CN111440855A - Near-zero thickness nanopore preparation and DNA sequencing method - Google Patents
Near-zero thickness nanopore preparation and DNA sequencing method Download PDFInfo
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Abstract
The invention relates to a near-zero thickness nanopore preparation and DNA sequencing method, and belongs to the technical field of nanometer. The method comprises the following steps: s1: preparing a nanopore with near-zero thickness; s2: near-zero thickness nanopore DNA single base recognition. The method has high spatial resolution of sub-nanopores based on the near-zero thickness nanopores, and can be realized by a large-scale semiconductor production process, so the method has strong innovation and practicability, can greatly promote the development of a DNA sequencing technology and a protein detection technology based on the nanopores, and has great basic research value and potential practical application prospect.
Description
Technical Field
The invention belongs to the technical field of nanometer, and relates to a method for preparing a nanopore with near zero thickness and a DNA sequencing method.
Background
DNA (deoxyribose nucleic acid) sequences are metaphorically "blueprints" of life because they contain genetic information about the organism. Correctly reading the genetic information in the DNA sequence has important significance in the fields of human health and disease diagnosis, life exploration, molecular breeding and the like. Therefore, the development of accurate, rapid, low-cost, high-throughput and high-precision DNA sequencing technology can help people deepen the interpretation and effective utilization of the genetic information of the organism.
To date, DNA sequencing has undergone three generations of technological changes. The earliest used method of DNA sequencing was the dideoxynucleotide chain termination method (Sanger method). In addition, second-generation sequencing technologies that perform sequencing by synthesis and third-generation sequencing technologies that use a single molecule with zero mode waveguides have emerged, but have not been able to meet the ever-increasing personal medical needs. Nanopore sequencing is one of the mainstream technologies of fourth generation DNA sequencing, and mainly comprises biological nanopores and solid nanopores. Theoretically, the differences in the chemical properties of A, T, C, G four different bases can cause the variations in electrical parameters caused by their passage through the nanopore to be different, and the types of the corresponding bases can be obtained through the variations. Compared with other methods, nanopore sequencing does not need biological or chemical treatment on DNA, can directly read DNA sequences by a physical method, and has the advantages of low cost, long read length, easy integration and the like, thereby being concerned. Potentially combined with large scale semiconductor processing techniques, potentially reducing the cost to under $ 100, a person's genome sequence test can be completed. Due to factors such as fixed size of lipid bilayer membrane and biological nanopore and difficulty in fabricating large-scale array, the use of biological nanopore in wider field and extreme conditions is limited. In 2001, solid nanopores with a diameter of 1.8 nm were completed on silicon nitride films by the JeneGoovchenko project group of the university of Harvard physics by an argon ion beam with an energy of 3 KeV. The solid nanopore can overcome the defects of the biological nanopore, and can be potentially combined with a large-scale semiconductor production process to form large-scale production, so that the production cost is further reduced.
However, the sensitivity and spatial resolution of solid-state nanopores mainly depend on the size and thickness of the nanopores, and currently, research on graphene, boron nitride and molybdenum disulfide nanopores with atomic layer thickness has high sensitivity and theoretical spatial resolution of 0.5 nm, and has demonstrated the resolving power of a single base in a molybdenum disulfide nanopore system, but these thin film materials are very fragile and not suitable for operation, and need to be made by a manual method (transmission electron microscope, focused helium ion beam), and nanopores formed on them have low repeatability, low efficiency and slow speed; and due to the defects of stability, repeatability and the like of the material, the requirements of large-scale production and practical application cannot be met. And the pore diameter of the nanopore gradually increases along with the change of time due to the defects of the stability and the repeatability of the material in a liquid environment. Thus, nanopore DNA sequencing faces the problem of how to improve the sensitivity and spatial resolution of nanopore measurement DNA to identify single bases.
Disclosure of Invention
In view of the above, the present invention provides a near-zero thickness nanopore preparation and DNA sequencing method.
In order to achieve the purpose, the invention provides the following technical scheme:
a near-zero thickness nanopore preparation and DNA sequencing method comprises the following steps:
s1: preparing a nanopore with near-zero thickness;
s2: near-zero thickness nanopore DNA single base recognition.
Optionally, the S1 specifically includes:
preparing a silicon nitride insulating film substrate;
forming a silicon nanowire on the silicon nitride insulating film by using an ashing process or a side wall process by using an electron beam lithography technology, wherein the size of the silicon nanowire determines the size of the nanopore; wherein, the thickness of the silicon nanowire is 10-20 nanometers, the width is 5-10 nanometers, and the length is 50 nanometers-200 nanometers;
then, growing a layer of silicon dioxide film on the desalted silicon insulating film with the silicon nanowires, and covering the silicon nanowires, wherein the thickness of the silicon dioxide film is 15-25 nanometers; exposing the silicon nanowire by using a chemical mechanical polishing method;
forming a second layer of silicon nanowires in the vertical direction of the exposed silicon nanowires by using the electron beam lithography technology; wherein, the thickness of the silicon nanowire is 10-20 nanometers, the width is 5-10 nanometers, and the length is 50 nanometers-200 nanometers; growing a layer of silicon dioxide on the silicon nanowire to cover the silicon nanowire;
then, forming a vent hole in the upper silicon nanowire area of the chip by using optical or electron beam lithography; a window is formed on the silicon nitride film at the bottom by reactive ion etching, and the size of the window is 10-20 microns;
finally, xenon difluoride XeF is used2And etching the two layers of silicon nanowires to manufacture the nanopore with the thickness close to zero.
Optionally, the S2 specifically includes:
the prepared near-zero thickness nanopore is used for integrating a nanopore detection device, the concentration of a buffer solution, the pH value, the solution temperature and the driving voltage are preferably selected, the patch clamp technology is adopted, the weak current signal detection of DNA during the transmission of the near-zero thickness nanopore is realized, the chemometrics method is used for extracting DNA transmission characteristic information, and the Bayesian inference and neural network method are combined to realize the identification of DNA single base.
Optionally, the DNA transmission characteristic information includes a blocking time, a blocking current, and a baseline current.
The invention has the beneficial effects that:
the invention realizes the research of the nanopore with sub-nanometer high spatial resolution and the DNA transmission characteristic thereof based on a large-scale processing mode, the structure is formed by mutually vertical upper and lower nanometer-level channels, and the middle crossed region is the effective region of the nanopore. The molecular dynamics theory simulation indicates that the ionic current passing through the structure is only related to the external voltage and the size of the nanopore, and has no great dependence on the thickness, so that the structure is called a near-zero-thickness nanopore, the effective thickness approaches to zero, and the characteristic of single-layer graphene and molybdenum disulfide nanopore is similar, so that the molecular dynamics theory simulation has sub-nanometer spatial resolution, and can be used for carrying out experimental study on the transmission characteristic of DNA passing through the zero-thickness nanopore. Meanwhile, the nanopore with the near-zero thickness has high spatial resolution of a sub-nanopore and can be realized by a large-scale semiconductor production process, so that the nanopore has strong innovation and practicability, can greatly promote the development of a DNA sequencing technology and a protein detection technology based on the nanopore, and has great basic research value and potential practical application prospect.
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 flow diagram of a process for preparing a nanopore of near-zero thickness;
FIG. 2 is a 3D block diagram and cross-sectional illustration of a near-zero thickness nanopore; FIG. 2(a) is a 3D overall effect plot of a near-zero thickness nanopore, the middle white portion being the effective area of the nanopore; (b) is a cross-sectional view along red line BB; (c) is a cross-sectional view along the black line CC.
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.
Wherein the showings are for the purpose of illustrating the invention only and not for the purpose of limiting the same, and in which there is shown by way of illustration only and not in the drawings in which there is no intention to limit the invention thereto; to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of an actual product; it will be understood by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted.
The same or similar reference numerals in the drawings of the embodiments of the present invention correspond to the same or similar components; in the description of the present invention, it should be understood that if there is an orientation or positional relationship indicated by terms such as "upper", "lower", "left", "right", "front", "rear", etc., based on the orientation or positional relationship shown in the drawings, it is only for convenience of description and simplification of description, but it is not an indication or suggestion that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes, and are not to be construed as limiting the present invention, and the specific meaning of the terms may be understood by those skilled in the art according to specific situations.
FIG. 1 is a flow diagram of a process for preparing a nanopore of near-zero thickness; FIG. 2 is a 3D block diagram and cross-sectional illustration of a near-zero thickness nanopore; FIG. 2(a) is a 3D overall effect plot of a near-zero thickness nanopore, the middle white portion being the effective area of the nanopore; (b) is a cross-sectional view along red line BB; (c) is a cross-sectional view along the black line CC.
The invention provides the following technical scheme:
1. near zero thickness nanopore preparation
Preparing a silicon nitride insulating film substrate;
and (3) forming the silicon nanowire on the silicon nitride insulating film by using an ashing process or a side wall process by using an electron beam lithography technology, wherein the size of the silicon nanowire determines the size of the nanopore. Wherein, the thickness of the silicon nanowire is 10-20 nanometers, the width is 5-10 nanometers, and the length is 50 nanometers-200 nanometers;
then, growing a layer of silicon dioxide film on the desalted silicon insulating film with the silicon nanowires, and covering the silicon nanowires, wherein the thickness of the silicon dioxide film is 15-25 nanometers; exposing the silicon nanowire by using a chemical mechanical polishing method;
forming a second layer of silicon nanowires in the vertical direction of the exposed silicon nanowires by using the electron beam lithography technology; wherein, the thickness of the silicon nanowire is 10-20 nanometers, the width is 5-10 nanometers, and the length is 50 nanometers-200 nanometers; similarly, a layer of silicon dioxide is grown on the silicon nanowire to cover the silicon nanowire;
then, forming a vent hole in the upper silicon nanowire area of the chip by using optical or electron beam lithography; a window is formed on the silicon nitride film at the bottom by reactive ion etching, and the size of the window is 10-20 microns;
finally, xenon difluoride (XeF) is used2) Etching off the two layers of silicon nanowires to manufacture a nanopore with the thickness close to zero;
2. near-zero thickness nanopore DNA single base identification method
The prepared near-zero thickness nanopore is used for integrating a nanopore detection device, buffer solution concentration, pH value, solution temperature and driving voltage are optimized, the patch clamp technology is adopted, weak current signal detection of DNA during near-zero thickness nanopore transmission is achieved, DNA transmission characteristic information (blocking time, blocking current and base line current) is extracted by a chemometrics method, and the recognition of DNA single base is achieved by combining Bayesian inference and a neural network method.
Example one
1. Near zero thickness nanopore preparation
Preparing a silicon nitride insulating film substrate;
and (3) forming the silicon nanowire on the silicon nitride insulating film by using an electron beam lithography technology and an ashing process, wherein the size of the silicon nanowire determines the size of the nanopore. Wherein, the thickness of the silicon nanowire is 10 nanometers, the width is 5 nanometers, and the length is 50 nanometers;
then, growing a layer of silicon dioxide film on the desalted silicon insulating film with the silicon nanowires, and covering the silicon nanowires, wherein the thickness of the silicon dioxide film is 15 nanometers; exposing the silicon nanowire by using a chemical mechanical polishing method;
forming a second layer of silicon nanowires in the vertical direction of the exposed silicon nanowires by using the electron beam lithography technology; wherein the silicon nanowire has a thickness of 10 nm, a width of 5 nm and a length of 50 nm; similarly, a layer of silicon dioxide is grown on the silicon nanowire to cover the silicon nanowire;
then, forming a vent hole in the silicon nanowire area at the upper end of the chip by using electron beam lithography; a window is formed on the silicon nitride film at the bottom by reactive ion etching, and the size of the window is 10 microns;
finally, utilize twoFluorinated xenon (XeF)2) Etching off the two layers of silicon nanowires to manufacture a nanopore with the thickness close to zero;
2. near-zero thickness nanopore DNA single base identification method
The prepared near-zero thickness nanopore is used for integrating a nanopore detection device, buffer solution concentration, pH value, solution temperature and driving voltage are optimized, the patch clamp technology is adopted, weak current signal detection of DNA during near-zero thickness nanopore transmission is achieved, DNA transmission characteristic information (blocking time, blocking current and base line current) is extracted by a chemometrics method, and the recognition of single DNA base is achieved by combining a Bayesian inference method.
Example two
1. Near zero thickness nanopore preparation
Preparing a silicon nitride insulating film substrate;
and forming a silicon nanowire on the silicon nitride insulating film by using an electron beam lithography technology and a side wall process, wherein the size of the silicon nanowire determines the size of the nanopore. Wherein, the thickness of the silicon nanowire is 20 nanometers, the width is 10 nanometers, and the length is 200 nanometers;
then, growing a layer of silicon dioxide film on the desalted silicon insulating film with the silicon nanowires, and covering the silicon nanowires, wherein the thickness of the silicon dioxide film is 25 nanometers; exposing the silicon nanowire by using a chemical mechanical polishing method;
forming a second layer of silicon nanowires in the vertical direction of the exposed silicon nanowires by using the electron beam lithography technology; wherein the silicon nanowire has a thickness of 20 nm, a width of 10 nm and a length of 200 nm; similarly, a layer of silicon dioxide is grown on the silicon nanowire to cover the silicon nanowire;
then, forming a vent hole in the upper silicon nanowire area of the chip by using optical lithography; a window is formed on the silicon nitride film at the bottom by reactive ion etching, and the size of the window is 20 microns;
finally, xenon difluoride (XeF) is used2) Etching off the two layers of silicon nanowires to manufacture a nanopore with the thickness close to zero;
2. near-zero thickness nanopore DNA single base identification method
The prepared near-zero thickness nanopore is used for integrating a nanopore detection device, the concentration of a buffer solution, the pH value, the solution temperature and the driving voltage are preferably selected, the patch clamp technology is adopted, the weak current signal detection of DNA during the transmission of the near-zero thickness nanopore is realized, the chemometrics method is used for extracting DNA transmission characteristic information (blocking time, blocking current and baseline current), and the recognition of DNA single base is realized by combining the neural network method.
In conclusion, the near-zero thickness nanopore preparation technology and the near-zero thickness nanopore-based DNA sequencing method provided by the invention realize the identification of DNA single base.
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 (4)
1. The near-zero thickness nanopore preparation and DNA sequencing method is characterized by comprising the following steps: the method comprises the following steps:
s1: preparing a nanopore with near-zero thickness;
s2: near-zero thickness nanopore DNA single base recognition.
2. The method of near-zero thickness nanopore preparation and DNA sequencing of claim 1, wherein: the S1 specifically includes:
preparing a silicon nitride insulating film substrate;
forming a silicon nanowire on the silicon nitride insulating film by using an ashing process or a side wall process by using an electron beam lithography technology, wherein the size of the silicon nanowire determines the size of the nanopore; wherein, the thickness of the silicon nanowire is 10-20 nanometers, the width is 5-10 nanometers, and the length is 50 nanometers-200 nanometers;
then, growing a layer of silicon dioxide film on the desalted silicon insulating film with the silicon nanowires, and covering the silicon nanowires, wherein the thickness of the silicon dioxide film is 15-25 nanometers; exposing the silicon nanowire by using a chemical mechanical polishing method;
forming a second layer of silicon nanowires in the vertical direction of the exposed silicon nanowires by using the electron beam lithography technology; wherein, the thickness of the silicon nanowire is 10-20 nanometers, the width is 5-10 nanometers, and the length is 50 nanometers-200 nanometers; growing a layer of silicon dioxide on the silicon nanowire to cover the silicon nanowire;
then, forming a vent hole in the upper silicon nanowire area of the chip by using optical or electron beam lithography; a window is formed on the silicon nitride film at the bottom by reactive ion etching, and the size of the window is 10-20 microns;
finally, xenon difluoride XeF is used2And etching the two layers of silicon nanowires to manufacture the nanopore with the thickness close to zero.
3. The method of near-zero thickness nanopore preparation and DNA sequencing of claim 1, wherein: the S2 specifically includes:
the prepared near-zero thickness nanopore is used for integrating a nanopore detection device, the concentration of a buffer solution, the pH value, the solution temperature and the driving voltage are preferably selected, the patch clamp technology is adopted, the weak current signal detection of DNA during the transmission of the near-zero thickness nanopore is realized, the chemometrics method is used for extracting DNA transmission characteristic information, and the Bayesian inference and neural network method are combined to realize the identification of DNA single base.
4. The method of near-zero thickness nanopore preparation and DNA sequencing of claim 3, wherein: the DNA transmission characteristic information includes a blocking time, a blocking current, and a baseline current.
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| CN112198194A (en) * | 2020-10-15 | 2021-01-08 | 中国科学院重庆绿色智能技术研究院 | Method for preparing near-zero thickness nanopore by double-sided helium ion beam etching, product and application thereof |
| CN114572931A (en) * | 2022-02-28 | 2022-06-03 | 中国科学院重庆绿色智能技术研究院 | Preparation method of mortise and tenon structure nano hole with controllable thickness |
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