Nanopore preparation and detection method and detection device

Abstract

Provided are a nanopore preparation and detection method and a detection device thereof. The method uses a plurality of protein monomers to aggregate to form a nanopore, wherein the narrow channel portion of the nanopore forms the signal detection region of the nanopore; a positive charge cluster is formed in the signal detection region of the nanopore, and the charge interaction between the positive charge cluster and the negatively charged single-molecule analyte passing through the nanopore can extend the residence time of the single-molecule analyte in the nanopore. Effective detection of analytes at the single-molecule level is achieved, so that single-molecule analytes that cannot generate effective detection signals due to too short an interaction time with the nanopore can be effectively detected, significantly improving the detection rate of single-molecule analytes, and being applicable to different analytes and detection requirements.

Description

Technical Field

[0001] The present disclosure belongs to the field of gene sequencing, and in particular, relates to a nanopore preparation and detection method and a detection device thereof. Background Art

[0002] Nanopores are a type of nanoscale ion channels. When voltage is applied across a nanopore, the electrolyte ions in the solution will form an electric current through the nanopore under the action of the electric field driving force. Some small molecules can interact with nanopores, usually by entering the nanopore channel and staying there for a short time, thereby affecting the magnitude of the current passing through the nanopore and generating a characteristic blocking current. Using the characteristic blocking current signal, nanopores can be used as a detector at the single-molecule level in different scenarios, including the study of the mechanism of chemical reactions and the sequence determination of nucleic acids.

[0003] However, nanopores still have great limitations as single-molecule detectors. In addition to meeting certain size and charge characteristics, analytes that can be detected by nanopores also need to interact with the nanopores for a long enough time so that their corresponding characteristic blocking current signals can be effectively captured and detected by electronic components. Existing nanopores for nucleic acid sequencing, including α-bacterial toxins, protein nanopores such as MspA and CsgG, or solid-state artificial nanopores made of materials such as graphene, are difficult to directly detect nucleotide signals at the single-molecule level. Whether it is a monophosphate, diphosphate or triphosphate nucleotide molecule, the speed of passing through the nanopore under the action of the electric field driving force is very fast, and the time of interaction with the nanopore is usually only a few microseconds or even shorter. In this process, even if the nucleotides generate a blocking current signal, the too short signal duration is far beyond the detection limit of all electronic components.

[0004] An existing improved scheme obtains the ability to detect single-molecule nucleotides by covalently modifying β-cyclodextrin at specific positions of α-bacterial toxins. However, the scheme of covalently modifying nanopores with cyclodextrin has two obvious defects: 1. An additional process is required to prepare β-cyclodextrin that can be used to modify α-bacterial toxins, and because the modification site is inside the pore size of the nanopore, the efficiency of the chemical reaction is difficult to guarantee, and it is also difficult to effectively separate the modified and unmodified nanopores, which affects subsequent detection; 2. The pore size of the modified nanopore is reduced from 1.4nm to 0.8nm, and the opening current is also reduced to about 40% of the unmodified time, which greatly reduces the signal window of the characteristic blocking current and the detection range of the nanopore. In addition, for Roche's sequencing system, since the nucleotides are labeled, the overly narrow nanopore pore size is likely to cause the label to block the nanopore, making the entire system unable to work properly.

[0005] Chinese invention patent CN102317310B proposes a method for enhancing the displacement of charged analytes through transmembrane protein pores. The patent increases the frequency of nucleic acid chain displacement through the pore by adding positive charge inside the nanopore, and reduces the threshold voltage of the nucleic acid chain displacement through the pore. At the same time, the solution can reduce the displacement speed of the nucleic acid chain through the pore, thereby increasing the residence time of the analyte in the nanopore. However, the positive charge sites introduced into the nanopore by the patent are dispersed at various positions in the nanopore. The main purpose is to increase the displacement frequency of the analyte and achieve the capture efficiency of the nanopore for the analyte. There is no data or examples showing that the nanopore it has modified can have the ability to detect single-molecule nucleotides. Summary of the invention

[0006] The embodiments of the present disclosure provide a nanopore preparation and detection method and a detection device thereof, which are used to achieve effective detection of single-molecule analytes and improve detection accuracy.

[0007] In a first aspect, the present disclosure provides a method for preparing a nanopore, comprising:

[0008] Aggregating a plurality of protein monomers to form a nanopore, wherein the narrow channel portion of the nanopore forms a signal detection region of the nanopore;

[0009] Positively charged clusters are formed in the signal detection region of the nanopore, and the charge interaction between the positively charged clusters and the negatively charged single-molecule analyte passing through the nanopore can prolong the residence time of the single-molecule analyte in the nanopore.

[0010] In an optional embodiment, the forming of the positively charged clusters in the signal detection region of the nanopore comprises: introducing positively charged amino acid residues into the signal detection region of the nanopore by protein engineering to form the positively charged clusters.

[0011] In an alternative embodiment, the amino acid residue comprises: lysine, arginine or histidine.

[0012] In an optional embodiment, the forming of the positively charged cluster in the signal detection region of the nanopore comprises: introducing non-natural amino acids with positive charge into the signal detection region of the nanopore by biochemical means to form the positively charged cluster.

[0013] In an optional embodiment, the protein monomer includes any one of α-hemolysin, MspA, CsgG, and OmpF.

[0014] In alternative embodiments, the single molecule analyte comprises nucleotides with different numbers of phosphates.

[0015] In a second aspect, an embodiment of the present disclosure provides a nanopore detection device, comprising:

[0016] a test chamber having a nanopore and containing an electrolyte solution; and

[0017] A detection circuit connected to the test cavity; wherein the narrow channel portion of the nanopore forms a signal detection region of the nanopore, the signal detection region has a positive charge cluster, and when a negatively charged single-molecule analyte in an electrolyte solution passes through the nanopore under the action of a driving voltage of the nanopore, the charge interaction between the positive charge cluster and the negatively charged single-molecule analyte can prolong the residence time of the single-molecule analyte in the nanopore.

[0018] In an alternative embodiment, the charge number of the positively charged clusters can be controlled to adjust the residence time of the single molecule analyte within the nanopore.

[0019] In an alternative embodiment, the driving voltage of the nanopore can be controlled to adjust the residence time of the single molecule analyte within the nanopore.

[0020] In an alternative embodiment, the concentration of the electrolyte solution can be controlled to adjust the residence time of the single molecule analyte within the nanopore.

[0021] In an alternative embodiment, the nanopore comprises a plurality of protein monomers.

[0022] In an optional embodiment, the protein monomer includes any one of α-hemolysin, MspA, CsgG, and OmpF.

[0023] In alternative embodiments, the single molecule analyte comprises nucleotides with different numbers of phosphates.

[0024] In a third aspect, the present disclosure provides a nanopore detection method, which is applied to the nanopore detection device as described in the above embodiment, including:

[0025] The charge number of the positively charged clusters in the signal detection region 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.

[0026] In an optional embodiment, controlling the charge number of the positively charged clusters in the signal detection region of the nanopore to adjust the residence time of the negatively charged single-molecule analyte in the nanopore when passing through the nanopore comprises:

[0027] The charge number of the positively charged clusters in the signal detection region of the nanopore is increased to prolong the residence time of the negatively charged single molecule analyte in the nanopore when the single molecule analyte passes through the nanopore.

[0028] In an optional embodiment, it also includes:

[0029] 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.

[0030] In an optional embodiment, the step of 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 comprises:

[0031] 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.

[0032] In an optional embodiment, it also includes:

[0033] The concentration of the electrolyte solution is controlled to adjust the residence time of the negatively charged single molecule analyte in the nanopore when passing through the nanopore.

[0034] In an optional embodiment, 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 comprises:

[0035] The concentration of the electrolyte solution is reduced to prolong the residence time of the negatively charged single-molecule analyte in the nanopore when passing through the nanopore.

[0036] The disclosed embodiment introduces positive charge clusters into the signal detection region of the nanopore, and through the interaction between the charges, significantly prolongs the interaction time between the negatively charged single-molecule analyte and the nanopore, so that the characteristic blocking current signal detected in the signal detection region can be accurately detected by the electronic component. Compared with the existing scheme, the disclosed embodiment has at least the following beneficial effects: 1. It realizes the effective detection of analytes at the single-molecule level, especially the single-molecule nucleotides in the scenario related to nucleic acid sequencing, and significantly improves the detection rate of single-molecule analytes; 2. The nanopore prepared by the present disclosure is used in the detection device, and the duration of the detection signal can be flexibly adjusted according to the charge number of the positive charge cluster, the driving voltage, and the concentration of the electrolyte solution, so that the detection device can be suitable for different analytes and detection requirements, and has a broader application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0038] Figure 1A and 1B are side views and top views of a three-dimensional model of an R4 nanopore prepared according to an embodiment of the present disclosure;

[0039] Figure 2 is a schematic structural diagram of a nanopore detection device according to an embodiment of the present disclosure;

[0040] Figure 3A It is a schematic diagram of the SDS electrophoresis detection results of the R4 nanopore;

[0041] Figure 3B It is a schematic diagram of the molecular sieve detection results of R4 nanopore;

[0042] Figure 4A This is the detection signal diagram of nucleotide triphosphate by R4 nanopore under the conditions of 100 mV voltage and 400 mM KCl solution;

[0043] Figure 4B This is the detection signal diagram of natural α-hemolysin to nucleotide triphosphate under the same conditions;

[0044] Figure 4C is a statistical analysis of the nucleotide triphosphate signals detected by the R4 nanopore;

[0045] Figure 4D is the detection signal diagram of R4 nanopore to monophosphate nucleotide;

[0046] Figure 5A It is the detection signal diagram of nucleotide triphosphate by R5 nanopore under the conditions of 100mV voltage and 300mM KCl solution;

[0047] Figure 5B This is the detection signal diagram of nucleotide triphosphates by R4 nanopore under the same conditions;

[0048] Figures 6A-6C is the detection signal diagram of nucleotide triphosphate by R4 nanopore at 1M KCl, 500mM KCl, and 300mM KCl solution concentrations;

[0049] Figures 7A-7FThese are the detection signal diagrams of nucleotide triphosphates by the R4 nanopore at voltages of 180 mV, 160 mV, 140 mV, 120 mV, 100 mV, and 80 mV, respectively. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure.

[0051] In the present disclosure, it should be understood that terms such as "include" or "have" are intended to indicate the presence of features, numbers, steps, behaviors, components, parts, or a combination thereof disclosed in the present specification, and are not intended to exclude the possibility that one or more other features, numbers, steps, behaviors, components, parts, or a combination thereof exist or are added.

[0052] As mentioned above, the existing nanopores used for the detection of analytes have insufficient detection capabilities for single-molecule analytes. In particular, the effective detection of nucleotides at the single-molecule level is of great significance for the development of new nucleic acid sequencing schemes or for improving the accuracy of existing sequencing schemes.

[0053] The disclosed embodiment first proposes a method for preparing a nanopore, which uses multiple protein monomers to aggregate to form a nanopore, wherein the narrow part of the channel of the nanopore forms the signal detection area of ​​the nanopore; a positive charge cluster is formed in the signal detection area of ​​the nanopore, and the charge interaction between the positive charge cluster and the negatively charged single molecule analyte passing through the nanopore can extend the residence time of the single molecule analyte in the nanopore. The method forms a positive charge cluster in the signal detection area (constriction site) of the nanopore. When the negatively charged single molecule analyte enters the nanopore under the action of an electric field, the charge interaction between the positive charge cluster and the negatively charged single molecule analyte can significantly extend the residence time of the single molecule analyte in the nanopore, thereby increasing the duration of the characteristic current signal blocked by the nanopore. When the nanopore with such characteristics prepared by the method is used in a detection device, the residence time of the single molecule analyte passing through the nanopore is significantly extended, and the residence time can reach the millisecond level, so that the characteristic blocking current signal generated by the nanopore can be effectively captured by the electronic component, so that the single molecule analyte that could not be detected due to the short residence time can be effectively detected, thereby improving the detection rate of the single molecule analyte.

[0054] In some embodiments, the nanopore comprises a protein nanopore. Optional protein nanopores include, but are not limited to, bacterial toxin α-hemolysin, MspA, CsgG, OmpF, etc. These protein nanopores are widely used in molecular detection, including many scenarios of sequencing, and have known high-resolution three-dimensional structures, which can easily locate their signal detection regions and nearby amino acid residues. Therefore, introducing positive charge mutations in the signal detection regions of these nanopores by genetic engineering means is highly targeted and feasible.

[0055] In some embodiments, single molecule analytes may include, but are not limited to, nucleotides with varying numbers of phosphates.

[0056] Take the nanopore of bacterial toxin α-hemolysin as an example. Bacterial toxin α-hemolysin is a natural protein monomer containing 293 amino acids, and its primary protein structure sequence is as follows:

[0057] ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVI

[0058] RTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEY

[0059] MSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVI

[0060] FNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFS

[0061] PDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSER

[0062] YKIDWEKEEMTN (SEQ ID NO: 1)

[0063] In the presence of detergents, phospholipids or other amphiphilic molecules, seven α-hemolysin protein monomers will spontaneously aggregate to form nanopores. The side view and top view of the three-dimensional model structure are shown in Figure 2. Figure 1A and 1B As shown. Among them, Figure 1A and 1BThe spherical model in the figure shows the narrowest part of the nanopore channel composed of 7 monomers E111, M113 and K147, which becomes the signal detection area (constriction site) where the analyte causes a characteristic blockade current.

[0064] In some embodiments, by means of protein engineering, 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, and the nanopore formed by the mutant will form a positively charged cluster with high positive charge density near the signal detection region. The positive charge mutation on each protein monomer corresponds to 7 additional positive charges on the nanopore. In some embodiments, the amino acid residues may include: lysine (Lys), arginine (Arg) or histidine (His) protonated at low pH. In some embodiments, mutants with multiple mutation sites, such as E111R, M113R, T115R, G143R, K147R, can be prepared by the same means as the wild-type protein to obtain a high-purity heptamer nanopore with a positively charged cluster.

[0065] In other embodiments, protein monomers with corresponding positive charge mutations can be mixed with protein monomers without mutations to form a hybrid multimer, so that the positive charge density in the nanopore signal detection area can be more finely adjusted, rather than simply adding 7 positive charges for each mutation introduced. For example, a hybrid heptamer formed by 3 wild-type α-hemolysin monomers and 4 monomers with the M113R mutation will have 4 positive charges at site 113 in the nanopore.

[0066] In other embodiments, non-natural amino acids can also be introduced into the signal detection region of the nanopore by specific biochemical means. The non-natural amino acids can be different from natural lysine (Lys), arginine (Arg) or histidine (His), but can carry different numbers of charges, so that the nanopore formed by the introduction into the protein monomer has a richer and adjustable positive charge cluster.

[0067] The disclosed embodiment also provides a nanopore detection device. After the preparation of the nanopore with positively charged clusters is completed, the modified nanopore protein can be embedded in a specific phospholipid bilayer membrane or an artificial bilayer membrane with similar physical and chemical properties to form a nanopore. Figure 2 The nanopore detection device. The modified nanopore protein is purified by biochemical means similar to wild nanopores, and its biochemical activity can be well retained. The purified and prepared nanopore protein can be spontaneously embedded in a phospholipid bilayer membrane or an artificial bilayer membrane due to its own lipophilic properties.

[0068] The nanopore detection device includes a test cavity embedded with a prepared nanopore and a microcurrent detection circuit connected thereto. The test cavity is separated by a first compartment and a second compartment by a phospholipid bilayer membrane or an artificial bilayer membrane, and an electrolyte solution is contained in the first compartment and the second compartment. The analyte is located in the electrolyte solution of the first compartment. Under the voltage applied to the electrodes at both ends of the test cavity, the analyte with a negative charge, such as but not limited to monophosphate nucleotide (NMP or dNMP), diphosphonic acid nucleotide (NDP or dNDP), triphosphate nucleotide (NTP or dNTP), polyphosphate nucleotide (with 4 or more phosphate groups) and some nucleotide derivatives, passes through the nanopore from one side of the bilayer membrane into the second compartment on the other side. In this process, the negatively charged analyte is attracted and captured by the positive charge cluster introduced into the signal detection area of ​​the nanopore, thereby staying for a longer time, allowing the characteristic blocking current signal generated by it to be effectively captured by the microcurrent detection circuit.

[0069] The nanopore detection device of the disclosed embodiment can use the electrodes at both ends of the test cavity to apply a voltage, such as 50-300mV, including but not limited to 75-275mV, 100-250mV, 125-225mV, 150-200mV, or 100mV, 125mV, 150mV, 175mV, 200mV, so as to form a transmembrane potential gradient along the inner diameter of the nanopore. The electrolyte moves in a directional manner in the nanopore under the drive of the potential gradient to form a current. When negatively charged analytes, including nucleotide molecules with different numbers of phosphates, approach the nanopore opening under the dual effects of free diffusion and electrophoretic movement, they will be captured by the potential gradient along the nanopore, thereby passing through the nanopore under the drive of the electric field force. For the vast majority of natural nanopore proteins, including α-hemolysin, MspA, CsgG, OmpF, etc., the speed at which nucleotide molecules pass through is in the range of 1-10μs, and the resulting blocking current signal is only in the pA order of magnitude. Such weak signal intensity and short signal duration cannot support effective detection using electronic components. The nanopore detection device of the disclosed embodiment introduces positively charged clusters into the nanopore protein. The high-density positively charged clusters can effectively slow down the speed at which negatively charged analytes pass through the nanopore, prolonging the duration of the signal, so that the characteristic blocking current signal of the single-molecule analyte that was originally undetectable can be effectively captured, thereby obtaining the ability to detect single-molecule analytes. In particular, nanopores that have obtained the ability to detect single-molecule nucleotides can be used in different detection scenarios, including for sequencing or to assist in improving sequencing accuracy.

[0070] In some embodiments, the phospholipids constituting the phospholipid bilayer membrane may include 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). However, 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 the structure of the nanopore protein can be supported to form a stable current channel, it can be used in the embodiments of the present disclosure.

[0071] The disclosed embodiment also provides a nanopore detection method applicable to the above nanopore detection device, which controls the charge number of positive charge clusters in the signal detection region of the nanopore to adjust the residence time of a negatively charged single molecule analyte in the nanopore when passing through the nanopore.

[0072] In some embodiments, increasing the number of charges of the positively charged clusters in the signal detection region of the nanopore can significantly extend the duration of the corresponding signal of the analyte passing through the nanopore. For example, adding one more positive charge to the preferred position of the monomer of α-hemolysin will correspondingly increase 7 positive charges on the heptamer nanopore. The positively charged clusters with more charges significantly enhance the capture ability of negatively charged analytes. In some embodiments, the time for nucleotide triphosphates to pass through the nanopore with more positive charges can be further extended by nearly an order of magnitude.

[0073] In some embodiments, the method can further control 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.

[0074] In some embodiments, reducing the driving voltage of the nanopore can extend the residence time of a single molecule analyte with a negative charge in the nanopore when passing through the nanopore. For example, when the driving voltage is reduced from 180mV to 120mV, 100mV or 80mV, the electric field driving force of the negatively charged analyte, such as a nucleotide, will be significantly reduced, and the time it takes to pass through the nanopore will increase significantly. In some embodiments, when the driving voltage is reduced from 180mV to 80mV, the corresponding signal duration of the nucleotide passing through the nanopore can be greatly extended from less than 100μs to more than 1ms, thereby achieving more effective detection of the analyte.

[0075] In some embodiments, the method can further control the concentration of the electrolyte solution of the detection device to adjust the residence time of the negatively charged single molecule analyte in the nanopore when passing through the nanopore.

[0076] In some embodiments, reducing the concentration of the electrolyte solution of the detection device can prolong the residence time of the negatively charged single molecule analyte in the nanopore when passing through the nanopore. For example, potassium chloride solution is a commonly used electrolyte solution in nanopore detection. The concentration of potassium chloride solution has a very significant effect on the duration of the analyte passing through the nanopore. In some embodiments, simply reducing the potassium chloride concentration from 1M to 300mM is sufficient to prolong the time for nucleotide triphosphates to pass through the nanopore by more than an order of magnitude.

[0077] The nanopore detection method of the disclosed embodiment demonstrates the ability to effectively adjust the duration of the target signal of the analyte, thereby enriching the detection means and the corresponding signal characteristics, so that the nanopore detection of analytes represented by single-molecule nucleotides can be applied to more different scenarios and adapt to different analytes and detection needs.

[0078] The following is a further description of the specific application examples of the present disclosure.

[0079] Example 1 Preparation of α-hemolysin mutants

[0080] In this example, the natural α-hemolysin gene was obtained by direct synthesis based on the sequence NC_007795.1 included in Genebank. The synthesized cDNA sequence is as follows:

[0081] gcagattctgat attaatatta aaaccggtac tacagatatt

[0082] ggaagcaata ctacagtaaa aacaggtgat ttagtcactt atgataaaga aaatggcatg

[0083] cacaaaaaag tattttatag ttttatcgat gataaaaatc ataataaaaa actgctagtt

[0084] attagaacga aaggtaccat tgctggtcaa tatagagttt atagcgaaga aggtgctaac

[0085] aaaagtggtt tagcctggcc ttcagccttt aaggtacagt tgcaactacc tgataatgaa

[0086] gtagctcaaa tatctgatta ctatccaaga aattcgattg atacaaaaga gtatatgagt

[0087] actttaactt atggattcaa cggtaatgtt actggtgatg atacaggaaa aattggcggc

[0088] cttattggtg caaatgtttc gattggtcat acactgaaat atgttcaacc tgatttcaaa

[0089] acaattttag agagcccaac tgataaaaaa gtaggctgga aagtgatatt taacaatatg

[0090] gtgaatcaaa attggggacc atatgataga gattcttgga acccggtata tggcaatcaa

[0091] cttttcatga aaactagaaa tggctctatg aaagcagcag ataacttcct tgatcctaac

[0092] aaagcaagtt ctctattatc ttcagggttt tcaccagact tcgctacagt tattactatg

[0093] gatagaaaag catccaaaca acaaacaaat atagatgtaa tatacgaacg agttcgtgat

[0094] gactaccaat tgcactggac ttcaacaaat tggaaaggta ccaatactaa agataaatgg

[0095] atagatcgtt cttcagaaag atataaaatc gattgggaaa aagaagaaat gacaaattaa(SEQID NO:2)

[0096] The synthesized α-hemolysin was directly cloned into the expression vector pET26b. Agilent's quick change kit was used to introduce site-directed mutations into the above gene, and a mutant strain with four mutation sites, E111R / M113R / T115R / K143R, was prepared, named R4 nanopore. The structure of the R4 nanopore is shown in Figure 1. All mutation sites are concentrated near the signal detection area (constriction site) of α-hemolysin. The R4 nanopore can be obtained in the same way as the natural α-hemolysin nanopore. The SDS electrophoresis detection results of its heptamer are shown in Figure 1. Figure 3A As shown, the molecular sieve test results are as follows Figure 3B As shown. Among them, Figure 3A Lane 1 in the middle represents a standard molecular weight marker, lane 2 represents a small amount of heptamers of the R4 mutant strain dissociated into monomers during electrophoresis, and lane 3 represents the wild-type α-bacterial toxin. Figure 3B In the figure, the upper side is the molecular sieve detection result of the purified R4 nanopore, and the lower side is the molecular sieve spectrum control of the wild-type α-bacterial toxin. Using a similar method, a mutant strain E111R / M113R / T115R / G143R / K147R with a higher number of positive charges can also be prepared, which can be named R5 nanopore.

[0097] On the basis of preparing the modified nanopore, a compartment system of the test chamber that meets the requirements can be selected, for example, a compartment system with an opening diameter of 50 microns between the left and right compartments, and a hydrophobic material suitable for phospholipid attachment near the opening. Add 0.3M KCl solution to the left and right compartments respectively, and the liquid level is lower than the position of the opening. Select 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DphPC) and dissolve it in the organic solvent pentane to a final concentration of 1mg / ml. Add this phospholipid solution to the existing KCl solution in the compartment to form an organic phase covering the KCl liquid surface. Add 0.3M KCl solution along the outer wall of the compartment, and the organic phase containing the phospholipid layer will submerge the small hole between the two compartments as the liquid level rises. In this process, the phospholipids combine with the hydrophobic material near the small hole and spontaneously form a phospholipid bilayer across the small hole.

[0098] Add R4 nanopore solution to the first compartment to a final concentration of 1 nM. At the same time, test the current between the two compartments. When the single-molecule nanopore spontaneously inserts into the phospholipid bilayer membrane, the system will detect the opening current of R4. Remove the excess R4 nanopore solution in time to ensure that there is only a single-molecule R4 nanopore channel on the phospholipid membrane. In this way, a phospholipid membrane with R4 nanopore protein can be prepared and assembled into a nanopore detection device of the embodiment of the present disclosure.

[0099] After obtaining a single-channel R4 nanopore, the driving voltage was set to 180mV, and the opening current through the R4 nanopore was recorded to be ~106pA. Compared with the opening current of ~110pA of wild-type α-hemolysin under the same conditions, there is no significant difference, indicating that after deep modification, the R4 protein with 28 positive charges introduced near the signal detection area still retains the current signal detection window similar to that of natural α-hemolysin. Correspondingly, the opening current of α-hemolysin covalently modified with β-cyclodextrin is reduced to about 40% of the unmodified nanopore, only ~45pA.

[0100] Example 2: Capture of different phosphate nucleotides by R4 nanopore

[0101] A final concentration of 1 μM nucleotide triphosphate (dCTP) was added to the electrolyte solution pool of the first compartment of the nanopore detection device assembled with the R4 nanopore. The voltage was set to 100 mV, and under the action of the electric field force, dCTP was captured by the R4 nanopore and transported from the first compartment to the second compartment. Figure 4A The figure shows the detection signal of the R4 mutant strain for nucleotide triphosphates under the conditions of 100 mV voltage and 400 mM KCl solution. It can be seen that dCTP interacts with the R4 nanopore and gives a clear blocking current signal. As a control, Figure 4B As shown, in the nanopore detection device composed of natural α-hemolysin, when dCTP is added to the first compartment, the system cannot detect an effective dCTP blocking current signal.

[0102] Statistical analysis of the duration of the dCTP current signal captured by R4 showed that the signal time conformed to an exponential distribution, which was consistent with the theoretically predicted model. Figure 4C It represents the statistical analysis of the nucleotide triphosphate signals detected by R4. The distribution of signal density at different durations is a random exponential distribution.

[0103] Similar to dCTP, when 1 μM dCMP was added to the first compartment of the R4 nanopore, a clear analyte-characteristic blocking current signal was also detected. Figure 4D It represents the detection signal of the R4 mutant to monophosphate nucleotide. Since the analyte is less charged, the signal duration is significantly shorter than the corresponding signal duration of triphosphate nucleotide.

[0104] Example 3 Capture of dCTP by R5 Nanopore with More Positively Charged Mutations

[0105] As in the above-mentioned embodiment, the R4 nanopore was replaced by the R5 nanopore using the same method, and the blocking current generated by the capture of dCTP by the R5 nanopore was recorded under the same conditions. Figure 5AIt represents the nucleotide triphosphate signal detected by R5 nanopore under the conditions of 100mV voltage and 300mM KCl solution. Figure 5B It represents the detection signal of nucleotide triphosphate by R4 nanopore under the same conditions. Since R5 nanopore introduces more positive charges near the signal detection area, its interaction force 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 detection of nucleotide triphosphate by R5 mutant with more positive charges has a significantly longer signal duration than that detected by R4 nanopore, and the blocking current amplitude is also deeper.

[0106] Example 4 Effect of adjusting salt concentration on dCTP signal duration

[0107] The salt concentration of KCl in the solution pool in the nanopore detection device was adjusted between 0.3M and 1M, and the blocking current caused by dCTP was recorded respectively. The corresponding signal data obtained are as follows: Figures 6A-6C shown. Figures 6A-6C The following are the signal detection results of dCTP by R4 nanopore at 100mV voltage in 1M KCl, 500mM KCl, and 300mM KCl solution concentrations. The results show that the signal duration of dCTP is inversely correlated with the salt concentration. The lower the salt concentration, the longer the signal duration.

[0108] Example 5 Effect of adjusting driving voltage on dCTP signal duration

[0109] Different driving voltages, such as 60 mV, 70 mV, 80 mV, 90 mV, 100 mV, ..., 180 mV, 190 mV, 200 mV, etc., were applied to the nanopore detection device to record the blocking currents generated by the capture of dCTP by the R5 nanopore. Figures 7A-7F The voltages are 180mV, 160mV, 140mV, 120mV, 100mV and 80mV respectively at 300mM salt concentration, and the different voltages affect the signal of R4 nanopore detecting nucleotide triphosphates. The results show that the signal duration is very sensitive to the driving voltage. The lower the voltage, the longer the signal duration. When the voltage is <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.

[0110] The above examples and related data elaborate on the implementation methods and practical effects of the present disclosure. The highly adjustable duration of the analyte characteristic current signal also makes the specific application scenarios of the present disclosure more extensive. It should be noted that the present disclosure is not limited to the specific embodiments described herein, which are intended to be exemplary of various aspects of the present disclosure. Without departing from the spirit and scope of the present disclosure, many modifications and changes can be made to the present disclosure, which will be obvious to those skilled in the art. In addition to those listed herein, functionally equivalent methods and devices within the scope of the present disclosure will become apparent to those skilled in the art from the above description. These modifications and changes are intended to fall within the scope of the appended claims. The present disclosure is limited only by the terms of the appended claims and the full scope of the equivalents of these claims. It should be understood that the present disclosure is not limited to specific methods, reagents, compound compositions or biological systems that can certainly vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not restrictive.

Claims

1. A method for detecting a single molecule analyte using a nanopore detection device, characterized in that: The nanopore detection device comprises: a test chamber having a nanopore and containing an electrolyte solution; and A detection circuit connected to the test cavity; wherein the narrow channel portion of the nanopore forms a signal detection region of the nanopore, the signal detection region has a positive charge cluster, and when a negatively charged single-molecule analyte in an electrolyte solution passes through the nanopore under the action of a driving voltage of the nanopore, the charge interaction between the positive charge cluster and the negatively charged single-molecule analyte can prolong the residence time of the single-molecule analyte in the nanopore; wherein the nanopore comprises a plurality of protein monomers, wherein the protein monomers are mutants in which the amino acid sequence of α-hemolysin shown in SEQ ID NO: 1 is introduced with mutations E111R, M113R, T115R, G143R and K147R; The single molecule analytes are nucleotides with different numbers of phosphates.

2. The method according to claim 1, characterized in that The single molecule analyte includes nucleotides with a single phosphate, a double phosphate, a triphosphate or a higher number of phosphates.

3. The method according to claim 1, characterized in that Also includes: The charge number of the positively charged clusters in the signal detection region of the nanopore is increased to prolong the residence time of the negatively charged single molecule analyte in the nanopore when the single molecule analyte passes through the nanopore.

4. The method according to any one of claims 1 to 3, characterized in that: Also 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.

5. The method according to any one of claims 1 to 3, characterized in that: Also 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 passing through the nanopore.

6. A nanopore detection device for use in the method according to any one of claims 1 to 5, characterized in that: A test chamber having a nanopore and containing an electrolyte solution; as well as A detection circuit connected to the test cavity; wherein the narrow channel portion of the nanopore forms a signal detection region of the nanopore, the signal detection region has a positive charge cluster, and when a negatively charged single-molecule analyte in an electrolyte solution passes through the nanopore under the action of a driving voltage of the nanopore, the charge interaction between the positive charge cluster and the negatively charged single-molecule analyte can prolong the residence time of the single-molecule analyte in the nanopore; The nanopore comprises a plurality of protein monomers, wherein the protein monomers are mutants of α-hemolysin with the amino acid sequence as shown in SEQ ID NO: 1, in which mutations E111R, M113R, T115R, G143R and K147R are introduced.

7. The device according to claim 6, characterized in that The charge number of the positively charged clusters can be controlled to adjust the residence time of the single molecule analyte within the nanopore.

8. The device according to claim 6 or 7, characterized in that The driving voltage of the nanopore can be controlled to adjust the residence time of the single molecule analyte within the nanopore.

9. The device according to claim 6 or 7, characterized in that The concentration of the electrolyte solution can be controlled to adjust the residence time of the single molecule analyte within the nanopore.

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