CN110554079A - Antibody single molecule detection system and method based on nanochannel - Google Patents
Antibody single molecule detection system and method based on nanochannel Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
Abstract
the invention relates to an antibody single molecule detection system and method based on a nano channel, belonging to the field of molecular detection. The detection system comprises an electrolyte solution chamber, a supporting film with a nanopore and a current detection system; the current detection system comprises a power supply, an electrode I, an electrode II and an ammeter, wherein the electrode I, the electrode II and the ammeter are respectively connected with the power supply; the supporting film with the nano holes divides the electrolyte solution chamber into a chamber I and a chamber II, the electrode I is arranged in the chamber I, and the electrode II is arranged in the chamber II; the antibody reduction reaction system is arranged in a chamber I; the invention obtains signals that the antibody or fragments generated after the antibody is reduced (or hydrolyzed) penetrate through the nanopore and signals generated by the interaction of the antibody or fragments generated after the antibody is reduced (or hydrolyzed) and the nanopore by detecting by placing the to-be-detected antibody reduction (or hydrolysis) system in a chamber of the detection system, and has the characteristics of simple operation, effective improvement of the detection reliability of the nanopore and realization of high specificity and high sensitivity detection and analysis of the antibody.
Description
Technical Field
the invention belongs to the field of molecular detection, and particularly relates to an antibody single-molecule detection system and method based on a nano channel.
Background
at present, the antibody detection method based on immune reaction is complex to operate, long in reaction time and difficult to avoid the influence of fluorescent labeling; antibody detection based on gel electrophoresis methods does not allow for the discrimination of similar antibodies (e.g., different subclasses of homogeneous antibodies or antibody molecules of comparable molecular mass); in the existing direct antibody detection method based on the nanopore, antibody molecules are easy to adsorb on the surface of a solid-state pore, so that detection signals are unstable, the repeatability is poor, or the large molecules of the antibody easily cause the gating effect of biological pores, so that the pore blocking phenomenon is caused, and the detection sensitivity and reliability are reduced. How to realize the rapid and sensitive detection of different antibodies is a key technical difficulty faced by the current nanopore detection technology.
therefore, there is a need for a system and method that can reduce antibody molecule adsorption and pore blocking, ultimately enabling nanopore multiple antibody detection assays at the single molecule level.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a nanochannel-based antibody single-molecule detection system; the second objective of the present invention is to provide a preparation method of antibody single molecule detection system based on nano channel; the invention also aims to provide a detection method of the antibody single-molecule detection system based on the nano-channel.
In order to achieve the purpose, the invention provides the following technical scheme:
1. A nanochannel-based antibody single-molecule detection system comprising an electrolyte solution chamber, a support membrane with nanopores, and a current detection system; the current detection system comprises a power supply (6), an electrode I (8) and an electrode II (9) which are respectively connected with the power supply, and an ammeter (7) is connected between the power supply and the electrode II; the support film with the nano holes divides the electrolyte solution chamber into a chamber I (4) and a chamber II (5), the electrode I is arranged in the chamber I, and the electrode II is arranged in the chamber II.
Preferably, the support film with the nano holes contains one nano hole or a nano hole array consisting of a plurality of nano holes.
preferably, the nanopore is a biological nanopore, a molecular self-assembly nanopore, and a solid-state nanopore.
preferably, the biological nanopore or molecular self-assembly nanopore is formed by self-assembly of one or more natural or synthetic DNA, RNA, peptide chains (Peptides) or protein molecules.
Preferably, when the nanopore is a solid nanopore, the support film is a solid nanopore material, and the solid nanopore material is any one of a teflon film, silicon nitride, silicon carbide, aluminum oxide, molybdenum disulfide, tungsten disulfide, graphene/graphene oxide, a carbon nanotube or a glass microtube.
preferably, when the nanopore is a biological nanopore or a molecular self-assembly nanopore, the support film is composed of a solid-state nanopore material and a phospholipid bilayer adsorbed on the surface of the solid-state nanopore material.
Preferably, the electrode is any one of an Ag/AgCl electrode, a glass electrode, a gold electrode, or a platinum electrode.
2. The preparation method of the antibody single-molecule detection system based on the nano-channel comprises the following steps:
(1) Preparing a support film of the nano-pores: preparing nano holes on the supporting film to form the supporting film containing one nano hole or a nano hole array consisting of a plurality of nano holes, namely the supporting film with the nano holes;
(2) Preparing a current detection system: connecting the electrode I and the electrode II with the negative electrode and the positive electrode of a power supply respectively, and then connecting an ammeter between the electrode II and the positive electrode of the power supply to form a current detection system;
(3) preparing a detection system: and (2) placing the support film with the nano holes prepared in the step (1) in an electrolyte solution chamber, dividing the electrolyte solution chamber into a chamber I and a chamber II, placing an electrode I in the current detection system formed in the step (2) in the chamber I, placing an electrode II in the chamber II, and adding an electrolyte solution into the electrolyte solution chamber to form the antibody monomolecular detection system based on the nano channel.
Preferably, when the nanopore is a biological nanopore or a molecular self-assembly nanopore, the support film with the nanopore is prepared according to the following method: firstly, preparing a solid-state nanopore material with solid-state nanopores; secondly, adsorbing a phospholipid bilayer on the solid-state nano-pore material with the solid-state nano-pores to form the supporting film; and then the supporting film with the biological nano-pores or the molecular self-assembly nano-pores can be obtained when the supporting film forms the biological nano-pores or the molecular self-assembly nano-pores by a self-assembly method.
Preferably, the electrolyte solution is any one or more of a potassium chloride aqueous solution, a sodium chloride aqueous solution, a lithium chloride aqueous solution or an ionic liquid.
3. A detection method of a nanochannel-based antibody monomolecular detection system, the method using the above-mentioned nanochannel-based antibody monomolecular detection system for detection, the method comprising the steps of:
(1) Preparing a reduction or hydrolysis reaction system of the antibody to be detected: preparing a stock solution of an antibody to be detected, adding a disulfide bond reducing agent or hydrolase, placing the mixture into a table concentrator at 37 ℃ for reaction to obtain a reduction or hydrolysis reaction system of the antibody to be detected, and storing the system in a refrigerator at-20 ℃ for later use;
(2) adding the antibody to be detected prepared in the step (1) into a chamber I in the detection system, and collecting a current signal of an ammeter in the detection system;
(3) And analyzing and processing the collected current signals to obtain an analysis result of the antibody detection.
Preferably, the antibody is of murine, rabbit or human origin.
Preferably, the antibody to be detected is an immunoglobulin or a disease-specific antibody.
Preferably, the immunoglobulin is any one of immunoglobulin g (igg), immunoglobulin M, immunoglobulin a, immunoglobulin D, or immunoglobulin E.
Preferably, the reducing agent is tris (2-carboxyethyl) phosphine (TCEP) or Dithiothreitol (DTT).
preferably, the hydrolase is either one or both of papain and pepsin
Preferably, the stock solution in step (1) uses an aqueous solution or a PBS buffer solution as a solvent.
Preferably, the aqueous solution is any one of pure water, an aqueous solution containing dnase i (dnase), an aqueous solution of RNase (RNase), or an aqueous solution of proteolytic enzyme (Protease).
preferably, the mass molar ratio of the antibody to be detected to the disulfide bond reducing agent is 5:3, g: mmol.
preferably, the current signal in step (3) comprises a signal generated by the antibody passing through the nanopore, a signal generated by the fragment passing through the nanopore after the antibody is reduced or hydrolyzed, a signal generated by the antibody interacting with the nanopore, or a signal generated by the fragment interacting with the nanopore after the antibody is reduced or hydrolyzed.
The invention has the beneficial effects that:
1. The invention discloses an antibody single molecule detection system based on a nanochannel, wherein a supporting film with a nanopore divides an electrolyte solution chamber into a chamber I and a chamber II, the nanopore allows a specific structure fragment formed by reduction or hydrolysis of a disulfide bond reducing agent or hydrolase in an antibody to be detected to pass through but not allow the antibody to be detected to pass through, and simultaneously the antibody or the reduced or hydrolyzed fragment of the antibody can generate interaction with the nanopore and acquire different current signals in an ammeter, so that the antibody to be detected is detected, the problems of unstable detection signals, poor repeatability and low detection sensitivity caused by the phenomenon that antibody molecules are easy to adsorb on the surface of a solid pore and the biological pore is blocked caused by the antibody macromolecules are solved, and high specificity and high sensitivity detection and analysis are realized;
2. the invention discloses a method for detecting by adopting an antibody single molecule detection system based on a nano channel, which comprises the steps of firstly reducing an antibody to be detected by using a disulfide bond reducing agent outside the detection system or hydrolyzing the antibody to be detected by using hydrolytic enzyme to form a specific structure fragment, then adding the specific structure fragment into a cavity in the detection system, and distinguishing different antibody molecules or different functional fragments in the antibody through the change of characteristic current signals generated by a nano hole by using a 'cut-up' specific structure fragment, thereby realizing the purpose of detection.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.
drawings
For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic structural diagram of a single-molecule detection system for antibody based on nanochannels, prepared in example 3 of the present invention;
FIG. 2 is a schematic diagram of the single molecule detection principle of the antibody based on the nanochannel prepared in example 3 of the present invention;
FIG. 3 is a diagram showing the results of a gel electrophoresis experiment performed on the reduction reaction system for an antibody to be detected in example 4 of the present invention;
FIG. 4 is a histogram of the current signal detected by the antibody single molecule detection system based on the nanochannel in example 4 of the present invention, where a is the characteristic peak of IgG antibody and b is the characteristic peak formed by the reduction product of IgG by TCEP;
FIG. 5 is a graph showing the results of further analysis of specific fragment-specific current signals of different structures detected by the antibody single-molecule detection system for nanochannels in example 4 of the present invention;
wherein: 1-nanopore, 2-lipid bilayer, 3-support membrane, 4-solution chamber I (cis), 5-solution chamber II (tras), 6-power supply, 7-amperemeter, 8-electrode I, 9-electrode II, 10-antibody molecule, 11-reducing agent, 12-antibody reduction I and 13-antibody reduction II.
Detailed Description
the embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It should be noted that the drawings provided in the following embodiments are only for illustrating the basic idea of the present invention in a schematic way, and the features in the following embodiments and examples may be combined with each other without conflict.
Example 1
Preparing a nano-channel-based antibody single-molecule detection system:
(1) preparing a support film of the nano-pores: selecting silicon nitride as a material of the supporting film, preparing and forming a single nanopore on the silicon nitride, and forming the supporting film containing one nanopore, namely the supporting film with the nanopore;
(2) Preparing a current detection system: taking Ag/AgCl electrodes as electrode materials of an electrode I and an electrode II, respectively connecting the two electrodes with a negative electrode and a positive electrode of a power supply, and then connecting an ammeter between the electrode II and the positive electrode of the power supply to form a current detection system;
(3) Preparing a detection system: and (2) placing the support film with the nano holes prepared in the step (1) in an electrolyte solution chamber, dividing the electrolyte solution chamber into a chamber I and a chamber II, placing the electrode I in the current detection system formed in the step (2) in the chamber I, placing the electrode II in the chamber II, and adding a potassium chloride aqueous solution into the electrolyte solution chamber to serve as an electrolyte solution, so that the antibody monomolecular detection system based on the nano channel can be formed.
Example 2
Preparing a nano-channel-based antibody single-molecule detection system:
(1) Preparing a support film of the nano-pores: selecting molybdenum disulfide as a solid-state nanopore material, preparing and forming a nanopore array formed by a plurality of nanopores on aluminum oxide, adsorbing a phospholipid bilayer on the surface of the solid-state nanopore material of the molybdenum disulfide, and preparing and forming biological nanopores on a support film by adopting a natural protein molecule self-assembly mode to form the support film containing the biological nanopore array as the support film with the nanopores;
(2) Preparing a current detection system: the glass electrode is used as the electrode material of the electrode I and the electrode II, the two electrodes are respectively connected with the negative electrode and the positive electrode of a power supply, and then an ammeter is connected between the electrode II and the positive electrode of the power supply to form a current detection system;
(3) Preparing a detection system: and (2) placing the support film with the nano holes prepared in the step (1) in an electrolyte solution chamber, dividing the electrolyte solution chamber into a chamber I and a chamber II, placing an electrode I in the current detection system formed in the step (2) in the chamber I, placing an electrode II in the chamber II, and adding a lithium chloride aqueous solution into the electrolyte solution chamber to serve as an electrolyte solution, so that the antibody monomolecular detection system based on the nano channel can be formed.
Example 3
Preparing a nano-channel-based antibody single-molecule detection system:
(1) Preparing a support film of the nano-pores: selecting a Teflon film as a solid-state nanopore material, preparing and forming a single micropore on the Teflon film, adsorbing a phospholipid bilayer on the surface of the Teflon film solid-state nanopore material, preparing and forming a molecular self-assembly nanopore on a support film by adopting a synthetic protein self-assembly mode, and forming the support film containing the molecular self-assembly nanopore as the support film with the nanopore;
(2) preparing a current detection system: the gold electrode is used as electrode materials of an electrode I and an electrode II, the two electrodes are respectively connected with the negative electrode and the positive electrode of a power supply, and then an ammeter is connected between the electrode II and the positive electrode of the power supply to form a current detection system;
(3) Preparing a detection system: and (2) placing the support film with the nano holes prepared in the step (1) in an electrolyte solution chamber, dividing the electrolyte solution chamber into a chamber I and a chamber II, placing the electrode I in the current detection system formed in the step (2) in the chamber I, placing the electrode II in the chamber II, and adding a sodium chloride aqueous solution into the electrolyte solution chamber to serve as an electrolyte solution, so that the antibody monomolecular detection system based on the nano channel can be formed.
The array formed by the single nanopore or the plurality of nanopores formed in the above embodiment can be replaced as required, and the solid-state nanopore material of the support film can be replaced in a teflon film, silicon nitride, silicon carbide, aluminum oxide, molybdenum disulfide, tungsten disulfide, graphene/graphene oxide, a carbon nanotube or a glass microtube as required; the electrode material can be replaced among Ag/AgCl electrodes, glass electrodes, gold electrodes or platinum electrodes according to the requirement; the electrolyte solution can be continuously replaced among a potassium chloride aqueous solution, a sodium chloride aqueous solution, a lithium chloride aqueous solution or an ionic liquid according to the requirement; the same nanopore on the supporting membrane can be a solid-state nanopore (formed by directly using a solid-state nanopore material as a supporting membrane and then preparing the supporting membrane), or a biological nanopore or molecular self-assembly nanopore (formed by self-assembly of one or more natural or artificially synthesized DNA, RNA, peptide chains (Peptides) or protein molecules on the supporting membrane, wherein the supporting membrane consists of the solid-state nanopore material and phospholipid bimolecules adsorbed on the solid-state nanopore material).
Example 4
antibody IgG was detected using the nanochannel-based antibody single molecule detection system prepared in example 3, the detection system comprising: the detection method comprises the following steps of (1) nanopore 1, phospholipid bilayer 2, support film 3, solution chamber I (cis)4, solution chamber II (tras)5, power supply 6, ammeter, electrode I8 and electrode II9, wherein the detection principle is shown in figure 2, and the specific detection method is as follows:
(1) preparing a reduction reaction system of an antibody to be detected: dissolving immunoglobulin G (IgG) of an antibody to be detected in PBS buffer solution with the pH value of 8.0 to prepare stock solution, wherein the content of the IgG in the stock solution is 5mg/ml, adding a disulfide bond reducing agent tris (2-carboxyethyl) phosphine (TCEP) according to the mass molar ratio of 5:3(g: mmol) of the IgG to the IgG, placing the mixture in a shaking table at 37 ℃ for reaction to obtain an antibody reduction reaction system to be detected, reducing the antibody to be detected by the disulfide bond reducing agent to form fragments, namely shearing the disulfide bond, wherein the antibody reduction reaction system to be detected contains an antibody molecule 10, a reducing agent 11, an antibody reduction product I12 and an antibody reduction product II13, storing the antibody reduction reaction system to be detected in a refrigerator at-20 ℃ for later use, verifying the prepared antibody reduction reaction system to be detected by an electrophoresis method, and the result is shown in figure 3, wherein a strip No. 1 is Marker, and a strip No. 2, No. 3 is the product after TCEP reduces IgG, and proves that TCEP partially reduces IgG antibody;
(2) Adding the antibody to be detected reduction reaction system prepared in the step (1) into a chamber I in the detection system prepared in the embodiment 3, collecting a current signal of an ammeter in the detection system, and amplifying the signal by using a low-noise current amplifier (AxoAxoxpatch 200B);
(3) And analyzing the collected current signal to obtain an analysis result of antibody detection, wherein the obtained data is processed and analyzed by utilizing analytical instrument software (Clampfit), and the main analysis object is the amplitude (the magnitude of the blocking current) and the signal duration of IgG and reduction products thereof passing through the nanopore. Fig. 4 is a histogram of the signals detected by the antibody and its reduction product with TCEP in the alpha-hemolysin protein nanopore, where a is the characteristic peak of the detected antibody, and b is the characteristic peak of the reduction product with TCEP, and comparison with the characteristic peak of the standard IgG antibody and its reduction product can achieve effective antibody detection.
(4) The reduced characteristic current signal is further analyzed, and the result is shown in fig. 5, which illustrates that the detection method of the present invention can be used to distinguish different functional fragments of antibodies or to distinguish or different antibody molecules according to the difference of specific fragment structures and the change of the characteristic current signal generated by the nanopore. Compared with other methods (such as ELISA, single-molecule fluorescence, gel electrophoresis and the like), the detection method disclosed by the invention can realize label-free, rapid and instant detection and analysis, and can avoid the defects that similar functional fragments have similar molecular weights and cannot be distinguished by gel electrophoresis.
the same detection system and detection method prepared by the invention can realize the corresponding detection of other antibodies (such as immunoglobulin M (IgM), immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE) or specific antibodies (such as HIV) of related diseases) from mouse sources, rabbit sources or human sources, and any one of pure water, an aqueous solution containing deoxyribonuclease I (DNase), an aqueous solution of ribonuclease (RNase) or an aqueous solution of proteolytic enzyme (Protease) can be used as a solvent when preparing a stock solution to be detected, and Dithiothreitol (DTT) can be selected as a corresponding reducing agent; meanwhile, a hydrolytic enzyme can be used for preparing a hydrolysis reaction system of the antibody to be detected, and the corresponding hydrolytic enzyme is papain or pepsin.
In summary, the following steps: the invention discloses an antibody single molecule detection system based on a nanochannel, wherein a supporting film with a nanopore divides an electrolyte solution chamber into a chamber I and a chamber II, the nanopore allows a specific structure fragment formed by reduction of a disulfide bond reducing agent or hydrolysis of hydrolytic enzyme in an antibody to be detected to pass through but not allow the antibody to be detected to pass through, so that different current signals are acquired in an ammeter, the antibody to be detected is detected, the problems of unstable detection signals, poor repeatability, low detection sensitivity and the like caused by the biological pore blocking phenomenon caused by antibody macromolecules due to easy adsorption of antibody molecules on the surface of a solid pore are solved, and high specificity and high sensitivity detection and analysis are realized; the invention also discloses a method for detecting by adopting the antibody single molecule detection system based on the nano channel, firstly, the detection system is externally applied with a disulfide bond reducing agent or hydrolase to reduce or hydrolyze the antibody to be detected to form a specific structure fragment, then the specific structure fragment is added into a chamber in the detection system, and the specific structure fragment which is cut up can be used for distinguishing different antibody molecules or different functional fragments in the antibody through the change of characteristic current signals generated by the nano hole, thereby realizing the purpose of detection.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.
Claims (10)
1. A nanochannel-based antibody single-molecule detection system, comprising an electrolyte solution chamber, a support membrane with nanopores, and a current detection system; the current detection system comprises a power supply (6), an electrode I (8) and an electrode II (9) which are respectively connected with the power supply, and an ammeter (7) is connected between the power supply and the electrode II; the support film with the nano holes divides the electrolyte solution chamber into a chamber I (4) and a chamber II (5), the electrode I is arranged in the chamber I, and the electrode II is arranged in the chamber II.
2. the antibody single-molecule detection system based on the nano-channel, according to claim 1, wherein the support film with the nano-channel comprises a nano-hole or a nano-hole array composed of a plurality of nano-holes, the nano-holes are biological nano-holes, molecular self-assembly nano-holes and solid-state nano-holes, and the biological nano-holes or the molecular self-assembly nano-holes are formed by self-assembly of one or more of natural or artificially synthesized DNA, RNA, peptide chains or protein molecules.
3. the antibody single-molecule detection system based on the nanochannel according to claim 2, wherein when the nanopore is a solid nanopore, the supporting film is a solid nanopore material, and the solid nanopore material is any one of a teflon film, silicon nitride, silicon carbide, aluminum oxide, molybdenum disulfide, tungsten disulfide, graphene/graphene oxide, a carbon nanotube, or a glass microtubule; when the nano-pores are biological nano-pores or molecular self-assembly nano-pores, the supporting film is composed of a solid nano-pore material and a phospholipid bilayer adsorbed on the surface of the solid nano-pore material.
4. The antibody single molecule detection system based on the nano-channel, according to claim 1, wherein the electrode is any one of Ag/AgCl electrode, glass electrode, gold electrode or platinum electrode.
5. The method for preparing a nano-channel based antibody single molecule detection system according to any one of claims 1 to 4, wherein the method comprises the following steps:
(1) Preparing a support film of the nano-pores: preparing nano holes on the supporting film to form the supporting film containing one nano hole or a nano hole array consisting of a plurality of nano holes, namely the supporting film with the nano holes;
(2) Preparing a current detection system: connecting the electrode I and the electrode II with the negative electrode and the positive electrode of a power supply respectively, and then connecting an ammeter between the electrode II and the positive electrode of the power supply to form a current detection system;
(3) Preparing a detection system: and (2) placing the support film with the nano holes prepared in the step (1) in an electrolyte solution chamber, dividing the electrolyte solution chamber into a chamber I and a chamber II, placing an electrode I in the current detection system formed in the step (2) in the chamber I, placing an electrode II in the chamber II, and adding an electrolyte solution into the electrolyte solution chamber to form the antibody monomolecular detection system based on the nano channel.
6. The method for preparing the antibody single-molecule detection system based on the nanochannel according to claim 5, wherein the electrolyte solution is any one or more of potassium chloride aqueous solution, sodium chloride aqueous solution, lithium chloride aqueous solution or ionic liquid.
7. A method for detecting antibody single molecule based on nanometer channel, which is characterized in that the method adopts the antibody single molecule detection system based on nanometer channel as described in any one of claims 1-4, and the method comprises the following steps:
(1) preparing a reduction or hydrolysis reaction system of the antibody to be detected: preparing a stock solution of an antibody to be detected, adding a disulfide bond reducing agent or hydrolase, placing the mixture into a table concentrator at 37 ℃ for reaction to obtain a reduction or hydrolysis reaction system of the antibody to be detected, and storing the system in a refrigerator at-20 ℃ for later use;
(2) adding the antibody to be detected prepared in the step (1) into a chamber I in the detection system, and collecting a current signal of an ammeter in the detection system;
(3) and analyzing and processing the collected current signals to obtain an analysis result of the antibody detection.
8. The detection method according to claim 7, wherein the antibody is derived from a murine source, a rabbit source or a human source, the antibody to be detected is an immunoglobulin or a disease-specific antibody, the immunoglobulin is any one of immunoglobulin G, immunoglobulin M, immunoglobulin A, immunoglobulin D or immunoglobulin E, and the disease-specific antibody is human immunodeficiency virus; the reducing agent is tri (2-carboxyethyl) phosphine or dithiothreitol, and any one or two of hydrolase papain or pepsin.
9. the detection method according to claim 7, wherein an aqueous solution or PBS buffer solution is used as a solvent in the stock solution in the step (1), wherein the aqueous solution is any one of pure water, an aqueous solution containing DNase I, an aqueous solution of RNase or an aqueous solution of proteolytic enzyme; the mass molar ratio of the antibody to be detected to the disulfide bond reducing agent is 5:3, g: mmol.
10. The detection method according to claim 7, wherein the current signal in step (3) comprises a signal generated by the antibody passing through the nanopore, a signal generated by the fragment passing through the nanopore after the antibody is reduced or hydrolyzed, a signal generated by the antibody interacting with the nanopore, or a signal generated by the fragment interacting with the nanopore after the antibody is reduced or hydrolyzed.
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Inventor after: Wang Liang Inventor after: Wang Han Inventor after: Wang Deqiang Inventor after: Liu Qianshan Inventor after: Wang Binjiao Inventor before: Wang Liang Inventor before: Wang Deqiang Inventor before: Liu Qianshan Inventor before: Wang Binjiao Inventor before: Wang Han |