CN109680052B - Nanopore film, gene sequencing device and preparation method of nanopore film - Google Patents

Nanopore film, gene sequencing device and preparation method of nanopore film Download PDF

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CN109680052B
CN109680052B CN201910032354.7A CN201910032354A CN109680052B CN 109680052 B CN109680052 B CN 109680052B CN 201910032354 A CN201910032354 A CN 201910032354A CN 109680052 B CN109680052 B CN 109680052B
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pore
nanopore
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CN109680052A (en
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董水浪
王利忠
刘清召
赵磊
卢鑫泓
党宁
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BOE Technology Group Co Ltd
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Abstract

The present disclosure provides a nanopore membrane, a gene sequencing device, and a method for preparing the nanopore membrane, the nanopore membrane comprising: the detection device comprises a substrate, a film body arranged on one surface of the substrate and a nanopore penetrating through the film body, wherein the substrate is provided with a through hole corresponding to the nanopore, and the nanopore is provided with a detection part which is used for accommodating single nucleic acid molecules to pass through and can change the moving direction of the nucleic acid molecules so as to reduce the moving speed of the nucleic acid molecules. According to the nanopore film disclosed by the invention, the nanopore is provided with the detection part which can only accommodate a single nucleic acid molecule to pass through, the detection part can reduce the moving speed of the nucleic acid molecule by changing the moving direction of the nucleic acid molecule, the speed of the nucleic acid molecule passing through the detection part is relatively low, the data volume of an obtained detection signal can be improved, and the accuracy of gene sequencing can be further improved.

Description

Nanopore film, gene sequencing device and preparation method of nanopore film
Technical Field
The disclosure relates to the technical field of gene sequencing, in particular to a nanopore film, a gene sequencing device using the nanopore film and a preparation method of the nanopore film.
Background
Since the 70's of the last century, three generations of DNA sequencing technologies have been developed. The first generation of DNA sequencing technology was based on the Sanger method, taking 15 years to complete the human genome project, costing directly about $ 30 billion. Second generation DNA sequencing technologies feature high throughput, requiring only a week, costing less than 100 ten thousand dollars to complete sequencing of human genomes. In recent years, DNA single molecule probe analysis based on solid-state nanopore devices is considered to be one of the most promising technical routes for realizing third-generation rapid low-cost human gene sequencing, and becomes a hot spot for current research and application exploration, and the method can realize single-person genome sequencing within 24 hours and cost less than $ 1000. Single molecule detection and analysis capabilities of nanopore-based devices can be achieved by electrophoretically driving molecules through a nanoscale pore in solution. A large number of molecules can be rapidly analyzed in a limited space of the nanopore by various means, and when the high polymer molecules pass through the nanopore, the structural information of the high polymer molecules and the detected signal characteristics have a one-to-one correspondence relationship. By utilizing the characteristic, single-stranded DNA molecules with the length of thousands of base pairs can be directly characterized, the preparation link of amplification or labeling experiments is avoided, and the rapid low-cost DNA sequencing technology becomes possible.
The current experimental results show that the speed of DNA passing through the nanopore is about 102 to 105base/ms, for example, under the action of 120mV applied voltage, the speed of DNA passing through the alpha-hemolysin nano channel without external constraint is about 103base/ms, which far exceeds the sampling frequency (<250kHz) of the existing commercial patch clamp detection system. Therefore, the main problems of the existing nanopore sensor in realizing single base identification are as follows: the speed of DNA molecules passing through the nanopore is too high, so that effective data points acquired by the existing patch clamp or other signal acquisition systems are too few, and each base cannot be distinguished according to signals blocking current.
Disclosure of Invention
In view of the above problems in the prior art, an object of the present invention is to provide a nanopore membrane, a gene sequencing device using the nanopore membrane, and a method for preparing the nanopore membrane, which can make the speed of passing through the nanopore of the nanopore membrane by a nucleic acid molecule slower, so as to improve the accuracy of gene sequencing.
According to a first aspect of the present disclosure, there is provided a nanoporous film comprising: the detection device comprises a substrate, a film body arranged on one surface of the substrate and a nanopore penetrating through the film body, wherein the substrate is provided with a through hole corresponding to the nanopore, and the nanopore is provided with a detection part which is used for accommodating single nucleic acid molecules to pass through and can change the moving direction of the nucleic acid molecules so as to reduce the moving speed of the nucleic acid molecules.
In some embodiments, the nanopore has a first pore section proximate one face of the membrane body, a second pore section proximate another face of the membrane body, and a third pore section between the first and second pore sections, the third pore section forming the detection portion.
In some embodiments, the first and second bore sections are arranged in an offset arrangement, and the third bore section is connected between the first and second bore sections to form the detection portion.
In some embodiments, the film body includes a first film layer and a second film layer in a stacked arrangement.
In some embodiments, the first aperture section is located within the first membrane layer, the second aperture section is located within the second membrane layer, and the third aperture section is disposed along a direction of a contact surface of the first membrane layer and the second membrane layer.
In some embodiments, a side of the second membrane layer adjacent to the first membrane layer has a sink communicated with the second hole section, and the sink and the first membrane layer together form the third hole section.
In some embodiments, the third hole segment is formed by lift-off of a deposition layer deposited between the first and second film layers.
In some embodiments, the first pore section has a pore diameter of 50nm to 100 nm; the pore diameter of the second pore section is 50nm to 100 nm; the pore diameter of the third pore section is 0.5nm to 2 nm; the third pore segment has a length of 5nm to 10 nm.
In some embodiments, the first film layer has a thickness of 5nm to 20 nm; the thickness of the second film layer is 5nm to 20 nm.
According to a second aspect of the present disclosure, there is provided a gene sequencing apparatus comprising: the container is used for containing a solution containing nucleic acid molecules, the nanopore film is arranged in the container to divide the container into two chambers, and the detection units are respectively electrically connected with the solutions in the two chambers to generate a detection signal representing the base sequence of the nucleic acid molecules when the nucleic acid molecules pass through the detection part.
According to a third aspect of the present disclosure, there is provided a method for preparing a nanoporous thin film, comprising:
forming a film body on a substrate;
stripping off part of the substrate to form a through hole;
and stripping the film body with the part corresponding to the through hole to form a nanopore with a detection part, wherein the detection part is used for accommodating single nucleic acid molecules and can change the moving direction of the nucleic acid molecules so as to reduce the moving speed of the nucleic acid molecules.
In some embodiments, the forming a thin film body on the substrate includes:
forming a first film layer on the substrate;
forming a deposition layer on a part of the first film layer through a deposition process;
a second film layer is formed on the first film layer and the deposited layer.
In some embodiments, the peeling off the thin film body having the portion corresponding to the through hole to form a nanopore having a detection portion includes:
stripping off a portion of the second film layer corresponding to the deposited layer to form a second pore segment of the nanopore;
stripping the deposited layer to form a third pore section of the nanopore;
and stripping off a part of the first film layer corresponding to the deposition layer to form a first pore section which is staggered with the second pore section of the nanometer pore channel.
In some embodiments, the forming a thin film body on the substrate further comprises:
stripping off a portion of the first membrane layer to form a first pore segment of the nanopore;
filling the first pore segment of the nanopore to form a planar surface for depositing the deposition layer.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.
This section provides a general summary of various implementations or examples of the technology described in this disclosure, and is not a comprehensive disclosure of the full scope or all features of the disclosed technology.
Drawings
To more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below, and it is apparent that the drawings in the following description relate only to some embodiments of the present disclosure and are not limiting to the present disclosure.
FIG. 1 is a schematic structural diagram of a nanoporous thin film according to an embodiment of the invention;
FIG. 2 is an enlarged view of a portion A of the nanoporous membrane of FIG. 1;
FIG. 3 is a schematic structural diagram of a gene sequencing apparatus according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of the current change detected by the detection unit when the nucleic acid molecule passes through the nanopore;
FIG. 5 is a flow chart of a first embodiment of a method for preparing a nanoporous thin film in accordance with embodiments of the present invention;
fig. 6 is a flowchart of a method for manufacturing a nanoporous thin film according to a second embodiment of the invention.
Reference numerals:
1-a substrate; 2-a film body; 3-a first film layer; 4-a second film layer; 5-depositing a layer; 6-a nanopore; 7-a first bore section; 8-a second pore section; 9-a third pore section; 10-a through hole; 11-a filler; 12-a nanoporous film; 13-a container; 14-a detection unit; 15-a chamber; 16-a nucleic acid molecule.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present disclosure more clear, the technical solutions of the embodiments of the present disclosure will be described below clearly and completely with reference to the accompanying drawings of the embodiments of the present disclosure. It is to be understood that the described embodiments are only a few embodiments of the present disclosure, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the disclosure without inventive step, are within the scope of protection of the disclosure.
Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this disclosure belongs. The use of "first," "second," and the like in this disclosure is not intended to indicate any order, quantity, or importance, but rather is used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items.
To maintain the following description of the embodiments of the present disclosure clear and concise, a detailed description of known functions and known components have been omitted from the present disclosure.
Referring to fig. 1, an embodiment of the present invention provides a nanopore membrane, including: the film comprises a substrate 1, a film body 2 arranged on one surface of the substrate 1 and a nanometer pore channel 6 penetrating through the film body 2, wherein the substrate 1 is used for bearing the film body 2, and the substrate 1 is provided with a through hole 10 corresponding to the nanometer pore channel 6; the nanopore 6 has a detection section for accommodating passage of a single nucleic acid molecule and capable of changing the moving direction of the nucleic acid molecule to reduce the moving speed of the nucleic acid molecule. The nucleic acid molecule may be a DNA molecule or an RNA molecule.
By adopting the nanopore film with the structure, the nanopore 6 is provided with the detection part which can only accommodate a single nucleic acid molecule to pass through, the detection part can reduce the moving speed of the nucleic acid molecule by changing the moving direction of the nucleic acid molecule, the speed of the nucleic acid molecule passing through the detection part is low, the data quantity of the obtained detection signal can be improved, and the accuracy of gene sequencing can be further improved.
Referring to fig. 2, in some embodiments, the nanopore 6 may have a first hole segment 7, a second hole segment 8 and a third hole segment 9, wherein the first hole segment 7 is disposed near one surface of the membrane body 2, the second hole segment 8 is disposed near the other surface of the membrane body 2, the third hole segment 9 is disposed between the first hole segment 7 and the second hole segment 8, and the third hole segment 9 forms the detection portion. Thus, the nucleic acid molecule can pass through the third pore section 9 under the guidance of the first pore section 7 or the second pore section 8, and can move out of the nanopore 6 under the guidance of the second pore section 8 or the first pore section 7 after passing through the third pore section 9, which is beneficial to the smooth proceeding of gene sequencing. Specifically, the first pore section 7 and the second pore section 8 may have a pore diameter of 50nm to 100nm, the third pore section 9 may have a pore diameter of 0.5nm to 2nm, and the third pore section 9 may have a length of 5nm to 10 nm.
In some embodiments, the first and second bore sections 7, 8 are arranged in an offset arrangement, and the third bore section 9 is connected between the first and second bore sections 7, 8 to form the detection portion. The nucleic acid molecule needs to change direction when passing through the first pore section 7 to the third pore section 9, and still needs to change direction when passing through the third pore section 9 to the second pore section 8, so that the moving speed of the nucleic acid molecule can be effectively reduced in the moving direction changing process. Specifically, the first hole section 7 and the second hole section 8 may be completely staggered by 5nm to 10nm, that is, the nearest positions of the first hole section 7 and the second hole section 8 are staggered by 5nm to 10nm, and taking the circular hole sections of 50nm as an example for both the first hole section 7 and the second hole section 8, the central line of the first hole section 7 is staggered by 55nm to 60nm with the central line of the second hole section 8.
In some embodiments, the film body 2 may include a first film layer 3 and a second film layer 4 stacked, the first film layer 3 and the second film layer 4 may each have a thickness of 5nm to 20nm, and the first film layer 3 and the second film layer 4 may each be formed by an electrodeposition or chemical deposition process. Wherein the first pore section 7 is located in the first membrane layer 3 and the second pore section 8 is located in the second membrane layer 4. The third hole segment 9 is formed after peeling off the deposition layer 5 deposited between the first film layer 3 and the second film layer 4. Specifically, after the first film layer 3 is formed on the substrate 1, the deposition layer 5 is formed on a part of the surface of the first film layer 3 by, for example, an atomic deposition process, then the second film layer 4 is deposited on the first film layer 3 and the deposition layer 5, then a patterning process is used to strip off a part of the first film layer 3 to form the first hole section 7, a part of the second film layer 4 is stripped to form the second hole section 8, and the deposition layer 5 is stripped to form the third hole section 9. Thus, after the deposition layer 5 is stripped, a sinking groove is formed on one side of the second film layer 4 close to the first film layer 3, the sinking groove and the first film layer 3 form a third hole section 9, and the third hole section 9 is arranged along the third hole section 9The contact surface direction of the first film layer 3 and the second film layer 4 is arranged. The atomic deposition process (ALD) can control the thickness of the deposition layer 5 very well, and the aperture of the third hole segment 9 formed after the deposition layer 5 is stripped is also precisely controlled. The method for forming the third hole segment 9 can be integrated in a semiconductor process, has strong operability, and is suitable for quantitative production. Specifically, the material of the deposition layer 5 may be Al2O3Or Cu or the like. With Al2O3For example, the deposition speed can be set to be one atomic layer deposited every 12s, the thickness of each layer is about 0.1nm, the thickness of the deposition layer 5 can be precisely controlled by controlling the deposition time, and further, the pore diameter of the third pore section 9 is precisely controlled to be 0.5nm to 2nm, so that the third pore section 9 is matched with the diameter of a single-stranded nucleic acid molecule (the diameter of the single-stranded nucleic acid molecule is about 1nm), the speed of the nucleic acid molecule passing through the nanopore 6 can be effectively reduced, and the accuracy of gene sequencing is improved. The nanopore 6 is not limited to the above-described structure, and is not limited to the above-described process, as long as the pore diameter of the detection portion thereof is set to allow only a single nucleic acid molecule to pass therethrough, and the structure of the detection portion thereof is set to change the moving direction of the nucleic acid molecule, thereby reducing the moving speed of the nucleic acid molecule.
Fig. 3 is a schematic structural diagram of a gene sequencing apparatus according to an embodiment of the present invention, and referring to fig. 3, the gene sequencing apparatus according to the embodiment of the present invention includes: a container 13, a detection unit 14 and the nanopore membrane 12 as described above, wherein the container 13 is used for containing a solution containing the nucleic acid molecule 16, the nanopore membrane 12 is disposed in the container 13 to divide the container 13 into two chambers 15, and the detection unit 14 is used for being electrically connected with the solutions in the two chambers 15 respectively to generate a detection signal representing the base sequence of the nucleic acid molecule 16 when the nucleic acid molecule 16 passes through the detection part.
The sequencing method using the gene sequencing device comprises the following steps: the nucleic acid molecules 16 are placed in a chamber 15 connected with the negative electrode of the detection unit 14, a driving voltage is applied between the positive electrode and the negative electrode of the detection unit 14, the nucleic acid molecules 16 are driven to move along the nanometer pore canal 6, the driving voltage can be 100-300 mV, the electrolyte can be a potassium chloride solution, the molar concentration of the electrolyte can be 0.1-3.2mol/L, and the pH value of the electrolyte can be 8-10. When a nucleic acid molecule 16 such as a DNA molecule passes through the nanopore 6, the ion flux is reduced by the blocking effect, and the current value changes, as shown in fig. 4. Different bases have different current value changes when passing due to different size directions and sizes, and the base sequence can be determined by detecting the current value changes. Due to the application of the nanopore film 12, the movement speed of the nucleic acid molecules 16 passing through the nanopore 6 is low, the data volume of the acquired detection signals is large, the signal to noise ratio can be improved, and the accuracy of gene detection is further improved.
Fig. 5 is a flowchart of a first embodiment of a method for preparing a nanopore membrane according to an embodiment of the present invention, and referring to fig. 5, the method for preparing a nanopore membrane according to an embodiment of the present invention specifically includes the following steps:
s101, a thin film body 2 is formed on a substrate 1. The substrate 1 is used as a carrier for carrying the film body 2, and a silicon substrate 1 can be used. The thin film body 2 may be formed on the substrate 1 by, for example, chemical deposition, electrodeposition, or other processes.
S102, a portion of the substrate 1 is peeled off to form the through-hole 10. The through-hole 10 may be formed by a process such as laser engraving or etching.
And S103, stripping off the film body 2 with a part corresponding to the through hole 10 to form a nanopore 6 with a detection part, wherein the detection part is used for accommodating single nucleic acid molecules to pass through and can change the moving direction of the nucleic acid molecules so as to reduce the passing speed of the nucleic acid molecules. The nanopores 6 may be formed by a patterning process such as photolithography or etching, or may be formed by a composite process including photolithography and etching. In step S102 and step S103, step S102 may be performed first, step S103 may be performed first, or the steps may be performed alternately.
According to the nanopore thin film prepared by the method, the nanopore is provided with the detection part which can only accommodate a single nucleic acid molecule to pass through, the detection part can reduce the moving speed of the nucleic acid molecule by changing the moving direction of the nucleic acid molecule, the speed of the nucleic acid molecule passing through the detection part is low, the data quantity of the obtained detection signal can be improved, and the accuracy of gene sequencing can be further improved. In addition, the preparation method has strong operability and is suitable for quantitative production.
Fig. 6 is a flowchart of a second embodiment of a method for manufacturing a nanoporous film according to an embodiment of the invention, and referring to fig. 6, the method for manufacturing a nanoporous film according to an embodiment of the invention is used for manufacturing the nanoporous film, and specifically includes the following steps:
s201, forming a first film 3 on the substrate 1, and peeling off a portion of the first film 3 to form a first pore segment 7 of the nanopore 6. The substrate 1 is used as a carrier for carrying the film body 2, and a silicon substrate 1 can be used. The first film 3 may be made of Si3N4The thickness of the first film 3 may be 5nm to 20 nm. The first layer 3 may be formed on the substrate 1 by, for example, chemical deposition, electrodeposition, or other processes. The first hole segment 7 may be formed using a patterning process such as a deep ultraviolet etching process, and the diameter of the first hole segment 7 may be 50nm to 100 nm.
S202, filling the first pore section 7 of the nanopore 6 with a filler 11 to form a flat surface on the first membrane layer 3. Specifically, the filler 11 in the first hole segment 7 of the nanopore 6 may be Cu deposited by, for example, an atomic deposition process to fill the first hole segment 7 of the nanopore 6, and then, the surface of the first film layer 3 may be planarized by a planarization process such as Chemical Mechanical Polishing (CMP) to form a planarized surface on the first film layer 3 for deposition of the deposition layer 5 in a subsequent step.
S203, a deposition layer 5 is formed on the first film layer 3 corresponding to the first pore section 7 of the nanopore 6 through a deposition process. The deposition layer 5 may be formed on the first film layer 3 by, for example, an atomic deposition (ALD) process, and the material of the deposition layer 5 may be Al2O3Or Cu, etc. With Al2O3For example, the deposition rate may be set to deposit one atomic layer every 12s, each layer being about 0.1nm thick, and the thickness of the deposited layer 5 may be accurately controlled to 0.5nm to 2nm by controlling the deposition time.
S204, on the first film layer 3 and the deposition layer 5A second film layer 4 is formed and portions of the second film layer 4 corresponding to the deposited layer 5 are peeled off to form a second pore segment 8 that is misaligned with the first pore segment 7 of the nanopore. The second film 4 may be Si3N4The thickness of the second film layer 4 may be 5nm to 20 nm. The second layer 4 may be formed on the substrate 1 by, for example, chemical deposition, electrodeposition, or other processes. The second hole segment 8 may also be formed by a patterning process such as a deep ultraviolet lithography process, and the diameter of the second hole segment 8 may be 50nm to 100 nm. The first hole section 7 and the second hole section 8 can be completely staggered by 5nm to 10nm, that is, the nearest positions of the first hole section 7 and the second hole section 8 are staggered by 5nm to 10nm, and taking a circular hole section with 50nm of both the first hole section 7 and the second hole section 8 as an example, the central line of the first hole section 7 is staggered by 55nm to 60nm with the central line of the second hole section 8.
S205, the deposited layer 5 is stripped to form a third hole segment 9 of the nanopore. The deposited layer 5 may be stripped using, for example, an etching process. After the deposition layer 5 is peeled off, a sink groove is formed on one side of the second film layer 4 close to the first film layer 3, and the sink groove and the first film layer 3 together form a third hole section 9. Since the nearest neighbors of the first and second pore segments 7 and 8 are staggered by 5nm to 10nm, the third pore segment 9 is formed to have a length of about 5nm to 10 nm. Since the thickness of the deposited layer 5 can be accurately controlled to 0.5nm to 2nm, the pore size of the third pore section 9 after peeling off the deposited layer 5 can also be accurately controlled to 0.5nm to 2nm to accommodate passage of only a single nucleic acid molecule.
S206, the substrate 1 corresponding to the nanopore 6 is peeled off to form the through hole 10. The through hole 10 can be formed by a process such as laser engraving or etching, and the diameter of the through hole 10 is much larger than the aperture of the nanopore 6, so as to avoid affecting the nucleic acid molecules to pass through the nanopore 6.
S207, the filler 11 in the first pore section 7 of the nanopore 6 is stripped to form a through nanopore 6. The filler 11 may be stripped using, for example, an etching process.
The nanopore 6 of the nanopore film prepared by the method is arranged in a zigzag shape, the moving direction of nucleic acid molecules can be changed to reduce the moving speed of the nucleic acid molecules, and the thickness of the deposition layer 5 can be accurately controlled to accurately control the aperture of the third pore section 9, so that the third pore section 9 can only accommodate a single nucleic acid molecule to pass through, and the moving speed of the nucleic acid molecules is further reduced. In addition, the method can be integrated in a semiconductor process, has strong operability and is suitable for quantitative production.
The above embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and the scope of the present invention is defined by the claims. Various modifications and equivalents of the invention may be made by those skilled in the art within the spirit and scope of the invention, and such modifications and equivalents should also be considered as falling within the scope of the invention.

Claims (10)

1. A nanoporous film, comprising: the detection device comprises a substrate, a film body arranged on one surface of the substrate and a nanopore penetrating through the film body, wherein the substrate is provided with a through hole corresponding to the nanopore, and the nanopore is provided with a detection part which is used for accommodating a single nucleic acid molecule to pass through and can change the moving direction of the nucleic acid molecule so as to reduce the moving speed of the nucleic acid molecule;
wherein the nanopore has a first pore section near one face of the membrane body, a second pore section near the other face of the membrane body, and a third pore section between the first and second pore sections, the third pore section forming the detection portion;
wherein the first and second pore segments are arranged in a staggered manner, and the third pore segment is connected between the first and second pore segments to form a zigzag-shaped nanopore, so that a nucleic acid molecule moves to the third pore segment after changing the moving direction through the first pore segment, and moves to the second pore segment after changing the moving direction again through the third pore segment.
2. The nanopore membrane of claim 1, wherein the membrane body comprises a first membrane layer and a second membrane layer arranged in a stack.
3. The nanopore membrane of claim 2, wherein the first pore segment is located within the first membrane layer, the second pore segment is located within the second membrane layer, and the third pore segment is disposed along a direction of a contact surface of the first membrane layer and the second membrane layer.
4. The nanopore membrane of claim 3, wherein a side of the second membrane layer adjacent to the first membrane layer has a sink connected to the second pore segment, the sink and the first membrane layer together forming the third pore segment.
5. The nanopore membrane of any of claims 2-4, wherein the third pore segment is formed after lift-off of a deposition layer deposited between the first membrane layer and the second membrane layer.
6. The nanoporous film according to any one of claims 1-4, wherein the first pore segment has a pore size of 50nm to 100 nm; the pore diameter of the second pore section is 50nm to 100 nm; the pore diameter of the third pore section is 0.5nm to 2 nm; the third pore segment has a length of 5nm to 10 nm.
7. The nanoporous film of any one of claims 2-4, wherein the first film layer has a thickness of 5nm to 20 nm; the thickness of the second film layer is 5nm to 20 nm.
8. A gene sequencing device, comprising: a container for holding a solution comprising a nucleic acid molecule, a detection unit disposed within the container to divide the container into two chambers, and the nanopore membrane of any one of claims 1-7 electrically coupled to the two chambers respectively to generate a detection signal indicative of a base sequence of the nucleic acid molecule when the nucleic acid molecule passes through the detection portion.
9. A method for preparing a nanoporous film, comprising:
forming a film body on a substrate;
stripping off part of the substrate to form a through hole;
stripping off the film body with a part corresponding to the through hole to form a nanopore with a detection part, wherein the detection part is used for accommodating single nucleic acid molecules to pass through and can change the moving direction of the nucleic acid molecules so as to reduce the passing moving speed of the nucleic acid molecules;
wherein the forming of the thin film body on the substrate includes:
forming a first film layer on the substrate;
forming a deposition layer on a part of the first film layer through a deposition process;
forming a second film layer on the first film layer and the deposition layer;
wherein the film body corresponding to the through hole and the peeling part to form a nanopore with a detection part comprises:
stripping off a portion of the second film layer corresponding to the deposited layer to form a second pore segment of the nanopore;
stripping the deposited layer to form a third pore section of the nanopore;
and stripping off a part of the first film layer corresponding to the deposition layer to form a first hole section which is staggered with a second hole section of the nanometer hole channel, so that the first hole section, the second hole section and the third hole section form a zigzag-shaped nanometer hole channel, and the nucleic acid molecule is moved to the third hole section after changing the moving direction through the first hole section, and is moved to the second hole section after changing the moving direction through the third hole section again.
10. The method of claim 9, wherein forming a film body on the substrate further comprises:
stripping off a portion of the first membrane layer to form a first pore segment of the nanopore;
filling the first pore segment of the nanopore to form a planar surface for depositing the deposition layer.
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