WO2021208936A1 - 纳米孔制备和检测方法及其检测装置 - Google Patents

纳米孔制备和检测方法及其检测装置 Download PDF

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WO2021208936A1
WO2021208936A1 PCT/CN2021/087118 CN2021087118W WO2021208936A1 WO 2021208936 A1 WO2021208936 A1 WO 2021208936A1 CN 2021087118 W CN2021087118 W CN 2021087118W WO 2021208936 A1 WO2021208936 A1 WO 2021208936A1
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nanopore
residence time
analyte
signal detection
detection area
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English (en)
French (fr)
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邹耀中
苏云鹏
江鹏
王艳
李玉琴
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成都今是科技有限公司
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Priority to US17/996,295 priority Critical patent/US20230194499A1/en
Priority to CN202180028157.3A priority patent/CN115398008A/zh
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • 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 present disclosure belongs to the field of gene sequencing, and specifically relates to a nanopore preparation and detection method and a detection device thereof.
  • Nanopores are a type of ion channel with nanometer scale. When a voltage is applied across the nanopore, the electrolyte ions in the solution will form a current through the nanopore under the action of the electric field driving force. Some small molecular substances can interact with nanopores, usually by entering the nanopore channel and staying in it for a short time, thereby affecting the size of the current passing through the nanopore and generating a characteristic blocking current. Using the characteristic to block the current signal, the nanopore can be used as a single-molecule detector in different scenarios, including the study of the mechanism of chemical reactions and the sequence determination of nucleic acids.
  • nanopores still have great limitations as single-molecule detectors.
  • the analyte that can be detected by the nanopore in addition to meeting the requirements of certain size and charge characteristics, also needs to interact with the nanopore for a long enough time, and its corresponding characteristic blocking current signal can be effectively captured and detected by the electronic component.
  • the existing various nanopores used for nucleic acid sequencing including ⁇ -bacterial toxins, protein nanopores such as MspA and CsgG, or solid artificial nanopores made of graphene and other materials, are difficult to detect directly at the single-molecule level. Signal to nucleotides.
  • the speed of passing through the nanopore is very fast, and the interaction time with the nanopore is usually only a few microseconds or less. .
  • the nucleotide produces a blocking current signal, the too short signal duration is far beyond the detection limit of all electronic components.
  • An existing improvement scheme obtains the ability to detect single-molecule nucleotides by covalently modifying ⁇ -cyclodextrin at specific positions of ⁇ -bacterial toxins.
  • the scheme of using cyclodextrin to covalently modify nanopores has two obvious shortcomings: 1. Additional processes are required to prepare ⁇ -cyclodextrin that can be used to modify ⁇ -bacterial toxins, and because the modification sites are in the nanopores Within the pore size, it is difficult to guarantee the efficiency of chemical reactions, and it is difficult to effectively separate modified and unmodified nanopores, which affects subsequent detection; 2.
  • the pore size of modified nanopores is reduced from 1.4nm to 0.8nm, and the opening current is also reduced accordingly.
  • Chinese invention patent CN102317310B proposes a method to enhance the translocation of charged analytes through transmembrane protein pores.
  • This patent increases the positive charge inside the nanopore, increases the frequency of nucleic acid strands translocating through the pore, and reduces The threshold voltage for the nucleic acid strand to translocate through the pore, and at the same time, the solution can reduce the translocation speed of the nucleic acid strand to translocate through the pore, and increase the residence time of the analyte in the nanopore.
  • the positive charge sites introduced in the nanopore in the patent are dispersed in various positions in the nanopore. The main purpose is to increase the frequency of analyte displacement and achieve the capture efficiency of the analyte by the nanopore.
  • the data or examples show that the nanopore modified by it can have the ability to detect single-molecule nucleotides.
  • the embodiments of the present disclosure provide a nanopore preparation and detection method and a detection device thereof, which are used to realize effective detection of single-molecule analytes and improve detection accuracy.
  • embodiments of the present disclosure provide a method for preparing nanopores, including:
  • a plurality of protein monomers are aggregated to form a nanopore, wherein the narrow part of the channel of the nanopore forms the signal detection area of the nanopore;
  • a positively charged cluster is formed in the signal detection area of the nanopore, and the charge interaction between the positively charged cluster and the negatively charged single-molecule analyte passing through the nanopore can prolong the presence of the single-molecule analyte. State the residence time in the nanopore.
  • the forming positively charged clusters in the signal detection area of the nanopore includes: introducing positively charged amino acid residues into the signal detection area of the nanopore by protein engineering to form the Positively charged clusters.
  • amino acid residues include: lysine, arginine or histidine.
  • the forming positively charged clusters in the signal detection area of the nanopore includes: introducing a positively charged unnatural amino acid into the signal detection area of the nanopore by biochemical means to form the Positively charged clusters.
  • the protein monomer includes any one of ⁇ -hemolysin, MspA, CsgG, and OmpF.
  • the single-molecule analyte includes nucleotides with different numbers of phosphates.
  • nanopore detection device including:
  • test cavity with nanopores and containing an electrolyte solution
  • the detection circuit connected to the test cavity; wherein the narrow part of the nanopore channel forms the signal detection area of the nanopore, and the signal detection area has positively charged clusters.
  • the number of charges of the positively charged clusters can be controlled to adjust the residence time of the single-molecule analyte in the nanopore.
  • the driving voltage of the nanopore can be controlled to adjust the residence time of the single-molecule analyte in the nanopore.
  • the concentration of the electrolyte solution can be controlled to adjust the residence time of the single-molecule analyte in the nanopore.
  • the nanopore includes a plurality of protein monomers.
  • the protein monomer includes any one of ⁇ -hemolysin, MspA, CsgG, and OmpF.
  • the single-molecule analyte includes nucleotides with different numbers of phosphates.
  • embodiments of the present disclosure provide a nanopore detection method, which is applied to the nanopore detection device described in the foregoing embodiment, and includes:
  • the number of positive charge clusters in the signal detection area of the nanopore is controlled to adjust the residence time of the negatively charged single-molecule analyte in the nanopore when it passes through the nanopore.
  • control of the charge number of the positively charged clusters in the signal detection area of the nanopore is used to adjust the residence time of the negatively charged single-molecule analyte in the nanopore when passing through the nanopore include:
  • the number of positive charge clusters in the signal detection area of the nanopore is increased to extend the residence time of the negatively charged single-molecule analyte in the nanopore when it passes through the nanopore.
  • the driving voltage of the nanopore is controlled to adjust the residence time of the negatively charged single molecule analyte in the nanopore when passing through the nanopore.
  • controlling the driving voltage of the nanopore to adjust the residence time of the negatively charged single-molecule analyte in the nanopore when passing through the nanopore includes:
  • the driving voltage of the nanopore is reduced to prolong the residence time of the negatively charged single-molecule analyte in the nanopore when passing through the nanopore.
  • the concentration of the electrolyte solution is controlled to adjust the residence time of the negatively charged single-molecule analyte in the nanopore when it passes through the nanopore.
  • controlling the concentration of the electrolyte solution to adjust the residence time of the negatively charged single-molecule analyte in the nanopore when passing through the nanopore includes:
  • the concentration of the electrolyte solution is reduced to prolong the residence time of the negatively charged single-molecule analyte in the nanopore when it passes through the nanopore.
  • the embodiment of the present disclosure introduces positive charge clusters in the signal detection area of the nanopore, and through the interaction between the charges, the interaction time between the negatively charged single-molecule analyte and the nanopore is significantly prolonged, so that the signal detection area can detect The characteristic blocking current signal can be accurately detected by electronic components.
  • the embodiments of the present disclosure have at least the following beneficial effects: 1.
  • the effective detection of single-molecule nucleotides is realized at the single-molecule level, especially in scenarios related to nucleic acid sequencing. , Significantly improve the detection rate of single-molecule analytes; 2.
  • the nanopore prepared in the present disclosure is used in a detection device, and the duration of the detection signal can be flexibly adjusted according to the number of positive charge clusters, the driving voltage, and the concentration of the electrolyte solution.
  • the detection device can be adapted to different analytes and detection requirements, and has a wider application prospect.
  • 1A and 1B are a side view and a top view of a three-dimensional model of R4 nanopore prepared according to an embodiment of the present disclosure
  • Fig. 2 is a schematic structural diagram of a nanopore detection device according to an embodiment of the present disclosure
  • Figure 3A is a schematic diagram of SDS electrophoresis detection results of R4 nanopores
  • Figure 3B is a schematic diagram of the detection results of R4 nanopore molecular sieve
  • Figure 4A is a graph of the detection signal of the R4 nanopore under the conditions of a voltage of 100mV and a KCl solution of 400mM for nucleotide triphosphates;
  • Figure 4B is a graph of the detection signal of natural ⁇ -hemolysin for nucleotide triphosphate under the same conditions
  • Figure 4C is a statistical analysis of the signal of nucleotide triphosphates detected by the R4 nanopore
  • Figure 4D is a graph of the detection signal of monophosphate nucleotides by R4 nanopore
  • Figure 5A is a graph of the detection signal of the R5 nanopore under the conditions of a voltage of 100mV and a 300mM KCl solution for nucleotide triphosphates;
  • Figure 5B is a graph of the detection signal of the R4 nanopore for nucleotide triphosphate under the same conditions
  • Figures 6A-6C are graphs of the detection signal of nucleotide triphosphates at the concentration of 1M KCl, 500 mM KCl, and 300 mM KCl in the R4 nanopore;
  • Figures 7A-7F are graphs of the detection signals of the R4 nanopore at voltages of 180mV, 160mV, 140mV, 120mV, 100mV and 80mV, respectively.
  • the existing nanopores are used in the detection of analytes, and their ability to detect single-molecule analytes is insufficient.
  • the effective detection of nucleotides at the single-molecule level is of great significance to the development of new nucleic acid sequencing solutions or to improve the accuracy of existing sequencing solutions.
  • the embodiment of the present disclosure first proposes a nanopore preparation method, which adopts the aggregation of a plurality of protein monomers to form a nanopore, wherein the narrow part of the channel of the nanopore forms the signal detection area of the nanopore;
  • the signal detection area of the signal detection area forms a positively charged cluster, and the charge interaction between the positively charged cluster and the negatively charged single molecular analyte passing through the nanopore can prolong the single molecular analyte in the nanopore.
  • Residence time forms positively charged clusters in the signal detection site (constriction site) of the nanopore.
  • the negatively charged monomolecular analyte When the negatively charged monomolecular analyte enters the nanopore under the action of an electric field, it passes between the positively charged cluster and the negatively charged monomolecular analyte.
  • the charge interaction can significantly extend the residence time of the single-molecule analyte in the nanopore, thereby increasing the duration of the nanopore blocking characteristic current signals.
  • the residence time of a single-molecule analyte passing through the nanopore is significantly prolonged, and the residence time can reach milliseconds, so that the characteristic blocking current signal generated by the nanopore It can be effectively captured by electronic components, so that single-molecule analytes that cannot be detected because of too short residence time can be effectively detected, which improves the detection rate of single-molecule analytes.
  • the nanopore includes a protein nanopore.
  • Optional protein nanopores include, but are not limited to, the bacterial toxin ⁇ -hemolysin, MspA, CsgG, OmpF, and the like. These protein nanopores are widely used in molecular detection, including many scenes of sequencing, and have a known high-resolution three-dimensional structure, which can easily locate the signal detection area and nearby amino acid residues. Therefore, the introduction of positively charged mutations into the signal detection regions of these nanopores by genetic engineering means has a high degree of targeting and feasibility.
  • single-molecule analytes may include, but are not limited to, nucleotides with different numbers of phosphates.
  • Bacterial toxin ⁇ -hemolysin is a natural protein monomer, containing 293 amino acids, and its protein primary structure sequence is as follows:
  • FIG. 1A and 1B The side view and top view of the three-dimensional model structure are shown in Figures 1A and 1B, respectively.
  • the spherical model in Figures 1A and 1B represents the narrowest part of the nanopore channel composed of 7 monomers of E111, M113, and K147, which becomes the signal detection site for the characteristic blocking current caused by the analyte. .
  • mutations of positively charged amino acid residues are introduced in and near the signal detection region, for example including but not limited to: E111R, M113R, T115R, G143R, or any combination of these mutations .
  • the nanopores formed by the mutant strains will form positive charge clusters with high positive charge density near the signal detection area.
  • the positive charge mutation on each protein monomer corresponds to 7 additional positive charges on the nanopore.
  • the amino acid residue may include: lysine (Lys), arginine (Arg), or histidine (His) protonated at low pH.
  • mutant strains with multiple mutation sites such as E111R, M113R, T115R, G143R, K147R, can be prepared using the same preparation methods as wild-type proteins to obtain high-purity positively charged clusters of heptamer Body nanopores.
  • hybrid multimers it is also possible to mix protein monomers with corresponding positive charge mutations and protein monomers without mutations to form hybrid multimers, so that the positive charge of the nanopore signal detection area can be adjusted more finely. Density, rather than just adding 7 positive charges for each mutation introduced. For example, a hybrid heptamer formed by 3 wild-type ⁇ -hemolysin monomers and 4 monomers with M113R mutations will carry 4 positive charges at position 113 in the nanopore.
  • unnatural amino acids can also be introduced into the signal detection area of the nanopore through specific biochemical means.
  • the unnatural amino acid can be different from the natural lysine (Lys), arginine (Arg) or histidine (His), but it can carry a different number of charges. Nanopores have more abundant and adjustable positive charge clusters.
  • the embodiment of the present disclosure also provides a nanopore detection device.
  • the modified nanoporins can be embedded in specific phospholipid bilayer membranes or artificial bilayer membranes with similar physical and chemical properties to form a nanopore detection device as shown in FIG. 2.
  • the modified nanoporins are purified by biochemical methods similar to wild nanopores, and can retain their biochemical activity.
  • the purified nanoporins can be spontaneously embedded in phospholipid bilayer membranes or artificial bilayer membranes due to their lipophilic properties.
  • the nanopore detection device includes a test cavity embedded in the prepared nanopore and a micro current detection circuit connected with the test cavity.
  • the test chamber is a first compartment and a second compartment separated by a phospholipid double-layer membrane or an artificial double-layer membrane.
  • the first and second compartments contain an electrolyte solution, and the analyte is located in the electrolyte solution of the first compartment
  • negatively charged analytes such as but not limited to nucleotide monophosphate (NMP or dNMP), nucleotide diphosphonate (NDP or dNDP), Nucleotide triphosphates (NTP or dNTP), nucleotide polyphosphates (with 4 or more phosphate groups), and some nucleotide derivatives pass through the nanopore from one side of the bilayer membrane into the other The second compartment on one side.
  • the negatively charged analyte is attracted and captured by the positive charge clusters introduced in the signal detection area of the nanopore, thereby staying for a longer time, allowing the characteristic blocking current signal generated by it to be effective by the micro current detection circuit capture.
  • the nanopore detection device of the embodiment of the present disclosure can use the electrodes at both ends of the test cavity to apply voltage, such as 50-300mV, including but not limited to 75-275mV, 100-250mV, 125-225mV, 150-200mV, or 100mV, 125mV, 150mV , 175mV, 200mV, which will form a potential gradient across the membrane along the inner diameter of the nanopore.
  • the electrolyte moves directionally in the nanopore under the drive of the electric potential gradient to form an electric current.
  • negatively charged analytes including nucleotide molecules with different numbers of phosphoric acid, approach the opening of the nanopore under the dual effects of free diffusion and electrophoretic motion, they will be captured by the potential gradient along the nanopore.
  • the passing speed of nucleotide molecules is in the range of 1-10 ⁇ s, and the blocking current signal caused is only in the pA level. .
  • Such weak signal strength and short signal duration cannot support the effective detection of electronic components.
  • the nanopore detection device of the embodiment of the present disclosure introduces positive charge clusters in the nanoporins.
  • the high density of positive charge clusters can effectively slow down the speed of negatively charged analytes passing through the nanopores, prolong the duration of the signal, and make The characteristic blocking current signal of the single-molecule analyte that could not be detected is effectively captured, so as to obtain the detection ability of the single-molecule analyte.
  • nanopores that have the ability to detect single-molecule nucleotides can be used in different detection scenarios, including sequencing or assisting in improving sequencing accuracy.
  • the phospholipids constituting the phospholipid bilayer membrane may include 1,2-bisphytanyl-sn-glycero-3-phosphocholine (DPhPC).
  • DPhPC 1,2-bisphytanyl-sn-glycero-3-phosphocholine
  • the implementation of the present disclosure is not limited to specific phospholipids, including but not limited to other choline phospholipids or artificial amphiphilic molecules, as long as the electrolyte solution pool can be divided into two parts with electrodes and insulated from each other, and can support The structure of the nanoporin forms a stable current channel, which can be used in the embodiments of the present disclosure.
  • the embodiment of the present disclosure also proposes a nanopore detection method suitable for the aforementioned nanopore detection device.
  • the method controls the number of positive charge clusters in the signal detection area of the nanopore to adjust the residence time of the negatively charged single-molecule analyte in the nanopore when it passes through the nanopore.
  • increasing the number of positive charge clusters in the signal detection area of the nanopore can significantly extend the corresponding signal duration of the analyte passing through the nanopore. For example, if one more positive charge is added to the preferred position of the monomer of ⁇ -hemolysin, seven positive charges are correspondingly added to the heptameric nanopore. The ability of more charged positively charged clusters to capture negatively charged analytes is significantly enhanced. In some embodiments, the time for the nucleotide triphosphate to pass through the more positively charged nanopore can be further extended by nearly an order of magnitude.
  • the method can further control the driving voltage of the nanopore to adjust the residence time of the negatively charged monomolecular analyte in the nanopore when passing through the nanopore.
  • reducing the driving voltage of the nanopore can prolong the residence time of the negatively charged single-molecule analyte in the nanopore when passing through the nanopore. For example, if the driving voltage is reduced from 180mV to 120mV, 100mV, or 80mV, the driving force of the electric field on negatively charged analytes, such as nucleotides, will be significantly reduced, and the time it takes to pass through the nanopore will increase significantly. In some embodiments, the driving voltage is reduced from 180 mV to 80 mV, and the corresponding signal duration of the nucleotide passing through the nanopore can be greatly extended from less than 100 ⁇ s to more than 1 ms, thereby realizing more effective detection of the analyte.
  • the method can further control the concentration of the electrolyte solution of the detection device to adjust the residence time of the negatively charged monomolecular analyte in the nanopore when it passes through the nanopore.
  • reducing the concentration of the electrolyte solution of the detection device can prolong the residence time of the negatively charged monomolecular analyte in the nanopore when it passes through the nanopore.
  • potassium chloride solution is an electrolyte solution commonly used in nanopore detection.
  • the concentration of the potassium chloride solution has a very significant effect on the duration of the analyte passing through the nanopore.
  • simply reducing the potassium chloride concentration from 1 M to 300 mM is sufficient to extend the time for nucleotide triphosphates to pass through the nanopore by more than an order of magnitude.
  • the nanopore detection method of the embodiments of the present disclosure demonstrates the effective adjustment ability of the target signal duration of the analyte, thereby enriching the detection means and corresponding signal characteristics, so that the nanopore can be used for analytes represented by single molecule nucleotides. Detection can be applied to more different scenarios to adapt to different analytes and detection needs.
  • the natural ⁇ -hemolysin gene was obtained by direct synthesis according to the sequence NC_007795.1 included in Genebank.
  • the synthesized cDNA sequence is as follows:
  • the synthesized ⁇ -hemolysin was directly cloned into the expression vector pET26b.
  • Agilent quick change kit to introduce site-directed mutations in the above genes
  • a mutant strain with four mutation sites E111R/M113R/T115R/K143R was prepared and named R4 nanopore.
  • the structure of the R4 nanopore is shown in Figure 1. All mutation sites are concentrated near the signal detection site of ⁇ -hemolysin.
  • the R4 nanopore can be obtained in the same way as the natural ⁇ -hemolysin nanopore.
  • the SDS electrophoresis detection result of the heptamer is shown in Figure 3A, and the molecular sieve detection result is shown in Figure 3B. Wherein, lane 1 in FIG.
  • lane 3A indicates the standard molecular weight marker
  • lane 2 indicates that a few heptamers of the R4 mutant strain are dissociated into monomers during electrophoresis
  • lane 3 indicates the wild-type ⁇ -bacterial toxin.
  • the upper side is the molecular sieve detection result of the purified R4 nanopore
  • the lower side is the comparison of the wild-type ⁇ -bacterial toxin molecular sieve map.
  • a mutant strain E111R/M113R/T115R/G143R/K147R with a larger number of positive charges can also be prepared, which can be named R5 nanopore.
  • a compartment system that meets the requirements of the test chamber can be selected, for example, a compartment system with an opening between the left and right compartments of 50 microns in size, and the vicinity of the opening is suitable for phospholipid attachment Hydrophobic material.
  • DphPC 1,2-diphytanoyl-sn-glycero-3-phosphocholine
  • This phospholipid solution is added to the existing KCl solution in the compartment to form an organic phase covering the KCl liquid surface.
  • the phospholipid membrane with R4 nanoporin can be prepared and assembled into the nanopore detection device of the embodiment of the disclosure.
  • the driving voltage was set to 180 mV, and the opening current through the R4 nanopore was recorded to be ⁇ 106 pA.
  • the opening current of ⁇ 110pA there is no significant difference in the opening current of ⁇ 110pA, indicating that after deep modification, 28 positively charged R4 proteins are introduced near the signal detection area, which still retains the natural ⁇ -hemolysis Element similar current signal detection window.
  • the opening current is reduced to about 40% of that of unmodified nanopores, which is only ⁇ 45pA.
  • Example 2 The capture of different phosphoric acid nucleotides by R4 nanopore
  • FIG. 4A shows the detection signal of the R4 mutant strain on the nucleotide triphosphate under the conditions of 100 mV voltage and 400 mM KCl solution. It can be seen that dCTP interacts with the R4 nanopore, giving a clear blocking current signal.
  • FIG. 4B in a nanopore detection device composed of natural ⁇ -hemolysin, dCTP is added to the first compartment, and the system cannot detect an effective dCTP blocking current signal.
  • FIG. 4D shows the detection signal of the R4 mutant strain for nucleotide monophosphate. Because the analyte is less charged, the signal duration is significantly shorter than the corresponding signal duration of nucleotide triphosphate.
  • the R4 nanopore was replaced with the R5 nanopore in the same way, and the blocking current generated by the R5 nanopore on the capture of nucleotide triphosphate dCTP was recorded under the same conditions.
  • Figure 5A shows the nucleotide triphosphate signal detected by the R5 nanopore under the conditions of 100 mV voltage and 300 mM KCl solution
  • Figure 5B shows the detection signal of the R4 nanopore under the same conditions. Because the R5 nanopore introduces more positive charges near the signal detection area, its interaction with dCTP is significantly increased, and the duration of the blocking current signal generated by dCTP is significantly increased. It can be seen that the capture and detection of nucleotide triphosphates by the more positively charged R5 mutants has a significantly longer signal duration than the signal detected by the R4 nanopore, and the blocking current amplitude is also deeper.
  • Example 4 The effect of adjusting salt concentration on the duration of dCTP signal
  • FIG. 6A-6C show the signal detection results of dCTP at the concentration of 1M KCl, 500 mM KCl, and 300 mM KCl under the voltage of 100 mV for the R4 nanopore.
  • the results show that the signal duration of dCTP is inversely related to the salt concentration. The lower the salt concentration, the longer the signal duration.
  • Example 5 The influence of adjusting the driving voltage on the duration of the dCTP signal
  • FIGS. 7A-7F respectively show the voltages of 180mV, 160mV, 140mV, 120mV, 100mV and 80mV at 300mM salt concentration, and the influence of different voltages on the signal of R4 nanopore detecting nucleotide triphosphate.
  • the results show that the signal duration is very sensitive to the driving voltage. The lower the voltage, the longer the signal duration. When ⁇ 100mV, the signal duration can be as long as several milliseconds or even tens of milliseconds. When it is greater than 150mV, the signal duration is already very short, with only sporadic signal spikes.

Abstract

提供一种纳米孔制备和检测方法及其检测装置。该方法采用多个蛋白单体聚集形成纳米孔，其中纳米孔的通道狭窄部分形成所述纳米孔的信号检测区域；在所述纳米孔的信号检测区域形成正电荷簇，所述正电荷簇与通过所述纳米孔的带负电荷的单分子分析物之间的电荷相互作用能够延长所述单分子分析物在所述纳米孔内的停留时间。实现了在单分子水平上对分析物的有效检测，使得因为和纳米孔相互作用时间太短而无法产生有效检测信号的单分子分析物可以被有效检测，显著提高单分子分析物的检出率，并且适用不同的分析物和检测需求。

Description

纳米孔制备和检测方法及其检测装置 技术领域
本公开属于基因测序领域，具体而言，涉及一种纳米孔制备和检测方法及其检测装置。
背景技术
纳米孔是一类具有纳米尺度的离子通道。在纳米孔两端施加电压时，溶液中的电解质离子会在电场驱动力的作用下通过纳米孔形成电流。一些小分子物质能与纳米孔相互作用，通常是以进入纳米孔通道并在其中短暂停留的方式，从而影响通过纳米孔的电流大小，产生特征阻断电流。利用特征阻断电流信号，纳米孔可以作为一种单分子水平上的检测器应用于不同场景中，包括化学反应的机制研究和核酸的序列测定。
然而，纳米孔作为单分子检测器仍旧存在很大的局限性。能被纳米孔所检测到的分析物，除了满足一定的尺寸和电荷特性的要求外，还需要与纳米孔相互作用的时间足够长，其相应的特征阻断电流信号才能被电子元件有效捕获检测。现有的用于核酸测序的各种纳米孔，包括α-细菌毒素，MspA和CsgG等蛋白纳米孔或者是由石墨烯等材料制备的固态人工纳米孔，都很难在单分子水平上直接检测到核苷酸的信号。无论是单磷酸，二磷酸还是三磷酸的核苷酸分子，在电场驱动力的作用下通过纳米孔的速度都非常快，和纳米孔相互作用的时间通常只有几个微秒甚至更短的时间。在此过程中核苷酸即使产生了阻断电流信号，过于短暂的信号持续时间也远远落在了一切电子元件的检测极限之外。
现有的一种改进方案通过在α-细菌毒素的特定位置共价修饰β-环糊精的方式，获得了对单分子核苷酸的检测能力。然而，利用环糊精共价修饰纳米孔的方案有两方面的明显缺陷：1、需要额外的工艺制备可用于修饰α-细菌毒素的β-环糊精，而且由于修饰位点在纳米孔的孔径内部，化学反应的效率难以保证，修饰成功和未修饰的纳米孔也难以有效分离，影响后续检测；2、修饰之后的纳米孔的孔径从1.4nm缩小为0.8nm,开孔电流也相应减小到未修饰时候的40％左右，极大地缩小了特征阻断电流的信号窗口，也缩小了纳米孔的检测范围，并且对于罗氏的测序系统而言，由于核苷酸带有标 签，过于狭窄的纳米孔孔径很可能造成标签堵塞纳米孔，使得整个系统无法正常工作。
中国发明专利CN102317310B提出了一种增强荷电分析物穿过跨膜蛋白孔的移位的方法，该专利通过在纳米孔内部增加正电荷，增加核酸链移位穿过所述孔的频率，降低所述核酸链移位穿过所述孔的阈值电压，同时该方案可以降低核酸链移位穿过所述孔的移位速度，增加了分析物在纳米孔中的停留时间。但是，该专利在纳米孔中引入的正电荷位点是分散在纳米孔内各个位置，主要目的是为了增加分析物的移位频率，实现纳米孔对分析物的捕获效率，并且并未有任何数据或者实施例展示它所改造的纳米孔可以具有检测到单分子核苷酸的能力。
发明内容
本公开实施例提供一种纳米孔制备和检测方法及其检测装置，用于实现对单分子分析物的有效检测，提高检测准确率。
第一方面，本公开实施例提供一种纳米孔制备方法，包括：
采用多个蛋白单体聚集形成纳米孔，其中所述纳米孔的通道狭窄部分形成所述纳米孔的信号检测区域；
在所述纳米孔的信号检测区域形成正电荷簇，所述正电荷簇与通过所述纳米孔的带负电荷的单分子分析物之间的电荷相互作用能够延长所述单分子分析物在所述纳米孔内的停留时间。
在可选的实施方式中，所述在所述纳米孔的信号检测区域形成正电荷簇包括：通过蛋白质工程在所述纳米孔的信号检测区域引入带有正电荷的氨基酸残基，形成所述正电荷簇。
在可选的实施方式中，所述氨基酸残基包括：赖氨酸、精氨酸或组氨酸。
在可选的实施方式中，所述在所述纳米孔的信号检测区域形成正电荷簇包括：通过生化手段在所述纳米孔的信号检测区域引入带有正电荷的非天然氨基酸，形成所述正电荷簇。
在可选的实施方式中，所述蛋白单体包括α-溶血素、MspA、CsgG、OmpF中任意一种。
在可选的实施方式中，所述单分子分析物包括带有不同磷酸数目的核苷酸。
第二方面，本公开实施例提供一种纳米孔检测装置，包括：
具有纳米孔、容置有电解质溶液的测试腔；以及
连接至所述测试腔的检测电路；其中，所述纳米孔的通道狭窄部分形成所述纳米孔的信号检测区域，所述信号检测区域具有正电荷簇，当电解质溶液中的带负电荷的单分子分析物在纳米孔的驱动电压的作用下通过所述纳米孔时，所述正电荷簇与所述带负电荷的单分子分析物之间的电荷相互作用能够延长所述单分子分析物在所述纳米孔内的停留时间。
在可选的实施方式中，所述正电荷簇的电荷数目能够被控制来调节所述单分子分析物在所述纳米孔内的停留时间。
在可选的实施方式中，所述纳米孔的驱动电压能够被控制来调节所述单分子分析物在所述纳米孔内的停留时间。
在可选的实施方式中，所述电解质溶液的浓度能够被控制来调节所述单分子分析物在所述纳米孔内的停留时间。
在可选的实施方式中，所述纳米孔包括多个蛋白单体。
在可选的实施方式中，所述蛋白单体包括α-溶血素、MspA、CsgG、OmpF中任意一种。
在可选的实施方式中，所述单分子分析物包括带有不同磷酸数目的核苷酸。
第三方面，本公开实施例提供一种纳米孔检测方法，应用于如前述实施方式所述的纳米孔检测装置，包括：
控制纳米孔的信号检测区域的正电荷簇的电荷数目，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
在可选的实施方式中，所述控制纳米孔的信号检测区域的正电荷簇的电荷数目，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间包括：
增加所述纳米孔的信号检测区域的正电荷簇的电荷数目，来延长带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
在可选的实施方式中，还包括：
控制所述纳米孔的驱动电压，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
在可选的实施方式中，所述控制所述纳米孔的驱动电压，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间包括：
减小所述纳米孔的驱动电压，来延长带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
在可选的实施方式中，还包括：
控制所述电解质溶液的浓度，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
在可选的实施方式中，所述控制所述电解质溶液的浓度，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间包括：
降低所述电解质溶液的浓度，来延长带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
本公开实施例在纳米孔的信号检测区域引入正电荷簇，通过电荷之间的相互作用，显著延长带负电荷的单分子分析物与纳米孔之间的相互作用时间，使得信号检测区域检测出的特征阻断电流信号可以被电子元件准确检测。与现有的方案相比，本公开实施例至少具有以下有益效果：1、实现了在单分子水平上对分析物，特别是在和核酸测序相关的场景中对单分子核苷酸的有效检测，显著提高单分子分析物的检出率；2、本公开制备的纳米孔用于检测装置中，可以灵活根据正电荷簇的电荷数量、驱动电压、电解质溶液浓度来调控检测信号的持续时间，使得检测装置可以适用不同的分析物和检测需求，具有更加广泛的应用前景。
附图说明
为了更清楚地说明本公开实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施例，对于本领域普通技术人员来说，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1A和1B是根据本公开实施例制备的R4纳米孔的三维模型的侧视图和俯视图；
图2是根据本公开实施例的纳米孔检测装置的结构示意图；
图3A是R4纳米孔的SDS电泳检测结果示意图；
图3B是R4纳米孔的分子筛检测结果示意图；
图4A是R4纳米孔在100mV电压、400mM的KCl溶液条件下对三磷酸核苷酸的检测信号图；
图4B是天然的α-溶血素在同样条件下对三磷酸核苷酸的检测信号图；
图4C是对R4纳米孔检测到的三磷酸核苷酸的信号统计分析；
图4D是R4纳米孔对单磷酸核苷酸的检测信号图；
图5A是R5纳米孔在100mV电压、300mM KCl溶液条件下对三磷酸核苷酸的检测信号图；
图5B是R4纳米孔在同样条件下对三磷酸核苷酸的检测信号图；
图6A-6C是R4纳米孔分别在1M KCl、500mM KCl、300mM KCl溶液浓度下对三磷酸核苷酸的检测信号图；
图7A-7F是R4纳米孔分别在电压180mV、160mV、140mV、120mV、100mV和80mV下对三磷酸核苷酸的检测信号图。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将结合本公开实施例中的附图，对本公开实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例是本公开的一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
在本公开中，应理解，诸如“包括”或“具有”等的术语旨在指示本说明书中所公开的特征、数字、步骤、行为、部件、部分或其组合的存在，并且不欲排除一个或多个其他特征、数字、步骤、行为、部件、部分或其组合存在或被添加的可能性。
如前所述，现有的纳米孔用于分析物的检测中，对单分子分析物的检测能力存在不足。特别地，在单分子水平上对核苷酸的有效检测，对开发新的核酸测序方案或者提高现有测序方案的准确率有着重要意义。
本公开实施例首先提出一种纳米孔制备方法，该方法采用多个蛋白单体聚集形成纳米孔，其中所述纳米孔的通道狭窄部分形成所述纳米孔的信号检测区域；在所述纳米孔的信号检测区域形成正电荷簇，所述正电荷簇与通过所述纳米孔的带负电荷的单分子分析物之间的电荷相互作用能够延长所述单分子分析物在所述纳米孔内的停留时间。该方法在纳米孔的信号检测区域(constriction site)形成正电荷簇，当带负电荷的单分子分析物在电场作用下进入纳米孔，通过正电荷簇与带负电荷的单分子分析物之间的电荷相互作用，可以显著延长单分子分析物在所述纳米孔内的停留时间，从而提 高纳米孔阻断特征电流信号的持续时间。该方法制备的具有这种特征的纳米孔用于检测装置时，单分子分析物通过纳米孔的停留时间被显著延长，该停留时间可以达到毫秒级，从而使得纳米孔产生的特征阻断电流信号可以有效地被电子元件捕获到，使得原本因为停留时间太短而无法被检测的单分子分析物可以被有效检测，提高单分子分析物的检出率。
在一些实施方式中，所述纳米孔包括蛋白纳米孔。可选的蛋白纳米孔包括但不限于细菌毒素α-溶血素(α-hemolysin)、MspA、CsgG、OmpF等。这些蛋白纳米孔被广泛用于分子检测，包括测序的众多场景，并且具有已知的高分辨率三维结构，可以很方便定位其信号检测区域及附近的氨基酸残基。因此，通过基因工程手段在这些纳米孔的信号检测区域引入正电荷突变具有高度的靶向性和可行性。
在一些实施方式中，单分子分析物可以包括但不限于带有不同磷酸数目的核苷酸。
以细菌毒素α-溶血素的纳米孔为例。细菌毒素α-溶血素属于天然的蛋白单体，含有293个氨基酸，其蛋白质一级结构序列如下：
Figure PCTCN2021087118-appb-000001
在去垢剂、磷脂或其他两性分子存在的条件下，七个α-溶血素的蛋白单体会自发聚集形成纳米孔，其三维模型结构的侧视图和俯视图分别如图1A和1B所示。其中，图1A和1B中由球状模型表示的是由7个单体的E111、M113和K147组成的纳米孔通道的最狭窄部分，成为分析物引起特征阻断电流的信号检测区域(constriction site)。
在一些实施方式中，通过蛋白质工程的方式，在该信号检测区域及附近引入带正电荷的氨基酸残基的突变，例如包括但不限于：E111R、M113R、T115R、G143R，或者这些突变的任意组合，突变株形成的纳米孔则会在信号检测区域附近形成高正电荷密度的正电荷簇。每一个蛋白单体上的正电荷突变，对应着纳米孔上的7个额外的正电荷。在一些实施方式中，该氨基酸残基可以包括：赖氨酸(Lys)、精氨酸(Arg)或低pH下质子化的组氨酸(His)。在一些实施方式中，带有多个突变位点的突变株， 如E111R、M113R、T115R、G143R、K147R，可以用与野生型蛋白同样的制备手段，获得高纯度的带正电荷簇的七聚体纳米孔。
在另一些实施方式中，还可以采用带有相应正电荷突变的蛋白单体与不带突变的蛋白单体混合形成杂合多聚体，从而可以更精细的调节纳米孔信号检测区域的正电荷密度，而不仅仅是每引入一个突变则增加7个正电荷。例如，3个野生型的α-溶血素单体和4个带有M113R突变的单体形成的杂合七聚体，将在纳米孔内113位点处带有4个正电荷。
在另一些实施方式中，也可以通过特定的生化手段在纳米孔的信号检测区域引入非天然氨基酸。非天然氨基酸可以不同于天然的赖氨酸(Lys)、精氨酸(Arg)或组氨酸(His)，但可以带有不同的电荷个数，从而在引入到蛋白单体上所形成的纳米孔具有电荷数更加丰富、可以调节的正电荷簇。
本公开实施例还提出一种纳米孔检测装置。在完成带有正电荷簇的纳米孔的制备以后，改造后的纳米孔蛋白可以嵌入特定的磷脂双层膜或理化性质类似的人工双层膜，形成如图2所述的纳米孔检测装置。改造后的纳米孔蛋白采用和野生纳米孔相似的生化手段进行纯化，并可以很好的保留其生化活性。纯化制备完毕的纳米孔蛋白由于自身的亲脂属性，可以自发的嵌入磷脂双层膜或人工双层膜。
该纳米孔检测装置包括嵌入所制备纳米孔的测试腔和与之连接的微电流检测电路。测试腔由磷脂双层膜或人工双层膜分隔的第一隔室和第二隔室，第一隔室和第二隔室中容置有电解质溶液，分析物位于第一隔室的电解质溶液中，在施加在测试腔两端的电极的电压作用下，带有负电荷的分析物，例如但不限于单磷酸核苷酸(NMP或dNMP)、双膦酸核苷酸(NDP或者dNDP)、三磷酸核苷酸(NTP或者dNTP)、多磷酸核苷酸(带有4个或4个以上的磷酸基团)以及一些核苷酸衍生物从双层膜的一侧穿过纳米孔进入另一侧第二隔室。在此过程中，带负电荷的分析物被纳米孔的信号检测区域引入的正电荷簇所吸引和捕获，从而停留更长的时间，允许其产生的特征阻断电流信号被微电流检测电路有效捕获。
本公开实施例的纳米孔检测装置可以利用测试腔两端的电极施加电压，例如50-300mV，包括但不限于75-275mV、100-250mV、125-225mV、150-200mV，或者100mV、125mV、150mV、175mV、200mV，从而将沿纳米孔内径形成跨膜的电势梯度。电解质在电势梯度的驱动下在纳米孔内定向运动形成电流。当带负电荷的分析 物，包括带不同数目磷酸的核苷酸分子在自由扩散和电泳运动的双重作用下靠近纳米孔开口时，将被沿纳米孔的电势梯度所俘获，从而在电场力的驱动下通过纳米孔。对于绝大多数的天然纳米孔蛋白，包括α-溶血素、MspA、CsgG、OmpF等,核苷酸分子穿过的速度都在1-10μs范围，引起的阻断电流信号也仅在pA量级。这样微弱的信号强度以及短暂的信号持续时间是无法支持利用电子元件对其进行有效检测的。本公开实施例的纳米孔检测装置在纳米孔蛋白中引入正电荷簇，高密度的正电荷簇可以有效地减慢带负电荷的分析物通过纳米孔的速度，延长了信号的持续时间，使得原本无法被检测到的单分子分析物的特征阻断电流信号被有效捕获，从而获得对单分子分析物的检测能力。特别是，获得单分子核苷酸检测能力的纳米孔，可用于不同的检测场景，包括用于测序或辅助提升测序准确率。
在一些实施方式中，构成磷脂双层膜的磷脂可以包括1,2-二植烷酰-sn-甘油-3-磷酸胆碱(DPhPC)。但本公开的实施并不限于特定的磷脂，包括但不限于其他一些胆碱类磷脂或者人工两性分子，只要能把电解质溶液池分割为分别带有电极并相互绝缘的两个部分，而且能支持纳米孔蛋白的结构形成稳定的电流通道，都可以在本公开实施方式中使用。
本公开实施例还提出一种适用于上述纳米孔检测装置的纳米孔检测方法。该方法控制纳米孔的信号检测区域的正电荷簇的电荷数目，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
在一些实施方式中，增加纳米孔的信号检测区域的正电荷簇的电荷数目可以显著延长分析物通过纳米孔的相应信号持续时间。例如，在α-溶血素的单体优选的位置上多增加一个正电荷，则在七聚体纳米孔上相应增加7个正电荷。带有更多电荷的正电荷簇对带负电荷的分析物的捕获能力显著增强。在一些实施方式中，三磷酸核苷酸通过带有更多正电荷的纳米孔的时间能进一步延长接近一个数量级。
在一些实施方式中，该方法还可以进一步控制所述纳米孔的驱动电压，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
在一些实施方式中，减小所述纳米孔的驱动电压，可以延长带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。例如，驱动电压从180mV减小到120mV、100mV或80mV，带负电荷的分析物，例如核苷酸，受到的电场驱动力会明显减小，其通过纳米孔的时间会随之显著上升。在一些实施方式中，驱动电压从 180mV减小到80mV,核苷酸通过纳米孔的相应信号持续时间可以从小于100μs大幅延长到超过1ms，从而实现对分析物更有效的检测。
在一些实施方式中，该方法还可以进一步控制所述检测装置的电解质溶液的浓度，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
在一些实施方式中，降低所述检测装置的电解质溶液的浓度，可以延长带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。例如，氯化钾溶液是纳米孔检测中常用的电解质溶液。氯化钾溶液的浓度对分析物通过纳米孔的持续时间有非常显著的影响。在一些实施方式中，单纯把氯化钾浓度从1M降低到300mM就足以延长三磷酸核苷酸通过纳米孔的时间一个数量级以上。
本公开实施例的纳米孔检测方法展示了对分析物的目标信号持续时间的有效调节能力，从而丰富了检测手段和相应的信号特点，使得纳米孔对以单分子核苷酸为代表的分析物检测可以运用于更多的不同场景，适应不同的分析物和检测需求。
以下结合本公开具体应用的实施例示例进行进一步的说明。
实施例1α-溶血素突变株的制备
本实施例中，天然α-溶血素基因根据Genebank已收录序列NC_007795.1，通过直接合成的方式获得，所合成的cDNA序列如下：
Figure PCTCN2021087118-appb-000002
所合成的α-溶血素直接克隆入表达载体pET26b。利用安捷伦公司quick change试剂盒在上述基因引入定点突变，制备出带有E111R/M113R/T115R/K143R共四个突变位点的突变株，命名为R4纳米孔。R4纳米孔的结构如图1所示，所有的突变位点均集中在α-溶血素的信号检测区域附近(constriction site)。R4纳米孔可以用制备天然α-溶血素纳米孔同样的方式获得，其七聚体的SDS电泳检测结果如图3A所示，分子筛检测结果如图3B所示。其中，图3A中泳道1表示标准分子量标记，泳道2表示R4突变株的少许七聚体在电泳过程中解离为单体，泳道3表示野生型α-细菌毒素。图3B中，上侧为纯化后R4纳米孔的分子筛检测结果，下侧为野生型α-细菌毒素分子筛图谱对照。用类似的方法，还可制备带有更多正电荷数目的突变株E111R/M113R/T115R/G143R/K147R，可以命名为R5纳米孔。
在制备出改造后的纳米孔的基础上，可以选用符合要求的测试腔的隔室系统，例如选择左右隔室之间的开口孔径为50微米尺寸的隔室系统，开口附近为适合磷脂附着的疏水材质。在左右隔室分别添加0.3M的KCl溶液，液面低于开口的位置。选用1,2-diphytanoyl-sn-glycero-3-phosphocholine(DphPC)，溶于有机溶剂戊烷，终浓度为1mg/ml。将此磷脂溶液添加到隔室已有的KCl溶液中，形成覆盖在KCl液面的有机相。沿隔室外壁添加0.3M的KCl溶液，含有磷脂层的有机相将随液面升高而没过两隔室之间的小孔。在此过程中磷脂和小孔附近的疏水材质结合，并自发形成跨小孔的磷脂双分子膜。
在第一隔室中添加R4纳米孔溶液至终浓度为1nM。同时，测试两隔室之间的电流。当单分子纳米孔自发插入磷脂双层膜后，系统将会检测到R4的开孔电流。及时移除多余的R4纳米孔溶液，确保磷脂膜上只有单分子的R4纳米孔通道。由此，可以制备具有R4纳米孔蛋白的磷脂膜，组装为本公开实施例的纳米孔检测装置。
在获得单通道的R4纳米孔之后，设定驱动电压为180mV，记录到通过R4纳米孔的开孔电流为～106pA。和野生型α-溶血素相同条件下的开孔电流～110pA相比，没有显著差异，表明经过深度改造，在信号检测区域附近引入28个正电荷的R4蛋白，依旧保留了和天然α-溶血素相似的电流信号检测窗口。与之相对应，利用β-环糊精共价修饰的α-溶血素，开孔电流减小到未修饰纳米孔的40％左右，仅为～45pA。
实施例2R4纳米孔对不同磷酸核苷酸的捕获
在组装R4纳米孔的纳米孔检测装置的第一隔室的电解质溶液池加入终浓度为 1μM的三磷酸核苷酸(dCTP)。电压设为100mV，在电场力作用下，dCTP被R4纳米孔所捕获并从第一隔室转运到第二隔室。图4A表示R4突变株在100mV电压、400mM的KCl溶液条件下对三磷酸核苷酸的检测信号，可见dCTP与R4纳米孔相互作用，给出清晰的阻断电流信号。作为对照，如图4B所示，在天然的α-溶血素构成的纳米孔检测装置中，第一隔室加入dCTP，系统无法检测到有效的dCTP阻断电流信号。
对R4所捕获到的dCTP电流信号持续时间进行统计分析，表明信号时间符合指数分布，与理论预测的模型完全吻合。图4C表示对R4所检测到的三磷酸核苷酸信号统计分析，信号密度在不同持续时间上的分布为随机指数分布。
与dCTP类似，在R4纳米孔第一隔室加入1μM的单磷酸核苷酸(dCMP)，也能检测到明显的分析物特征阻断电流信号。图4D表示R4突变株对单磷酸核苷酸的检测信号，由于分析物带电少，信号持续时间比三磷酸核苷酸的相应信号持续时间明显缩短。
实施例3带有更多正电荷突变的R5纳米孔对dCTP的捕获
如以上所述实施例，用同样的方法将R4纳米孔替换为R5纳米孔，在同样的条件下记录R5纳米孔对三磷酸核苷酸dCTP捕获所产生的阻断电流。图5A表示R5纳米孔在100mV电压、300mM KCl溶液条件下所检测到的三磷酸核苷酸信号，图5B表示R4纳米孔在同样条件下对三磷酸核苷酸的检测信号。由于R5纳米孔在信号检测区域附近引入了更多的正电荷数目，其对dCTP的相互作用力明显增加，dCTP产生的阻断电流信号持续时间明显增加。可见，带有更多正电荷的R5突变株对三磷酸核苷酸的捕获检测，信号持续时间明显长于用R4纳米孔所检测到的信号，阻断电流幅度也更深。
实施例4调节盐浓度对dCTP信号持续时间的影响
在0.3M-1M之间调节纳米孔检测装置中溶液池中的KCL的盐浓度，分别记载dCTP引起的阻断电流。获得的相应信号数据如图6A-6C所示。图6A-6C分别表示R4纳米孔在100mV电压下分别在1M KCl、500mM KCl、300mM KCl溶液浓度下对dCTP的信号检测结果。结果显示，dCTP的信号持续时间与盐浓度反向相关，盐浓度越低，信号持续时间越长。
实施例5调节驱动电压对dCTP信号持续时间的影响
对纳米孔检测装置施加不同的驱动电压，例如60mV,70mV,80mV,90mV,100mV，…，180mV,190mV,200mV等，分别记载dCTP被R5纳米孔捕获所产生的阻 断电流。图7A-7F分别表示300mM盐浓度下电压分别为180mV,160mV,140mV,120mV,100mV和80mV，不同电压对R4纳米孔检测三磷酸核苷酸的信号影响。结果表明，信号持续时间对驱动电压非常敏感，电压越低，信号持续时间越长，在<100mV的时候,信号持续时间可以长达几毫秒甚至几十毫秒。在大于150mV的时候，信号持续时间已经非常短，只有零星的一些信号尖刺。
以上的实施例及相关数据详细的阐述了本公开的实施方式和实际效果。分析物特征电流信号持续时间高度可调也使得本公开的具体应用场景更加宽泛。应当注意，本公开不限于本文所描述的特定实施例，其旨在作为本公开的各个方面的示例性说明。在不脱离本公开的精神和范围的情况下，可以对本公开进行许多修改和变化，这对本领域技术人员是显而易见的。除了本文列举的那些之外，在本公开范围内的功能上等效的方法和装置对于本领域技术人员而言将从上述描述中变得显而易见。这些修改和变化旨在落在所附权利要求的范围内。本公开仅由所附权利要求的术语以及这些权利要求的等同物的全部范围来限制。应当理解，本公开不限于当然可以变化的特定方法、试剂、化合物组合物或生物系统。还应当理解，本文使用的术语仅用于描述特定实施例的目的，而不是限制性的。

Claims (19)

  1. 一种纳米孔制备方法，其特征在于，包括：
    采用多个蛋白单体聚集形成纳米孔，其中所述纳米孔的通道狭窄部分形成所述纳米孔的信号检测区域；
    在所述纳米孔的信号检测区域形成正电荷簇，所述正电荷簇与通过所述纳米孔的带负电荷的单分子分析物之间的电荷相互作用能够延长所述单分子分析物在所述纳米孔内的停留时间。
  2. 根据权利要求1所述的方法，其特征在于，所述在所述纳米孔的信号检测区域形成正电荷簇包括：通过蛋白质工程在所述纳米孔的信号检测区域引入带有正电荷的氨基酸残基，形成所述正电荷簇。
  3. 根据权利要求3所述的方法，其特征在于，所述氨基酸残基包括：赖氨酸、精氨酸或组氨酸。
  4. 根据权利要求1所述的方法，其特征在于，所述在所述纳米孔的信号检测区域形成正电荷簇包括：通过生化手段在所述纳米孔的信号检测区域引入带有正电荷的非天然氨基酸，形成所述正电荷簇。
  5. 根据权利要求1所述的方法，其特征在于，所述蛋白单体包括α-溶血素、MspA、CsgG、OmpF中任意一种。
  6. 根据权利要求1所述的方法，其特征在于，所述单分子分析物包括带有不同磷酸数目的核苷酸。
  7. 一种纳米孔检测装置，其特征在于，包括：
    具有纳米孔、容置有电解质溶液的测试腔；以及
    连接至所述测试腔的检测电路；其中，所述纳米孔的通道狭窄部分形成所述纳米孔的信号检测区域，所述信号检测区域具有正电荷簇，当电解质溶液中的带负电荷的单分子分析物在纳米孔的驱动电压的作用下通过所述纳米孔时，所述正电荷簇与所述带负电荷的单分子分析物之间的电荷相互作用能够延长所述单分子分析物在所述纳米孔内的停留时间。
  8. 根据权利要求7所述的装置，其特征在于，所述正电荷簇的电荷数目能够被控制来调节所述单分子分析物在所述纳米孔内的停留时间。
  9. 根据权利要求7所述的装置，其特征在于，所述纳米孔的驱动电压能够被控制来调节所述单分子分析物在所述纳米孔内的停留时间。
  10. 根据权利要求7所述的装置，其特征在于，所述电解质溶液的浓度能够被控制来调节所述单分子分析物在所述纳米孔内的停留时间。
  11. 根据权利要求7所述的装置，其特征在于，所述纳米孔包括多个蛋白单体。
  12. 根据权利要求11所述的装置，其特征在于，所述蛋白单体包括α-溶血素、MspA、CsgG、OmpF中任意一种。
  13. 根据权利要求7所述的装置，其特征在于，所述单分子分析物包括带有不同磷酸数目的核苷酸。
  14. 一种纳米孔检测方法，应用于如权利要求7-13中任一项所述的纳米孔检测装置，其特征在于，包括：
    控制纳米孔的信号检测区域的正电荷簇的电荷数目，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
  15. 根据权利要求14所述的方法，其特征在于，所述控制纳米孔的信号检测区域的正电荷簇的电荷数目，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间包括：
    增加所述纳米孔的信号检测区域的正电荷簇的电荷数目，来延长带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
  16. 根据权利要求14所述的方法，其特征在于，还包括：
    控制所述纳米孔的驱动电压，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
  17. 根据权利要求16所述的方法，其特征在于，所述控制所述纳米孔的驱动电压，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间包括：
    减小所述纳米孔的驱动电压，来延长带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
  18. 根据权利要求14所述的方法，其特征在于，还包括：
    控制所述电解质溶液的浓度，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
  19. 根据权利要求18所述的方法，其特征在于，所述控制所述电解质溶液的浓度，来调节带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间包括：
    降低所述电解质溶液的浓度，来延长带负电荷的单分子分析物通过所述纳米孔时在所述纳米孔内的停留时间。
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