于缓冲液；
    2)、在流体槽(9)溶液中加入氨基酸样品，样品在电场作用下通过纳米孔(8)；
    3)、通过电流放大器(2)记录电流穿孔图谱；
    4)、通过分析电流穿孔图谱和/或由电流穿孔图谱经过统计学分析得到的相对电流阻塞电导降和/或穿孔时间和/或捕获速率来分析检测氨基酸。
  14. 一种利用权利要求11所述的超灵敏纳米孔(8)分析装置用于纳米孔(8)快速蛋白质测序的方法，其特征在于，所述的纳米孔(8)的孔径在1-5nm的范围内，包括如下步骤：
    1)、设计并合成肽链与核苷酸链相连的复合物，引物链以及阻断链，在每次实验前将这三种物质按所需比例混合并退火，即得待测样品(10)；
    2)、在顺式侧腔室(11)中加入生物酶，脱氧核糖核苷三磷酸(dNTPs)，以及待测样品(10)；
    3)、施加电压，样品在电场作用下通过纳米孔(8)，通过电流放大器(2)记录电流穿孔图谱；
    4)、通过分析电流穿孔图谱解析蛋白质序列。
  15. 根据权利要求14所述的纳米孔(8)快速蛋白质测序的方法，其特征在于，所述的生物 酶包括phi29 DNA聚合酶、Tth DNA聚合酶、Pfu DNA聚合酶、Pfu HS DNA聚合酶、T4 DNA聚合酶、Bst DNA聚合酶、Taq DNA聚合酶、TaqⅡDNA聚合酶、Taq plus DNA聚合酶、LA Taq DNA聚合酶、VentR DNA聚合酶、Phusion DNA聚合酶、KOD DNA聚合酶、Iproof DNA聚合酶、Hel308解旋酶、T4解旋酶、DnaB解旋酶、Rep蛋白解旋酶、PcrA解旋酶、Tte UvrD解旋酶、Tth UvrD解旋酶、T4 Dda解旋酶、T4 gp41解旋酶、RecQ解旋酶、NS3 HCV解旋酶、elF4A解旋酶、WRN解旋酶、TRCF解旋酶、LTag解旋酶、E1解旋酶、T7 gp4解旋酶、Rho解旋酶、ClpX蛋白酶。
  16. 一种如权利要求6-9中任意一项所述的超灵敏纳米孔(8)芯片应用于纳米孔(8)在氨基酸检测，其特征在于，所述的纳米孔(8)的孔径在0.35-2nm的范围内，实现20种天然氨基酸的检测和区分。
  17. 一种如权利要求6-9中任意一项所述的超灵敏纳米孔(8)芯片应用于纳米孔(8)在在翻译后化学修饰氨基酸检测，其特征在于，所述的纳米孔(8)的孔径在1-5nm的范围内，实现翻译后修饰氨基酸的检测和区分。
  18. 一种如权利要求6-9中任意一项所述的超灵敏纳米孔(8)芯片应用于多肽检测，其特征在于，所述的纳米孔(8)的孔径在1-5nm的范围内，实现多肽的检测和杂多肽氨基酸序列的区分。
  19. 一种如权利要求6-9中任意一项所述的超灵敏纳米孔(8)芯片应用于蛋白质测序，其特征在于，所述的纳米孔(8)的孔径在1-5nm的范围内，实现蛋白质测序。
PCT/CN2023/088193 2022-07-29 2023-04-13 一种超灵敏纳米孔结构及芯片及分析装置及氨基酸和蛋白质检测方法及应用 WO2024021686A1 (zh)

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