CN112941160B

CN112941160B - Nanopore sequencing method based on nano manipulation

Info

Publication number: CN112941160B
Application number: CN202110114328.6A
Authority: CN
Inventors: 方绍熙; 王德强; 袁家虎; 何石轩; 殷博华; 谢婉谊; 石彪; 罗志勇; 唐鹏
Original assignee: Chongqing Institute of Green and Intelligent Technology of CAS
Current assignee: Chongqing Institute of Green and Intelligent Technology of CAS
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2023-07-07
Anticipated expiration: 2041-01-27
Also published as: CN112941160A

Abstract

The invention discloses a nanopore sequencing method based on nanometer manipulation, which can simultaneously realize free capture of DNA molecules and multiple-degree-of-freedom nanometer manipulation, is based on an in-situ nanometer manipulation nanopore preparation technology, prepares double nanopores with controllable aperture and interval, and controls the relative displacement of a prepared nanoprobe and the double nanopores; adding DNA molecules which can be captured and identified by the double nanopores, after the DNA molecules are freely captured by the double nanopores, manipulating the nanoprobes to stir the DNA molecules between the double nanopores, and performing nano manipulation according to sequencing requirements. And detecting current signals of the DNA molecules which are uniformly operated to enter and exit the nano holes, so as to analyze the sequence information of the DNA molecules to be detected. The method solves the problem of single molecule manipulation and further solves the problem of nanopore sequencing.

Description

Nanopore sequencing method based on nano manipulation

Technical Field

The invention belongs to the technical field of measurement, and relates to a nanopore sequencing method based on nano manipulation, which can be suitable for DNA molecular sequencing by using a nanopore.

Background

Nanopore testing technology arose in the 90 s of the 20 th century and was vigorously developed to achieve nanopore sequencing. In 1996, researchers have achieved via detection of DNA molecules using biological nanopores, which has been developed over 16 years, proved to be able to perform DNA molecular sequencing in 2012, and developed the first commercial DNA sequencer from oxford nanopores in 2014.

The core technology of the nanopore sequencer comprises stability control of the nanopore, and improvement of detection spatial resolution and time resolution. The biological nano-pore is pushed out of the sequencer in preference to the solid nano-pore, and the main reason is that the pore diameter is stable, so that the consistency of sequencing signals is ensured. And the time resolution is controlled by a molecular motor (polymerase or helicase), so that the via speed of the DNA molecule can be reduced to a level which can be recognized by the instrument.

However, the molecular biological motor control DNA molecules still have a series of problems, firstly, the molecular motor and the biological nano-pore are fixed on a fragile gel bilayer, are greatly influenced by factors such as environment and the like, and are easily damaged in the use process; secondly, the manipulation of DNA molecules by molecular motors is determined by their own biological forces, which, although can be addressed by adjusting temperature, PH and designing different types of molecular motors, can still have a significant impact on the final detection accuracy; finally, molecular motor control is the unwinding or polymerization of DNA molecules, which is speed controlled based on the mechanism of DNA unwinding or polymerization, with the limitations of DNA molecule selection.

Therefore, it is particularly necessary to use more direct nano-manipulation techniques to solve the problem of the speed of molecular sequence vias. Magnetic forceps, optical forceps, atomic force probes, optical fiber probes, silicon probes, dual nanopore systems, and the like are all some feasible methods. The magnetic forceps, the optical forceps, the atomic force probe, the optical fiber probe and the like have higher manipulation precision and degree of freedom, but the probability of capturing manipulation molecules is smaller; the silicon probe solves the problem of capture rate, but the interference of adjacent molecules cannot be removed; the double-nanopore system has a certain feasibility, but separate detection of the double-nanopore system cannot be realized in engineering.

Disclosure of Invention

Based on the technical problems existing in the prior art, the invention provides a nanopore sequencing method based on nano manipulation, which is based on an in-situ nano manipulation nanopore preparation technology, and the sequence information of a DNA molecule to be detected is analyzed by detecting current signals of the DNA molecule which is uniformly manipulated into and out of a nanopore; the method solves the problem of single molecule manipulation and further solves the problem of nanopore sequencing.

According to the technical scheme of the invention, a nanopore sequencing method based on nano manipulation is provided, and the method comprises the following steps.

Step S1, building a nanopore preparation system based on nano manipulation.

Step S2, designing DNA library molecules, namely, using the DNA library molecules as starting/stopping signal marks of DNA sequencing signals, as a judging basis of capturing the DNA molecules by the double nanopores, and also as a molecular target which is used for fixing the DNA molecules between the double nanopores and can be stirred by a probe.

S3, preparing a solution to be tested; the DNA molecules to be tested are modified onto a DNA library.

Step S4, monitoring the captured condition of the DNA molecules to be detected; and adding the modified DNA molecule solution, and observing the condition that the DNA molecules are captured by the double nano holes by detecting current signals.

Step S5, manipulating the nano-probe to contact with the DNA library molecules.

Step S6, manipulating the DNA molecules to exit and enter the nanopore.

Step S7, analyzing the nanopore DNA sequencing signal.

The nanopore preparation system comprises a three-dimensional nanometer operation platform, a nanometer probe electrode, a nanometer film chip, an electrode holder, a buffer pool, a light source, a microscope, a constant current source, a weak current detection system and a Faraday shielding cover, wherein the connection or the position relationship between each component is that the nanometer probe electrode is fixed on the electrode holder, and the electrode holder is fixed on the three-dimensional nanometer operation platform; the nanometer film chip is arranged in the buffer tank, and is connected with the constant current source and the negative end of the weak current detection system through the lower layer solution in the buffer tank; the nanometer probe electrode is connected to the positive end of the constant current source, and when the nanometer probe electrode contacts with the nanometer film chip, a current loop of the constant current source is formed; after the buffer solution is added into the upper layer of the buffer pool, the positive electrode of the weak current detection system is immersed into the solution to form a detection circuit path, and DNA library molecules and DNA molecules can perform Brownian motion in the solution; when a weak current detection system applies voltage, DNA library molecules and DNA molecules are partially captured by the first nano-pore and the second nano-pore; the microscope and the light source are positioned at the left side and the right side of the nano probe electrode to form a reflecting loop, so that the contact condition of the nano probe and the nano film chip can be observed conveniently; the Faraday shielding cover shields the three-dimensional nanometer operation platform, the nanometer probe electrode, the nanometer film chip, the electrode holder, the buffer pool, the light source, the microscope, the constant current source and the weak current detection system, and small holes are reserved for being used as wire holes for connecting the three-dimensional nanometer operation platform, the nanometer probe electrode, the nanometer film chip, the electrode holder, the buffer pool, the light source, the microscope, the constant current source and the weak current detection system with the circuit control system.

Preferably, the nanoprobe electrode is a nanoscale conductive electrode with a tip of less than 1 um.

More preferably, the nano-probe electrode is a nano-scale gold, platinum or silver electrode probe, or a gold-plated nano-scale tungsten steel needle or an optical fiber probe, or a nano-scale glass microtube filled with a conductive solution.

Preferably, the nano probe electrode is fixed on the three-dimensional nano operating platform through the electrode holder and is controlled by the platform, so that the high-precision nano motion with multiple degrees of freedom is realized.

In addition, the initiation/termination signal is labeled, which means that when the designed DNA molecule enters or exits the nanopore, the signal recognized by the detection circuit is known, and the site is the binding site between the molecule to be detected and the library molecule.

Preferably, the modification of the DNA molecule refers to the biological, physicochemical binding of the DNA molecule to be tested to the DNA library molecule.

Preferably, the via signal captured as a designed DNA molecule is detected by the current detection system and is captured by both nanopores.

Further, the exiting and entering of the DNA molecules into the nanopores refers to the movement condition that the DNA molecules to be detected are driven when the nanoprobe dials the DNA library molecules to move; the movement speed can be flexibly controlled by setting the control parameters of the nano-manipulation system.

Further, analyzing the nanopore DNA sequencing signal includes determining a start or stop signal of the DNA molecule to be detected entering and exiting the nanopore, a via signal of different nanopores of the dual nanopore system, and an accumulated signal of a differential signal of the dual nanopore system.

Compared with the prior art, the nanopore sequencing method based on nano manipulation can use the nanopore to sequence DNA molecules, and particularly can control the DNA molecules to enter and exit the nanopore, so as to realize direct sequencing of the DNA molecules. The sequencing method solves the problems of easy fatigue and selectivity of a molecular motor, improves the problem of low capture rate of the traditional nano-manipulation DNA molecules entering the nano holes, and can be especially expanded to the sequencing fields of RNA molecules, protein molecules and the like.

The nano-pore sequencing method based on nano-manipulation is based on an in-situ nano-manipulation nano-pore preparation technology, prepares double nano-pores with controllable pore diameters and spacing, and realizes the sequencing of DNA molecules by manipulating the DNA molecules to enter and exit a double nano-pore system through a nano probe. The method of the invention also has the following advantages.

1. The nano-manipulation is not influenced by environmental and time factors, and has the advantages of multiple degrees of freedom and high control precision.

2. The positions of the nano probe and the nano hole are accurate and controllable, and repeated positioning is not needed.

3. The aperture and the distance of the double-nanopore system are flexibly adjustable, and the resolution ratio of nanopore sequencing can be further improved by calculating the differential signal of the double nanopores under the same measuring environment.

4. The free movement of DNA molecules is combined with nano manipulation, so that the problem of low capture rate of DNA molecule manipulation fixed by the traditional method is solved.

5. The whole nano manipulation process is physical acting force, does not involve the influence of matching of chemical and biological actions, and can be directly extended to RNA and protein sequencing.

Drawings

FIG. 1 is a schematic diagram of DNA sequencing based on a nanopore sequencing method of nanomanipulation in accordance with the present invention.

FIG. 2 is a block diagram of a nanopore preparation and DNA sequencing instrument according to the present invention.

FIG. 3 is a block diagram of a split electrode dual nanopore sequencing in accordance with the present invention.

FIG. 4 is a graph of DNA sequencing via signals according to the present invention.

Wherein the reference numerals are as follows:

1: three-dimensional nanometer manipulation platform, 2: nanoprobe, 3: nano film chip, 4: first nanopore, 5: second nanopore, 6: DNA library molecules, 7: test DNA molecule, 101: electrode holder, 104: buffer pool, 102: light source, 103: microscope, 105: constant current source, 106: weak current detection system, 107: faraday shield, 201: signal of DNA molecule entry into nanopore, 202: the DNA molecule exits the nanopore signal.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Additionally, the scope of the invention should not be limited to the specific structures or components or specific parameters described below.

The invention provides a nanopore sequencing method based on nano manipulation, which is similar to a method for improving DNA molecule capture rate by a double-nano system and a DNA molecule nano manipulation method with high precision and multiple degrees of freedom such as an atomic force probe. The nanopore sequencing method based on nano manipulation can simultaneously realize free capture of DNA molecules and multi-degree-of-freedom nano manipulation, and is based on a process route of capturing before manipulating. The nano-pore sequencing method based on nano-manipulation is based on an in-situ nano-manipulation nano-pore preparation technology, prepares double nano-pores with controllable pore diameters and spacing, and controls the relative displacement of the prepared nano-probe and the double nano-pores; adding DNA molecules which can be captured and identified by the double nanopores, after the DNA molecules are freely captured by the double nanopores, manipulating the nanoprobes to stir the DNA molecules between the double nanopores, and performing nano manipulation according to the sequencing requirement; and detecting current signals of the DNA molecules which are uniformly operated to enter and exit the nano holes, so as to analyze the sequence information of the DNA molecules to be detected.

The invention is realized by the following design scheme:

(1) And building a nanopore preparation system based on nano manipulation. The system provides a hardware platform for a DNA sequencing instrument, and rapidly prepares double nano holes with controllable aperture and interval according to DNA sequencing requirements.

As shown in fig. 1 and 2, the nanopore preparation system based on nano-manipulation includes a three-dimensional nano-manipulation stage 1, a nano-probe electrode 2, a nano-thin film chip 3, an electrode holder 101, a buffer pool 104, a light source 102, a microscope 103, a constant current source 105, a weak current detection system 106, and a faraday shield 107.

Wherein, three-dimensional nanometer manipulation platform 1, it is used for fixing and controlling the nanometer level multiple freedom degree motion of nanometer probe electrode 2, and it is located faraday shield 107, and electrode holder 101 is fixed on nanometer manipulation platform 1 through hexagon socket head cap screw, and nanometer probe electrode 2 is fixed on electrode holder 101.

The nano probe electrode 2 is used for preparing a solid first nano hole 4, a solid second nano hole 5 and a nano manipulation DNA library molecule 6, is fixed on the electrode holder 101, is controlled to be contacted with the nano film chip 3 by the three-dimensional nano manipulation platform 1, and performs multi-degree-of-freedom movement between the prepared double nano holes to realize nano manipulation of the DNA library molecules 6 and the DNA molecules 7.

The nano-film chip 3 is used for preparing the first nano-hole 4 and the second nano-hole 5 to form a double-nano-hole sequencing system, the double-nano-hole sequencing system is arranged in the buffer pool 104, the nano-film chip 3 is connected with the constant current source 105 through a lower layer solution in the buffer pool 104, and the nano-film chip 3 is connected with the negative end of the weak current detection system 106; the nano probe electrode 2 is connected to the positive end of the constant current source 105, and when the nano probe electrode 2 is contacted with the nano film chip 3, a current loop of the constant current source 105 is formed, and a solid nano hole is prepared; after the buffer solution is added into the upper layer of the buffer pool 104, the positive electrode of the weak current detection system 106 is immersed into the solution to form a detection circuit path, and the DNA library molecules 6 and the DNA molecules 7 can perform Brownian motion in the solution; when a weak current detection system 106 applies a voltage, the DNA library molecules and the DNA molecules 7 are partially captured by the first nanopore 4 and the second nanopore 5, and DNA molecule sequencing is performed.

The electrode holder 101 is used for fixing the nano-probe electrode 2, is convenient for installing the nano-probe electrode 2, is fixed on the nano-operating platform 1 through an inner hexagon screw, and the nano-probe electrode 2 is fixed on the electrode holder.

A buffer cell 104 for mounting and fixing the nano-film chip 3 and storing buffer solution and sample molecules required for nanopore preparation and sequencing, which is located below the nanoprobe electrode 2 and on the observation light path of the microscope 103 and the light source 102.

A light source 102 for providing an illumination light source for microscopic observation of the nanoprobe electrode 2 and the nanofilm chip 3, which is located at one side of the nanoprobe electrode 2 while being fixed on the bottom plate of the faraday shield 107.

A microscope 103 for observing the nanoprobe electrode 2 and the nanofilm chip 3 for aiding in the observation of nanopore preparation and sequencing processes, which is located on one side of the nanoprobe electrode 2 and simultaneously fixed on the bottom plate of the faraday shield 107.

The constant current source 105 is used for providing a constant current source for preparing the solid first nano hole 4 and the solid second nano hole 5, and is used for preparing double nano holes with different apertures, the electrode parts of the constant current source are respectively connected with the nano probe electrode 2 and the solution at the lower end of the buffer pool 104 to form a solid nano hole preparation loop, and the instrument parts of the constant current source are arranged in a cabinet outside the Faraday shielding cover 107 and are connected with the electrode through a small connecting hole of the Faraday shielding cover 107.

The weak current detection system 106 is used for detecting current signals of DNA molecule through holes to realize DNA molecule sequencing, electrode parts of the weak current detection system are respectively connected with upper and lower end solutions of the buffer pool 104 to form a double-nanopore current detection loop, and instrument parts of the weak current detection system are arranged in a cabinet outside the Faraday shielding cover 107 and are connected with the electrodes through small connecting holes of the Faraday shielding cover 107.

The faraday shield 107, which is used to shield external electromagnetic wave interference and to improve the accuracy of nanopore preparation and nanopore sequencing signals, is located outside the whole sequencer, constitutes the main structural housing of the sequencer, and leaves small holes as the wire holes for these main structures to connect with its circuit control system.

Further, as shown in fig. 2, the nano-probe electrode 2 is a nano-scale conductive electrode with a needle tip smaller than 1um, and may be a nano-scale electrode probe of gold, platinum, silver, or the like, a nano-scale tungsten steel needle or an optical fiber probe subjected to gold plating treatment, or a nano-scale glass microtube filled with a conductive solution.

The nano-probe electrode 2 is fixed on the three-dimensional nano-manipulation platform 1 through the electrode holder 101, and can realize high-precision nano-motion with multiple degrees of freedom under the control of the platform.

The nano probe electrode 2 is electrically communicated with the positive electrode of the constant current source 105 power supply, and provides a communication electrode for ultra-high electric field and nano pore diameter test required by nano pore preparation.

The nano film chip 3 is fixed in a buffer pool 104, and a lower layer solution in the buffer pool is connected with a constant current source 105 and the negative end of a weak current detection system 106 to provide a circuit loop for nanopore preparation and DNA testing.

The light source 102 and the microscope 103 are externally added microscope observation systems, so that the positions of the nanopore chip 3 and the nanoprobe 2 can be observed in real time, and an initial positioning basis is provided.

The positive electrode of the weak current detection system 106 is connected with the DNA molecule solution to be detected which is added after the preparation of the nanopore is completed, and the detection of the current of the DNA molecule via hole is realized through communication.

The DNA sequencing requirement means that the nanopore system meets the DNA sequencing requirement, including three aspects of nanopore aperture, nanopore thickness and nanopore stability.

The rapid preparation of the nanopore means that the total preparation time of the nanopore needs to be kept at a second level, namely the whole preparation process needs to be completed within a few seconds, and the efficiency of whole sequencing is not affected.

The controllable aperture means that the diameter of the nano hole can be flexibly adjusted according to the test requirement, and the hole type can be flexibly adjusted; the adjustable range of the aperture of the nano-pore in the system is 0.5nm-60nm.

The controllable spacing means that the spacing between two nanopores can be flexibly adjusted, namely, the spacing can be flexibly adjusted in real time according to the requirements of DNA molecules and the capture rate, and the adjustable precision is 20nm, namely, the minimum controllable spacing is 20nm, which is far higher than the requirements of the optimal capture spacing of two nanopores of 100nm-500 nm.

The double-nanopore refers to two nanopore systems with independent sizes and positions, and a detection circuit of the two nanopores can be designed into a split electrode mode (shown in figure 3) and a common electrode mode. Correspondingly, when signal analysis is carried out, the DNA molecule via signals can be distinguished according to different pore sizes.

(2) The DNA library molecule 6 is designed, namely, the DNA library molecule is used as an initial/termination signal mark of a DNA sequencing signal, is used as a discrimination basis for capturing DNA molecules by double nanopores, and is also used as a molecular target for fixing the DNA molecules between the double nanopores and being stirred by a probe.

The initiation/termination signal mark refers to that when the designed DNA molecule enters or exits the nanopore, the signal identified by the detection circuit is known, and the site is the binding site of the molecule to be detected and the library molecule, so that the detection signal can be used as the initiation/termination point of the sequencing of the DNA molecule to be detected.

The basis of discrimination of the double-nanopore captured DNA molecule is consistent with the principle of taking the double-nanopore captured DNA molecule as a starting/ending point, the blocking signal of the designed DNA molecule entering two nanopores is known, and the captured situation of the DNA molecule can be monitored in real time through a detection signal.

The molecular target means that the designed DNA molecule diameter can be designed according to the stirring condition of the nano probe and can be larger than the aperture of the double nano holes, so that the DNA molecule can not directly pass through the nano holes after being captured, and the feasibility and the accuracy of nano manipulation can be improved.

(3) Preparing a solution to be tested. The DNA molecules to be tested are modified 7 onto the DNA library 6.

The DNA molecule modification refers to the combination of the DNA molecule 7 to be detected and the DNA library molecule 6 through biological and physical-chemical methods.

(4) The capture of the DNA molecule is monitored. And adding the modified DNA molecule solution, and observing the condition that the DNA molecules are captured by the double nano holes by detecting current signals.

The captured signal is the via signal of the designed DNA molecule, is detected by the current detection system, and is captured by both nanopores.

(5) The nanoprobe is manipulated and contacted with the DNA library molecules.

The control nano-probe is a probe 2 used in the preparation of the nano-hole, and the relative positions of the control nano-probe and the double nano-

holes

4 and 5 are precisely controlled in the preparation of the nano-hole, so that after the DNA molecule is determined to be freely captured by the nano-hole, the nano-probe is stirred, and the stress condition and the current detection system of the probe are detected. And then determining that the nano probe is contacted with the DNA library molecules and poking the DNA molecules to move so as to realize the entry and exit of the DNA molecules into and from the nano holes.

(6) The DNA molecules are manipulated out of and into the nanopore.

The exiting and entering of the DNA molecules into the nanopores refers to the movement condition that the DNA molecules to be detected are driven when the nanoprobe dials the DNA library molecules to move. The movement speed can be flexibly controlled by setting the control parameters of the nano-manipulation system.

(7) Analyzing the nanopore DNA sequencing signal.

The nanopore DNA sequencing signal is a current signal generated when a nanopore is accessed and exited by a nanopore manipulation DNA molecule, and analyzing the nanopore DNA sequencing signal comprises judging the starting or stopping signal of the access and the exit of the DNA molecule to be detected from the nanopore, the via hole signals of different nanopores of a double nanopore system and the accumulated signal of the differential signals of the double nanopore system. Through subsequent signal processing technology and analysis technology, the sequence signal of the DNA molecule can be effectively identified.

Furthermore, the nanopore sequencing method based on nano manipulation comprises two parts of nanopore preparation and DNA sequencing, and the nanopore preparation and sequencing work is completed through one-time parameter setting, and the specific steps are as follows.

(1) Preparation work

Preparing a system: preparing a nano film chip with a suspended window structure, fixing the chip in a flow cell, ensuring that a window opening part below is contacted with a solution at the lower end of the flow cell, and connecting electrodes; a nanoelectrode, such as a glass microtube drawn to 100nm-1um, is prepared, filled with 1M LiCl buffer solution, and mounted on a nano control platform above the nano film.

Sample preparation: adding the DNA molecule solution to be tested into the DNA library molecule solution according to the proportion, and carrying out mixed modification to ensure that the DNA molecule to be tested is connected to two ends of the DNA library molecule.

(2) Parameter setting

And setting system nanopore processing parameters and DNA sequencing parameters. The nanopore processing parameters comprise the thickness parameters of the nanopore chip (0.35 nm-200 nm), the aperture parameters of the nanopore processing target (0.5 nm-60 nm), the ultrahigh electric field of the nanopore processing (depending on the materials used, such as silicon nitride is higher than 1V/nm), the position of the nanopore (the planar position of the double nanopore on the nanopore, the control precision is 10 nm), and the distance between the double nanopores (the control precision depends on the size of the tip of the nanoelectrode, such as 20nm control precision can be realized by adopting a gold probe of 10 nm). The DNA sequencing parameters comprise the judgment value of the through hole current of DNA library molecules under the corresponding electric field, the capturing current of the nanopores, the DNA sequencing time length and the like.

(3) First nanopore preparation

According to the thickness of the nanometer film set in the parameter setting, the aperture parameter of the first nanometer hole, the position of the first nanometer hole, the instantaneous preparation nanometer hole processing electric field is applied, the nanometer manipulation nanometer electrode approaches the nanometer film, when the generation of the nanometer hole is detected, the software judges that the reaming pulse current is automatically generated, and the IV curve test is inserted, the aperture of the nanometer hole is judged in real time, and then the high-precision (less than 0.2 nm) rapid preparation (less than 10 s) of the first nanometer hole is realized.

And lifting the nano probe by 2um after the processing is finished, controlling the nano probe to be above the second nano hole at the horizontal position, and preparing the second nano hole.

(4) Second nanopore preparation

And the method is the same as that of processing the first nano hole, and according to the thickness of the nano film, the aperture parameter of the second nano hole and the position of the second nano hole which are set in parameter setting, an instantaneous nano hole processing electric field is applied, the nano control nano electrode approaches the nano film, when the generation of the nano hole is detected, a reaming pulse current is automatically generated by software judgment, and an IV curve test is inserted, the aperture of the nano hole is judged in real time, and then the high-precision (the diameter is smaller than 0.2 nm) rapid preparation (the time is smaller than 10 s) of the second nano hole is realized.

After the processing is finished, the nano probe is lifted by 2um, the movement of the nano probe between the first nano hole and the second nano hole is controlled at the horizontal position, and the nano probe is controlled to approach the nano film under the condition of no electric field.

(5) Nanometer probe positioning

According to the binding points of the DNA library molecules and the nano probes, the nano probes are controlled to flexibly move between the first nano holes and the second nano holes, so that preparation is made for the nano probes to stir the DNA molecules captured by the double nano holes.

(6) Adding the sample

And (3) adding the sample solution prepared in the step (1) to one side of the nano probe, applying an electric field to two ends of the double nano holes through a solution electrode, and recording the current change of the double nano holes.

(7) Capture of DNA molecules

And monitoring the current change of the double nanopores, and judging the capture of the DNA molecules by the double nanopores according to the current basis (theoretical and experimental combined empirical values) of the DNA library molecules captured by the nanopores in the parameter setting.

(8) Nano-manipulation DNA molecules

After the DNA library molecules are captured by the double nanopores, the nano-manipulation nano-probe moves linearly in the direction perpendicular to the connecting line of the double nanopores, and the change of the current of the nanopores is monitored in real time.

Repeatedly manipulating the DNA molecules to exit and enter the nanopore, and feeding back sequencing signals to the analysis software system in real time.

(9) Reading and analyzing DNA sequences

According to the signals of the DNA library molecules entering and exiting the nano holes, the starting point and the ending point of the DNA molecules to be detected are determined, and then the DNA sequence signals are analyzed once, finally the DNA sequence signals verified by repeated tests are obtained, and the sequencing of the DNA molecules is realized.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

1. A nanopore sequencing method based on nano manipulation, characterized in that: which comprises the following steps:

step S1, building a nanopore preparation system based on nano manipulation;

s2, designing DNA library molecules, wherein the DNA library molecules are used as starting/stopping signal marks of DNA sequencing signals, are used as a judging basis for capturing DNA molecules to be detected by the double nanopores, and are also used as molecular targets for fixing the DNA molecules to be detected between the double nanopores and being stirred by probes;

s3, preparing a solution to be detected, and modifying DNA molecules to be detected on a DNA library;

s4, monitoring the capturing condition of the DNA molecules to be detected, adding the modified DNA molecule solution to be detected, and observing the capturing condition of the DNA molecules to be detected by the double nanopores through detecting current signals;

s5, manipulating the nano-probe to contact with DNA library molecules;

s6, manipulating the DNA molecules to be detected to exit and enter the nano holes;

s7, analyzing a nanopore DNA sequencing signal;

the nanopore preparation system comprises a three-dimensional nanometer operation platform (1), a nanometer probe (2), a nanometer film chip (3), an electrode holder (101), a buffer pool (104), a light source (102), a microscope (103), a constant current source (105), a weak current detection system (106) and a Faraday shielding cover (107), wherein the nanometer probe (2) is fixed on the electrode holder (101), and the electrode holder (101) is fixed on the three-dimensional nanometer operation platform (1); the nano film chip (3) is arranged in the buffer tank (104) and is connected with the negative end of the weak current detection system (106) through a lower layer solution in the buffer tank (104); the nano probe (2) is connected to the positive end of the constant current source (105), and a current loop of the constant current source (105) is formed when the nano probe (2) is in contact with the nano film chip (3); after the buffer solution is added into the upper layer of the buffer pool (104), the positive electrode of the weak current detection system (106) is immersed into the solution to form a detection circuit path, and the DNA library molecules (6) and the DNA molecules (7) to be detected perform Brownian motion in the solution; when a weak current detection system (106) applies voltage, the DNA library molecules and the DNA molecules (7) to be detected are partially captured by the first nano-pore (4) and the second nano-pore (5); the microscope (103) and the light source (102) are positioned at the left side and the right side of the nano probe to form a reflecting loop, so that the contact condition of the nano probe (2) and the nano film chip (3) can be observed conveniently; the Faraday shielding cover (107) shields the three-dimensional nanometer operation platform (1), the nanometer probe (2), the nanometer film chip (3), the electrode holder (101), the buffer pool (104), the light source (102), the microscope (103), the constant current source (105) and the weak current detection system (106);

the nano probe (2) is a nano-scale conductive electrode with the needle point smaller than 1 micron.

2. The nanomanipulated nanopore sequencing method according to claim 1, wherein: the nano probe (2) is a nano gold, platinum and silver electrode probe, or a gold-plated nano tungsten steel needle or an optical fiber probe, or a nano glass microtube filled with conductive solution.

3. The nanomanipulated nanopore sequencing method according to claim 1, wherein: the nano probe (2) is fixed on the three-dimensional nano operating platform (1) through the electrode holder (101) and is controlled by the platform, so that the high-precision nano motion with multiple degrees of freedom is realized.

4. The nanomanipulated nanopore sequencing method of claim 2, wherein: the initiation/termination signal mark refers to that when the designed DNA molecule to be detected enters or exits the nanopore, the signal identified by the detection circuit is known.

5. The nanomanipulated nanopore sequencing method of claim 2, wherein: the modification of the DNA molecule to be detected means that the DNA molecule to be detected (7) and the DNA library molecule (6) are combined by biological, physical and chemical methods.

6. The nanomanipulated nanopore sequencing method according to claim 5, wherein: the exiting and entering of the DNA molecules to be detected into the nano holes refers to the movement condition that the DNA molecules to be detected are driven when the nano probes stir the DNA library molecules to move, and the movement speed is flexibly controlled by setting the control parameters of the nano control system.

7. The nanomanipulated nanopore sequencing method according to claim 5, wherein: analyzing the nanopore DNA sequencing signal comprises judging the starting or stopping signal of the DNA molecule to be detected entering and exiting the nanopore, the via hole signals of different nanopores of the double nanopore system and the accumulated signal of the differential signals of the double nanopore system.