CN115839982A - Nanopore detection method and device - Google Patents

Abstract

The invention discloses a method for detecting a structure of a biomolecule based on a nanopore (or a nanochannel) and a corresponding solid nanopore (or nanochannel) device, belonging to the technical field of nanometer. The method comprises two or more than two nanopores or nanochannels filled with electrolyte, wherein biomolecules or nanoparticles sequentially pass through the two or more than two nanopores or nanochannels according to a time sequence to form a physical measurement result of interaction of the biomolecules or nanoparticles and the nanopores or the nanochannels, the physical measurement result forms a flight time performance according to a physical distance between the two or more than two nanopores or the nanochannels, and the physical measurement result with the flight time performance is utilized for accurately analyzing the characteristics of the biomolecules or the nanoparticles.

Description

Nanopore detection method and device

Technical Field

The invention belongs to the technical field of nanometer, and relates to a method and a device for detecting a nanopore or a nanochannel, which can be used for sequencing single-molecule DNA (deoxyribonucleic acid), detecting a protein molecular structure, sequencing protein and detecting nanoparticles.

Background

With the development of nano-technology, the rise of nano-analysis technology provides a new development direction for the development of biomolecule detection technology. In organisms, DNA, RNA, proteins and other biological macromolecules move across membranes to enable intracellular and intercellular mass transfer and transfer to become a ubiquitous process that momentarily controls the life-sustaining processes. On one hand, the advent of low-cost, high-throughput DNA sequencing technologies has made possible the deciphering of life genetic codes; on the other hand, advances in artificial intelligence technology have enabled accurate prediction of protein molecule function given the known amino acid ordering. Accurate detection of biomolecules by technical means would benefit everyone in society.

Nanopore sequencing is an emerging molecular detection technology, and when biomolecules pass through nano-scale micropores, the conductance of a via electrolyte is changed. Finally, the identification of the biomolecules can be achieved by the change of the ion current signal. The nanopore can be made of biological materials (such as alpha-hemolysin and MpsA pore protein) or solid materials (such as graphene, silicon dioxide and silicon nitride). Although the bio-nanopore has the advantage of no need for processing, as a bio-protein, its structural stability is greatly affected by the environment, and the diameter of the channel is fixed and cannot be controlled. Compared with a biological pore, the solid nanopore overcomes the defect of instability, has the advantages of simple structure, easy preparation, easy integration and the like, and can realize batch production by combining with a semiconductor manufacturing process.

Disclosure of Invention

The main content of the present invention is to provide a nanopore (or nanochannel) detection method and device for accurate analysis of biomolecule or nanoparticle properties. In general, the present disclosure describes novel biomolecule detection techniques that provide massively parallel, accurate biomolecule detection by measuring electrical signals and transit time intervals of biomolecules through two or more nanopores or nanochannels connected by a sample flow channel based on two or more nanopores or nanochannels, and that can form nanopores or nanochannels and associated sample flow channels, sample chambers, etc. in specific materials using well-established semiconductor technologies such as thin film technology, photolithography, and etching.

In order to achieve the purpose, the invention provides the following technical scheme:

a nanopore or nanochannel detection method and apparatus, comprising the following:

s1: nanopore or nanochannel detection methods;

s2: nanopore or nanochannel detection device architecture.

Optionally, S1 specifically is:

and (3) integrating a nanopore detection peripheral device by using the prepared nanopore or nano channel device, and preferably selecting the ion concentration, the pH value, the solution temperature, the driving voltage and the like of the electrolyte. When the biological molecules or the nano particles sequentially pass through the two or more than two nano holes or the nano channels to form a physical measurement result of the interaction between the biological molecules or the nano particles and the nano holes or the nano channels, the physical measurement result forms a flight time performance according to a physical distance between the two or more than two nano holes or the nano channels, and the physical measurement result with the flight time performance can be combined with Bayesian inference, neural networks and other methods to realize the identification of the biological molecules.

Optionally, the S2 specifically is:

the nanopore or nanochannel detection device has multiple independent electrolyte solution chambers, which are a sample introduction chamber, a sample exit chamber, and a sample flow channel. Wherein adjacent sample introduction chambers are in communication with each other. The sample inlet chamber is communicated with the sample flowing channel through a nanopore (or a nanochannel), and the sample outlet chamber is communicated with the sample flowing channel through another nanopore (or a nanochannel) in the same way. Meanwhile, one electrode pair is respectively arranged on two sides of the sample flow channel and used for forming an electric field and changing the motion trail of the biological molecules or the nano particles in the channel.

Further, the nanopore (or nanochannel) has a diameter of 1 nm to 500 nm. The shape of the nanopore (or nanochannel) is circular, oval, square, or other polygonal shape.

Further, the sample flow channel connecting the two nanopores or nanochannels may be linear, curvilinear or a combination of shapes.

Further, the sample flow path has a length of 200 nm to 10000 nm, and a width and a thickness of 50 nm to 1000 nm.

Further, the shape of the sample inlet chamber and the sample outlet chamber can be triangular, rectangular, circular or a combination of various shapes.

The invention has the beneficial effects that: the method realizes the nano holes or nano channels with two or more sub-nanometer spatial resolutions based on a semiconductor processing technology, and can obtain more accurate detection results of biomolecules or nano particles through physical measurement results of flight time performance. By changing the specific semiconductor processing technology, the sizes and the shapes of each cavity and the nano hole or the nano channel can be effectively adjusted, and different use requirements can be met; the size of each chamber and the pore diameter of the nano-pore or the shape and the size of the nano-channel can be adjusted independently; the nano holes or nano channels with different numbers and the sample flow channels with different shapes can be combined and designed, thereby not only realizing the detection of different biomolecules or nano particles, but also realizing the screening of different biomolecules or nano particles in the detection process.

Drawings

The invention will be further explained with reference to the drawings, in which:

FIG. 1 is a schematic cross-sectional view of a base material before processing;

FIG. 2 is a schematic perspective view of the present invention;

FIG. 3 is a perspective view of the apparatus after capping;

FIG. 4 is a schematic top view of the device with the sample flow channel in a curved shape;

FIG. 5 is a schematic top view of an apparatus comprising a cylindrical support in a sample flow channel;

FIG. 6 is a schematic top view of an apparatus comprising rectangular supports in a sample flow channel;

figure 7 is a top schematic view of the device with the sample flow channel connected to the fluid flush channel.

Wherein: 1-substrate, 2-aluminum oxide, 3-polysilicon, 4-silicon dioxide, 5-silicon nitride, 6-sample introduction chamber, 7-nanopore (or nanochannel), 8-sample outlet chamber, 9-electrode pair, 10-sample flow channel, 11-support cylinder, 12-top cover, 13-liquid inlet, 14-liquid outlet, 15-circular flow resistance column, 16-rectangular flow resistance wall, and 17-scouring fluid channel.

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; also for the purpose of better illustrating embodiments of the present invention, the choice of certain film materials in the drawings does not represent a configuration 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 schematic cross-sectional view of a substrate material before processing, which is a stacked structure formed by sequentially growing different layers of thin film materials, including aluminum oxide 2, polysilicon 3, silicon dioxide 4, and silicon nitride 5, on a substrate 1 by epitaxial technique, chemical vapor deposition, physical vapor deposition, or the like, to form different thicknesses.

Fig. 2 is a finished solid-state lateral nanopore or nanochannel device structure, which consists of a sample introduction chamber 6, a nanopore or nanochannel 7, a sample exit chamber 8, an electrode pair 9, a sample flow channel 10, a support cylinder 11, and the like. The structure may be completed by a semiconductor manufacturing process.

The sample feeding cavity 6 and the sample discharging cavity 8 comprise supporting cylinders 11 distributed in an array mode, and the supporting cylinders 11 improve the mechanical stability of the cavity shape and prevent the cavity from collapsing. Meanwhile, adjacent structural units are communicated with each other. The sample inlet chamber 6 is in communication with the sample flow channel 10 via a nanopore 7, and the same sample outlet chamber 8 is in communication with the sample flow channel 10 via a nanopore. The electrode pairs 9 are respectively arranged at two sides of the sample flow channel 10, and the motion trail of the biomolecules or the nano particles in the sample flow channel is controlled by an electric field formed by bias voltage.

In this embodiment, the nanopore and each chamber are implemented by a semiconductor processing process, and the shape of the nanopore (or the nanopore) is circular, or may be elliptical, rectangular, or polygonal.

In this embodiment, the base material may include an organic material, an inorganic material, or both. Suitable exemplary solid materials include, for example, semiconductor materials, insulating materials (e.g., silicon nitride, aluminum oxide, and silicon oxide), some organic and inorganic polymers (e.g., polyamides, polytetrafluoroethylene), and glass.

Fig. 3 is a sealed solid-state lateral nanopore device, in which a sample introduction chamber, a sample flow channel, a sample discharge chamber, and the like are sealed by using a material such as glass or silicon nitride through a bonding technique. Meanwhile, the top covers above the sample inlet cavity and the sample outlet cavity are provided with openings comprising a liquid inlet 13 and a liquid outlet 14, so that a sample can be connected with an external microfluidic system.

The sequencing experiment of the biological molecules is carried out by the solid-state lateral nanopore device shown in fig. 3, and the basic process is as follows: firstly, a proper amount of sample solution to be tested is injected into the sample injection chamber 6 through the liquid inlet 13. When the test is started, a certain bias voltage is applied between the sample inlet chamber 6 and the sample outlet chamber 8, and the electrode pairs corresponding to the applied bias voltage can be arranged in the sample inlet chamber 6 and the sample outlet chamber 8 or in the liquid inlet 13 and the liquid outlet 14; driven by an electrostatic field, the biomolecules reach the sample flow channel 10 through the nanopore 7 and then reach the sample outlet chamber 8 from the sample flow channel 10; when a biomolecule traverses a nanopore, a change in electrolyte ionic current results. The different base structures that make up a DNA molecule are different, and the amino acid structures that make up a protein molecule are different, as are the changes in current that occur as they pass through the nanopore. Meanwhile, when the biological molecules move in the sample flow channel 10 between the two nanopores, the control of the track of the biological molecules or the nanoparticles is realized by adjusting the bias voltage on the electrode pair 9. Because the physical distance can form flight time performance, the test equipment can accurately record numerical values such as perforation signals, translocation time, blocking amplitude, blocking hole rate and the like, and the structure sequence of the biological molecules is calculated by combining methods such as Bayesian reasoning, neural network and the like.

In other embodiments, such as fig. 4, the sample flow path can be modified to a curvilinear shape by modifying the photolithographic mask area.

In other embodiments, as shown in fig. 5, circular support posts or rows of posts can be added to the sample flow channel to create or enhance electroosmotic flow by altering the photolithographic mask area.

In other embodiments, as shown in FIG. 6, rectangular support walls can be added to the sample flow channel to create or enhance electroosmotic flow by modifying the photolithographic mask area.

In other embodiments, such as fig. 7, the sample flow channel can be cleaned by modifying the lithographic mask area to communicate with other fluid channels on both sides of the sample flow channel.

It is obvious that the solid-state lateral nanopore or nanochannel device structure in the embodiment of fig. 2 may comprise only one nanopore or nanochannel, and a sample introduction chamber, a sample exit chamber, a support cylinder, etc. connected thereto.

Obviously, the above description is not a limitation of the embodiments, and it is obvious for a person skilled in the art that various modifications can be made on the above description, and these modified embodiments are still within the protection scope of the present invention.

Claims (15)

1. A nanopore or nanochannel technology method for biomolecule or nanoparticle detection, comprising two or more nanopores or nanochannels filled with an electrolyte, wherein the biomolecule or nanoparticle sequentially passes through the two or more nanopores or nanochannels in a time sequence, and forms a physical measurement result of the interaction of the biomolecule or nanoparticle and the nanopores or nanochannels, wherein the physical measurement result forms a time-of-flight property according to a physical distance between the two or more nanopores or nanochannels, and wherein the physical measurement result with the time-of-flight property is utilized for precise analysis of biomolecule or nanoparticle characteristics.

2. The method of claim 1, wherein the biomolecule comprises a gene, protein, or polypeptide.

3. The method of claim 1, the nanoparticles comprising organic or inorganic nanoparticles having a size between 1 and 500 nanometers.

4. The method of claim 1, the physical measurement being an ionic current.

5. The method of claim 1, wherein the properties of the biological molecule comprise sequence properties of a gene, sequence properties of a protein or polypeptide, and protein profiling properties.

6. A nanopore or nanochannel device for biomolecule or nanoparticle detection, the device comprising two or more nanopores or nanochannels, a sample introduction chamber, a sample flow channel connecting the two or more nanopores or nanochannels, a sample exit chamber, and the like.

7. The apparatus of claim 6, further comprising a cylindrical support structure, a cover plate, and electrode structures disposed in the sample introduction chamber and the sample discharge chamber.

8. The device of claims 6 and 7, wherein the nanopore or nanochannel, the sample inlet chamber, the sample connecting flow channel, the sample outlet chamber, and the columnar support structure are fabricated on a semiconductor substrate, the nanopore or nanochannel is fabricated laterally on the semiconductor substrate, the size of the laterally fabricated nanopore or nanochannel can be arbitrarily adjusted by a semiconductor fabrication process, and the semiconductor substrate is composed of a dielectric layer or a semiconductor layer.

9. The device of claim 6, wherein the nanopore or nanochannel has a size between 1 and 500 nm.

10. The device of claim 6, wherein the sample flow channel is linear, curvilinear or a combination thereof.

11. The device of claim 10, wherein the sample flow channel has a length of between 200 and 10000 nm, a width and a thickness of between 50 and 1000 nm.

12. The device of claim 6, wherein the sample flow channel is in communication with other fluid channels.

13. The device of claim 6, wherein the sample flow channel is provided with electrode pairs at two sides of its periphery, and the electrode pairs form an electric field which changes the motion performance of the biomolecule or nanoparticle to be detected.

14. A nanopore or nanochannel device for biomolecule or nanoparticle detection, the device comprising a lateral nanopore or nanochannel and an inlet chamber and an outlet chamber connected thereto.

15. The apparatus of claim 13, further comprising a cylindrical support structure, a cover plate, and electrode structures disposed in the sample introduction chamber and the sample discharge chamber.

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