A short-chain DNA probe assisted single molecule sequencing method based on double-layer nanopore

Abstract

The application discloses a short-chain DNA probe assisted single-molecule sequencing method based on a double-layer nanopore, and relates to the technical fields of biosensing, single-molecule detection and DNA sequencing. The application relates to a DNA single-molecule sequencing method based on a double-layer nanopore structure and an oligonucleotide probe hybridization strategy with a length of 3 nt. The application uses a double-layer nanopore chip with an upper nanopore, a lower nanopore and an intermediate cavity structure as a single-molecule detection platform, uses a 3-mer short-chain DNA probe with a known sequence as a sequence positioning marker, realizes positioning recognition and reconstruction of a target DNA sequence through multiple rounds of probe hybridization detection and continuous double-blocked ion current signal analysis. The application aims to improve the accuracy and stability of probe positioning in a probe assisted DNA sequencing process, enhance the distinguishing ability of a completely matched probe and a mismatched probe, and provide a new technical scheme for solid-state nanopore high-precision single-molecule DNA sequencing.

Description

Technical Field

[0001] This invention relates to the fields of biosensing, single-molecule detection and DNA sequencing technology, and in particular to a short-chain DNA probe-assisted single-molecule sequencing method based on bilayer nanopores. Background Technology

[0002] Deoxyribonucleic acid (DNA) sequencing technology is a fundamental technology in life sciences, precision medicine, genetic disease diagnosis, and pathogen detection. With the development of single-molecule detection technologies, nanopore sequencing has become an important research direction in DNA sequencing in recent years due to its advantages such as no amplification required, no fluorescent labeling required, fast detection speed, and the ability to perform long-fragment continuous sequencing. Based on the different nanopore materials, nanopores are mainly divided into two categories: biological nanopores and solid-state nanopores. Solid-state nanopores, with their advantages of high mechanical strength, good chemical stability, tunable pore size, and ease of integration and fabrication, have broad application prospects in single-molecule detection and DNA sequencing.

[0003] Currently, solid-state nanopore DNA detection typically employs a monolayer nanopore structure. Under an applied electric field, DNA molecules passing through the nanopore cause a momentary drop in ionic current. Detection of DNA molecules is achieved by analyzing characteristics such as the amplitude and duration of the blocking current. However, due to the small differences in size and electrical properties among the four bases of natural DNA, monolayer nanopores cannot directly achieve high-precision single-base identification. Therefore, researchers have proposed a short-chain probe-assisted nanopore sequencing (HANS) strategy. This method utilizes short-chain DNA probes with known sequences to hybridize with target DNA. By detecting the characteristic blocking signal generated when the probe passes through the nanopore, the probe's position in the target DNA is identified, and the target DNA sequence is inferred.

[0004] However, existing probe-assisted sequencing methods based on monolayer nanopores still have the following shortcomings: First, monolayer nanopores only generate a single blocking event, and there is a lack of secondary verification process when the probe passes through the nanopore. This limits the ability to distinguish between blocking signals generated by perfectly matched and mismatched probes, and the methods are easily affected by thermal motion, conformational changes, and noise interference, reducing probe localization accuracy. Second, probe hybridization stability directly affects the detection results. Mismatched probes or hybridization structures with low binding stability may still generate similar blocking signals when passing through monolayer nanopores, thereby increasing sequence identification errors. In addition, existing probe-assisted sequencing methods mainly focus on single probe identification and lack a unified positional analysis and sequence reconstruction mechanism for information obtained from multiple rounds of probe detection, making it difficult to further improve sequencing accuracy and reliability.

[0005] Bilayer nanopore structures, with their two nanopores at the top and bottom and an intermediate cavity, allow DNA molecules to pass through the two nanopores sequentially, creating a series of blocking events during detection. This has garnered significant attention in DNA molecule manipulation, transport regulation, and molecular dynamics studies. However, current applications of bilayer nanopore structures are primarily focused on DNA transpore dynamics regulation and single-molecule transport research. A method combining bilayer nanopore structures with short-chain probe-assisted sequencing is still lacking, and the continuous double-blocking events are not fully utilized for probe localization, mismatch screening, and DNA sequence reconstruction.

[0006] Therefore, in order to address the problems of low probe localization accuracy, insufficient mismatch identification capability and limited sequence reconstruction reliability in existing single-layer nanopore probe-assisted sequencing, there is an urgent need to propose a novel DNA single-molecule sequencing method that can improve probe identification accuracy by utilizing a continuous double-blocking detection mechanism of bilayer nanopores and combine it with multiple rounds of short probe detection to achieve target DNA sequence reconstruction. Summary of the Invention

[0007] The purpose of this invention is to provide a short-chain DNA probe-assisted single-molecule sequencing method based on bilayer nanopores, addressing the problems of insufficient probe localization accuracy, limited mismatch probe recognition capability, susceptibility of detection results to noise and molecular conformational changes, and lack of an effective sequence reconstruction mechanism in multi-round probe detection. This invention utilizes a bilayer nanopore chip with upper and lower nanopores and a central cavity as a single-molecule detection platform, using 3-mer short-chain DNA probes with known sequences as sequence localization markers. Through multi-round probe hybridization detection and continuous double-blocking ion current signal analysis, the localization, identification, and reconstruction of the target DNA sequence are achieved.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a short-chain DNA probe-assisted single-molecule sequencing method based on a bilayer nanopore structure. It utilizes a bilayer nanopore chip with upper and lower nanopores and a central cavity as a single-molecule detection platform, and uses 3-mer short-chain DNA probes with known sequences as sequence localization markers. Through multiple rounds of probe hybridization detection and continuous double-blocking ion current signal analysis, the localization, identification, and reconstruction of the target DNA sequence are achieved. The sequencing method specifically includes the following steps: S1. Hybridize a 3-mer short-chain DNA probe with the DNA sample to be sequenced to obtain a hybrid DNA mixture; S2. The hybrid DNA mixture is added to the double-layer nanopore detection cell. Electrolyte and Ag / AgCl electrodes are respectively placed on both sides of the double-layer nanopore, and a bias voltage is applied so that the DNA molecules pass through the double-layer nanopore chip in sequence under the drive of the electric field. The double-layer nanopore chip includes a first nanopore, an intermediate cavity and a second nanopore. S3. Correctly paired DNA molecules generate a first blocking event and a second blocking event respectively during the continuous passage of DNA through two nanopores; the ion current signal generated by the DNA passing through the double nanopores is collected in real time, and the continuous double blocking events are identified.

[0009] S4. Multi-round probe detection: After each probe detection is completed, the detection cell and electrode are cleaned, and the hybridization DNA sample corresponding to the next probe is added again. Steps S1-S3 are repeated. S5. After obtaining the localization information corresponding to each probe, establish the correspondence between the probe sequence and the DNA location to complete the reconstruction of the DNA sequence to be tested.

[0010] Furthermore, guide sequences are added to the 5' and 3' ends of the DNA sample to be sequenced before hybridization with the short-chain DNA probe.

[0011] Furthermore, the guide sequence is an oligonucleotide sequence consisting of no fewer than 12 consecutive T bases; the 3-mer short DNA probe is an oligonucleotide probe consisting of 3 bases.

[0012] Furthermore, in step S5, the positioning information corresponding to each probe includes the blocking current amplitude, duration, double blocking interval, and probe characteristic blocking signal; the position of the probe in the target DNA is determined by the positioning information corresponding to each probe.

[0013] Furthermore, in step S5, the target DNA sequence reconstruction involves obtaining all the localization information of each probe in the target DNA and establishing a probe localization matrix composed of probe types and target DNA locations; combining the complementary pairing relationships of each probe, the detection results of each probe are comprehensively analyzed to restore the base sequence of the target DNA and obtain the nucleotide sequence of the DNA to be tested.

[0014] The present invention also provides a dual-layer nanoporous chip, which includes a three-layer film structure comprising an upper silicon nitride thin film, a middle silicon dioxide cavity layer and a lower silicon nitride thin film; the upper and lower silicon nitride thin films each have a nanopore, and the upper and lower nanopores are coaxially arranged along the film thickness direction.

[0015] Furthermore, the intermediate silica cavity layer is selectively etched with HF solution to form a cavity structure with a size larger than the nanopore diameter, so that the upper and lower nanopores are connected through the intermediate cavity to form a double-layer nanopore detection channel; the upper and lower nanopores are formed by helium ion beam etching.

[0016] This invention also provides a method for preparing a bilayer nanoporous chip, which includes the following chip pretreatment steps: before nanopore processing, the three-layer film structure consisting of an upper silicon nitride film, a middle silicon dioxide cavity layer, and a lower silicon nitride film is washed in a piranha solution with a volume ratio of 3:1; then it is soaked and washed twice with deionized water, and dried in a vacuum drying oven; the piranha solution is prepared by mixing concentrated sulfuric acid and hydrogen peroxide in a volume ratio of 3:1.

[0017] This invention also provides the application of bilayer nanopore chips in DNA sequencing.

[0018] This invention also provides an application of a short-chain DNA probe-assisted single-molecule sequencing method based on bilayer nanopores in DNA sequencing.

[0019] The present invention discloses the following technical effects: This invention utilizes a bilayer nanopore chip with upper and lower nanopores and a central cavity structure, allowing target DNA molecules carrying 3-mer short-chain DNA probes to pass sequentially through the two nanopores. During the detection process, a continuous double-blocking ion current signal is generated, which, combined with the characteristic blocking signal generated by the probe, enables precise positioning of the probe in the target DNA. Furthermore, through multiple rounds of hybridization detection with different 3-mer short-chain DNA probes, a probe position matrix is ​​established to reconstruct the target DNA sequence.

[0020] Compared with existing probe-assisted DNA sequencing methods based on single-layer nanopores, this invention has the following beneficial technical effects: (1) Achieving continuous double-blocking detection and improving probe positioning accuracy. This invention uses a double-layer nanopore chip with upper and lower double nanopores and a middle cavity structure, so that the target DNA passes through the first nanopore, the middle cavity and the second nanopore in sequence, forming continuous double-blocking ion current events during the detection process. The continuous double-blocking detection mechanism provides dual time reference information for probe positioning, which can effectively improve the accuracy of probe position identification and detection stability compared with the detection method that only generates a single blocking event in single-layer nanopores. (2) Enhancing the identification ability of probe feature signals and improving detection reliability. This invention utilizes 3-mer short-chain DNA probes to form a local double-stranded structure with the target DNA. When the probe region passes through the double-layer nanopore, it generates a feature blocking signal that is different from that of single-stranded DNA. Combining continuous double-blocking events and signal features such as blocking current amplitude and duration, the probe position can be identified more accurately, reducing the influence of background noise and DNA conformational changes on the detection results and improving the reliability of single-molecule detection. (3) Improving the ability to distinguish between perfectly matched probes and mismatched probes. This invention utilizes the cavity in the middle of a double-layer nanopore and a continuous double-nanopore structure, allowing the target DNA to undergo two detection processes while continuously crossing the two nanopores, providing different cross-pore behavior characteristics for perfectly matched probes and mismatched probes with poor stability. Compared with existing single-layer nanopore detection methods, this method is beneficial for enhancing the ability to distinguish probes in different hybridization states, providing technical support for improving sequencing accuracy. (4) Establishing a DNA sequence reconstruction method based on multi-round probe detection. This invention establishes the correspondence between probe positioning information and target DNA position by performing hybridization detection on various 3-mer short-chain DNA probes with different sequences, and combines the results of multiple rounds of detection to achieve target DNA sequence reconstruction. Compared with existing methods that mainly rely on single detection results for analysis, this invention can make full use of the complementary information provided by different probes, improving the integrity and reliability of DNA sequence analysis.

[0021] This invention aims to improve the accuracy and stability of probe localization during probe-assisted DNA sequencing, enhance the ability to distinguish between perfectly matched and mismatched probes, improve the reliability of DNA sequence reconstruction, and provide a new technical solution for high-precision single-molecule DNA sequencing using solid-state nanopores. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of a double-layer nanoporous structure; Figure 2 This is a roadmap for sequencing methodologies; Figure 3 Design diagram for 3-mer probe hybridization DNA strand; Figure 4 This is a schematic diagram of the hybridization of a 3-mer probe with target DNA. Figure 5 Schematic diagram of DNA detection through a double-layer nanopore; Figure 6 Flowchart for probe positioning signal analysis; Figure 7 Sequencing results and ion current characteristics of DNA strands hybridized with different 3-mer probes. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” and “contain” used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] This invention provides a method for single-molecule DNA sequencing based on a 3-mer short-chain DNA probe using a bilayer nanopore structure. It utilizes a bilayer nanopore chip with upper and lower nanopores and a cavity in the middle as a single-molecule detection platform, uses 3-mer short-chain DNA probes with known sequences as sequence localization markers, and achieves the localization, identification and reconstruction of the target DNA sequence through multi-round probe hybridization detection and continuous double-blocking ion current signal analysis.

[0030] Example 1 Structure and fabrication method of bilayer nanoporous chip A. Double-layer nanoporous chip like Figure 1 As shown, the detection chip used in this invention is a three-layer Si3N4-SiO2-Si3N4 film structure, including an 8nm thick Si3N4 film on top, a 20nm thick SiO2 layer in the middle, and a 20nm thick Si3N4 film on the bottom. The upper and lower Si3N4 films are fabricated into nanopores using a helium ion microscope (HIM), with the two nanopores coaxially aligned along the film thickness direction. The middle SiO2 layer is selectively etched with HF solution to form a cavity structure larger than the nanopore diameter, connecting the upper and lower nanopores to form a double-layer nanopore detection channel. This structure not only serves as a DNA transmembrane transport channel but also allows DNA to continuously pass through the two nanopores, thereby generating a continuous double-blocking ion current signal.

[0031] B. Fabrication of bilayer nanoporous chips The fabrication method of the bilayer nanoporous chip is as follows: (1) Chip pretreatment: The Si3N4-SiO2-Si3N4 three-layer film chip was cleaned in a piranha solution (concentrated sulfuric acid: hydrogen peroxide) with a volume ratio of 3:1. Specifically, 6 mL of concentrated sulfuric acid and 2 mL of hydrogen peroxide were mixed to prepare the piranha solution. After immersing the chip, it was heated in a 90°C water bath for 30 min to remove organic contaminants from the chip surface. Subsequently, it was washed twice with deionized water, immersing for 10 min each time, and finally dried in a 100°C vacuum drying oven for 2 h.

[0032] (2) Nanopore fabrication: HIM was used to fabricate nanopores with an accelerating voltage of 30 kV and an ion gun pressure of 2 × 10⁻⁶ kV. - 6 Torr, operating temperature 74.0K, machining chamber vacuum degree 1.5×10 -7 Torr, beam current 5pA, dose 90nC / μm 2 The process involved scanning eight times to fabricate nanopores with a diameter of approximately 10 nm on both the upper and lower Si3N4 thin films.

[0033] (3) Formation of double-layer nanopores: The processed chip was immersed in anhydrous ethanol and deionized water for 10 min each to improve the wettability of the nanochannels; then the chip was placed in 10% HF solution to etch the middle SiO2 layer. During the etching process, the chip was slightly swung to improve the etching uniformity; after the etching was completed, the chip was immediately immersed in deionized water for 10 min to remove residual HF, and then immersed in anhydrous ethanol for 10 min to finally form a double-layer nanopore chip with upper and lower double nanopores and a middle cavity structure.

[0034] Example 2 A DNA single-molecule sequencing method based on a 3-mer short-chain DNA probe using a bilayer nanopore | (See sequencing method roadmap) Figure 2 The specific steps are as follows: S1, DNA sample and 3-mer short-chain DNA probe preparation like Figure 3 As shown, this DNA strand is the hybridization DNA strand of the 3-mer short-chain DNA probe designed for the experiment. Figure 4 As shown, the experiment used pre-designed single-stranded DNA and corresponding 3-mer short-stranded DNA probes. The dry powdered DNA samples were centrifuged at 500 rpm for 60 seconds to prevent powder loss. 1.5427 mL of deionized water was added to 61.09 μg of single-stranded DNA to prepare a 2 μM DNA solution; 0.209 mL of deionized water was added to 2.07 μg of 3-mer short-stranded DNA probes to prepare a 10 μM probe solution. The DNA and probe solutions were then mixed at a volume ratio of 1:2 to obtain the hybridization DNA mixture.

[0035] S2, probe hybridization Based on the preset 3-mer short-chain DNA probe sequence, a short-chain DNA probe with a known sequence is prepared and annealed with the target DNA to form a local double-stranded DNA structure at the complementary target site of the 3-mer short-chain DNA probe; for different probe sequences, independent hybridization is performed to obtain a hybrid DNA mixture.

[0036] like Figure 4 As shown, the bilayer nanoporous chip was mounted in the liquid cell, ensuring that the chip, rubber sealing ring, and center of the liquid cell were coaxial. The two sides of the liquid cell were rinsed sequentially with anhydrous ethanol and deionized water, followed by the addition of electrolyte. Then, 10 μL of hybridization DNA mixture was added to the cis-cell (Cis) liquid cell. The liquid cell was then cooled at 4°C for 5 min and incubated for another 30 min to allow the 3-mer short-strand DNA probe to fully hybridize with the target DNA. A low-temperature environment was maintained during the detection process to prevent probe denaturation.

[0037] S3, double-layer nanopore detection like Figure 5 As shown, Ag / AgCl electrodes are inserted on both sides of the liquid pool and connected to a patch-clamp detection system. Under an applied bias voltage, DNA is driven from the cis-cell liquid pool through the first nanopore, the intermediate cavity, and the second nanopore into the trans-cell liquid pool. Perfectly matched hybrid DNA sequentially generates two blocking events; the local double-stranded region carrying the 3-mer short-chain DNA probe generates a characteristic blocking signal due to spatial conformational changes; for mismatched hybrid DNA, due to the lower probe stability, the probe may dissociate in the intermediate cavity, generating only the first blocking event and not the second blocking event, thus distinguishing between perfectly matched and mismatched probes.

[0038] After each probe detection is completed, the liquid pool and electrodes are cleaned, and the hybridization DNA sample corresponding to the next probe is added again. The detection process is repeated until all probe detections are completed.

[0039] S4. Signal Acquisition and Analysis The system acquires ionic current signals generated by DNA crossing a double-layer nanopore in real time and identifies consecutive double-blocking events. Based on the blocking current amplitude, blocking duration, double-blocking time interval, and characteristic blocking signals generated by the probe, the probe's position within the target DNA is determined. Consecutive double-blocking events serve as a temporal localization reference, while the probe's characteristic blocking signals provide spatial localization, together achieving precise probe positioning.

[0040] S5, multi-round probe detection and sequence reconstruction like Figure 6 As shown, steps S3 to S5 were repeated for 64 different 3-mer short DNA probes to obtain complete localization information of each probe in the target DNA, and a probe localization matrix composed of probe type and target DNA location was established. By combining the complementary pairing relationships of each probe, the detection results of different probes were comprehensively analyzed to restore the base sequence of the target DNA and achieve target DNA sequence reconstruction.

[0041] like Figure 7 As shown in the figure, the DNA chain sequencing results and ion current characteristics of different 3-mer short DNA probes hybridized in the experiment are presented. For the ion current characteristic signals generated by different probes, the localization results of the corresponding probes in the target DNA can be extracted respectively. By combining the probe localization information obtained from multiple rounds of detection, the designed target DNA sequence can be analyzed step by step to finally obtain the complete sequence.

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for short-chain DNA probe-assisted single-molecule sequencing based on bilayer nanopores, characterized in that, A bilayer nanopore chip with upper and lower nanopores and a central cavity structure is used as a single-molecule detection platform. A 3-mer short-chain DNA probe with a known sequence is used as a sequence localization marker. Through multiple rounds of probe hybridization detection and continuous double-blocking ion current signal analysis, the localization, identification, and reconstruction of the target DNA sequence are achieved. The sequencing method specifically includes the following steps: S1. Hybridize a 3-mer short-chain DNA probe with the DNA sample to be sequenced to obtain a hybrid DNA mixture; S2. The hybrid DNA mixture is added to the double-layer nanopore detection cell. Electrolyte and Ag / AgCl electrodes are respectively placed on both sides of the double-layer nanopore, and a bias voltage is applied so that the DNA molecules pass through the double-layer nanopore chip in sequence under the drive of the electric field. The double-layer nanopore chip includes a first nanopore, an intermediate cavity and a second nanopore. S3. Correctly paired DNA molecules generate a first blocking event and a second blocking event respectively during the continuous passage of DNA through two nanopores; the ion current signal generated by the DNA passing through the double-layer nanopores is acquired in real time, and the continuous double blocking events are identified; S4. Multi-round probe detection: After each probe detection is completed, the detection cell and electrode are cleaned, and the hybridization DNA sample corresponding to the next probe is added again. Steps S1-S3 are repeated. S5. After obtaining the localization information corresponding to each probe, establish the correspondence between the probe sequence and the DNA location to complete the reconstruction of the DNA sequence to be tested.

2. The method for short-chain DNA probe-assisted single-molecule sequencing based on bilayer nanopores according to claim 1, characterized in that, Before hybridizing the DNA sample to be sequenced with the short-chain DNA probe, guide sequences are added to the 5' and 3' ends of the DNA to be sequenced.

3. The method for short-chain DNA probe-assisted single-molecule sequencing based on bilayer nanopores according to claim 1, characterized in that, The guide sequence is an oligonucleotide sequence consisting of no fewer than 12 consecutive T bases; the 3-mer short DNA probe is an oligonucleotide probe consisting of 3 bases.

4. The method for short-chain DNA probe-assisted single-molecule sequencing based on bilayer nanopores according to claim 1, characterized in that, In step S5, the positioning information corresponding to each probe includes the blocking current amplitude, duration, double blocking interval, and probe characteristic blocking signal; the position of the probe in the target DNA is determined by the positioning information corresponding to each probe.

5. The method for short-chain DNA probe-assisted single-molecule sequencing based on bilayer nanopores according to claim 1, characterized in that, In step S5, the target DNA sequence reconstruction involves obtaining all the localization information of each probe in the target DNA and establishing a probe localization matrix composed of probe types and target DNA locations. By combining the complementary pairing relationships of each probe, the detection results of each probe are comprehensively analyzed to restore the base sequence of the target DNA and obtain the nucleotide sequence of the DNA to be tested.

6. A bilayer nanoporous chip as described in claim 1, characterized in that, The dual-layer nanoporous chip comprises a three-layer film structure consisting of an upper silicon nitride thin film, a middle silicon dioxide cavity layer, and a lower silicon nitride thin film; each of the upper and lower silicon nitride thin films has a nanopore, and the upper and lower nanopores are coaxially arranged along the film thickness direction.

7. A bilayer nanoporous chip as described in claim 6, characterized in that, The middle silica cavity layer is selectively etched with HF solution to form a cavity structure with a size larger than the nanopore diameter, so that the upper and lower nanopores are connected through the middle cavity to form a double-layer nanopore detection channel; the upper and lower nanopores are formed by helium ion beam etching.

8. A method for fabricating a bilayer nanoporous chip, characterized in that, The preparation method includes the following chip pretreatment steps: before nanopore processing, the three-layer film structure consisting of an upper silicon nitride film, a middle silicon dioxide cavity layer, and a lower silicon nitride film is washed in a piranha solution with a volume ratio of 3:1; then it is soaked and washed twice with deionized water, and dried in a vacuum drying oven; the piranha solution is prepared by mixing concentrated sulfuric acid and hydrogen peroxide in a volume ratio of 3:

1.

9. The application of the bilayer nanopore chip as described in claim 6 in DNA sequencing.

10. Application of a short-chain DNA probe-assisted single-molecule sequencing method based on bilayer nanopores in DNA sequencing.

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