Method for resolving biomolecules and solid-state nanopore four-pore array device

Abstract

The application discloses a biomolecule resolution method and a solid-state nanopore four-hole array device. The solid-state nanopore four-hole array chip comprises nanopores penetrating through an insulating film, the nanopores are arranged in an array, the number of transverse holes and longitudinal holes is both 2, the distance between the centers of the two transverse holes and the two longitudinal holes is 30-200 nm, the diameter of each nanopore is 5-10 nm, and the depth of each nanopore is 20-50 nm. The solid-state nanopore four-hole array device is used for detection, the distribution form of an electric field is innovatively changed, the pulling force generated by the four holes on a molecule will play a clamping role on the molecule, the residence time of the molecule in the region is prolonged, the resolution of the technology is finally improved, and high-precision resolution of various proteins is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for resolving biomolecules and a solid-state nanopore four-pore array device thereof, and belongs to the technical field of solid-state nanopore resolution. BACKGROUND

[0002] With the in-depth study of biological structure and function, it is increasingly clear that there are differences in the transcriptome expression levels between cells in the same cell population (Nat. Methods 2013, 10, 186). Traditional methods of studying the genomes, transcriptomes, and epigenomes and proteomes of entire cell populations can only obtain the average value of gene expression, losing the heterogeneity information of each cell and failing to in-depth understand the relationship between the transcriptome expression and immune function in special cells (e.g. tumor cells). Single-cell analysis technology can detect the heterogeneity information that cannot be obtained in mixed samples, which is of great significance (Annu. Rev. Anal. Chem 2017, 10, 345). In October 2017, the first 38 projects of the Human Cell Atlas project, which is comparable to the Human Genome Project, were officially announced, marking the beginning of a new chapter in single-cell analysis. In single-cell analysis, single-cell sequencing technology has made great progress and has gradually been commercialized in the past decade; however, due to the lack of reliable amplification methods, the analysis technology of single-cell protein atlas is far behind. The existing and relatively mature single-cell proteome analysis technologies include fluorescent Edman degradation (Nat. Biotechnol. 2018, 36, 1076), single-molecule mass spectrometry (Mass Spectrom. Rev. 2017, 36, 715), but they require harsh acid-base conditions and complex instruments and equipment.

[0003] Nanopore technology, developed in the past 30 years, can be used to study the size, charge and mechanics of single molecules without labeling (Nat. Nanotechnol. 2007, 2, 209), greatly simplifying the experimental process and reducing the operation difficulty, and is expected to become a point-of-care testing (POCT) technology that can be applied anywhere, with great application prospects. Nanopore is a small hole in an insulating film. When a biomolecule passes through the nanopore under the driving of an electric field, the structural information of the biomolecule and the detected ion current signal characteristics have a one-to-one correspondence (J. Am. Chem. Soc. 2009, 131, 9287). Therefore, the mining of the current signal can obtain the physical, chemical properties and structural dynamics information of the charged molecules. Nanopore technology mainly uses two kinds of pores: biological pores, which are naturally formed protein channels embedded in a double-layer phospholipid membrane, and solid-state nanopores, which are small holes of nanometer scale processed on an insulating film by micro-nano processing technology. Currently, biological pores have achieved single-base resolution and have become the third-generation commercial DNA sequencing technology that has attracted much attention. Although biological pores have the advantages of good repeatability and uniform size, their small size and limited stability of the phospholipid membrane supporting the nanopore limit their transmission ability to larger protein molecules. The size of solid-state nanopores can be flexibly and accurately adjusted, making it easy to extend its detection range from the initial DNA molecules to other protein molecules of different sizes, and it is expected to become a useful tool for analyzing single-cell proteomics.

[0004] Currently, most research on solid-state nanopore electrical detection technology uses a single solid-state nanopore. The problem with this detection method is that under the action of an electric field in a single direction, the speed of small biomolecules passing through the nanopore is too fast. According to the one-dimensional diffusion model, this speed is much faster than the resolution of existing commercial current amplifiers, which is about 20 microseconds. Therefore, when using a single solid-state nanopore to detect small biomolecules, the current blockage signal (ΔI) generated is often truncated and submerged in noise, resulting in the phenomenon that no signal can be detected, which seriously affects the sensitivity of the technology. Experiments and molecular dynamics simulations have shown that prolonging the capture time (dt) of the molecule has an important influence on improving the resolution of the nanopore (Nat. Biotechnol. 2020, 38, 176). Therefore, how to reduce the passing speed of these small biomolecules and improve the detection sensitivity of solid-state nanopore technology is an important problem that needs to be solved in the development of single-cell proteomics based on this technology.

[0005] Current methods for reducing protein speed mainly reduce the size of the pore to match the size of the analyte (Nat. Biotechnol. 2020, 38, 176). However, such methods are only suitable for distinguishing two proteins with similar sizes or structural changes of the same protein, and cannot distinguish proteins with large size differences. Because a suitable nanopore for one protein may be too large or too small for another protein. In addition, pressure-driven technology is also used to reduce the speed of proteins, but due to the nanoscale size, the flow field generated by pressure is limited, and the effect of reducing the speed of proteins is not obvious (ACS Nano 2018, 12, 4494; Electrophoresis 2017, 38, 1130). Therefore, it is of great significance to develop a new speed reduction mechanism that is reliable and suitable for a wide range of analytes. SUMMARY

[0006] The purpose of the present application is to provide a method for distinguishing biomolecules and a solid-state nanopore four-pore array device, which can greatly extend the time of capturing molecules and improve the resolution of molecules by solid-state nanopore technology.

[0007] In a first aspect, the present application protects a solid-state nanopore four-pore array chip, comprising nanopores penetrating through an insulating film, the nanopores being arranged in an array, the number of lateral pores and longitudinal pores being both 2;

[0008] The distance between the centers of the two lateral pores and the two longitudinal pores is 30-200 nm;

[0009] The diameter of each nanopore is 5-10 nm, and the depth is 20-50 nm.

[0010] As an example, the nanopores are arranged in an array, the number of lateral pores and longitudinal pores being both 2;

[0011] The distance between the centers of the two lateral pores and the two longitudinal pores is 50 nm;

[0012] The diameter of each nanopore is 10 nm, and the depth is 50 nm.

[0013] In a second aspect, the present application protects a method for preparing the solid-state nanopore four-pore array chip, comprising the following steps: placing an insulating film in a helium ion microscope to etch a four-pore array, thereby obtaining the solid-state nanopore four-pore array chip.

[0014] Further, the etching conditions are as follows: beam current size is 1-1.5 pA (such as 1 pA), etching dose is 10-25 nC / μm 2 (such as 25 nC / μm 2 ), the beam current size and the etching dose are determined according to the thickness of the silicon nitride chip, i.e. the depth of the nanopores in the four-pore array.

[0015] In a third aspect, the present application protects the use of the solid-state nanopore four-pore array chip in at least one of the following 1)-3):

[0016] 1) prolonging the time of capturing biomolecules;

[0017] 2) improving the resolution of biomolecules;

[0018] 3) distinguishing biomolecules of different sizes.

[0019] In the above use, the biomolecules can be nucleic acids or proteins;

[0020] The nucleic acids can be DNA or RNA, and the DNA can be double-stranded DNA or single-stranded DNA.

[0021] In a fourth aspect, the present application protects a solid-state nanopore array detection device, which comprises an electrolytic cell provided with a positive electrode and a negative electrode, and a solid-state nanopore chip separating the positive electrode and the negative electrode of the electrolytic cell, wherein the solid-state nanopore chip is the solid-state nanopore four-pore array chip.

[0022] In a fifth aspect, the present application protects a method for single-molecule resolution of biomolecules, which comprises the following steps: placing the biomolecules to be tested in the positive electrode chamber of the solid-state nanopore array detection device containing an electrolyte, and when measuring, introducing an external electric field to the positive electrode chamber and the negative electrode chamber as a driving field for the capture of biomolecules by the nanopore array, while monitoring the current blockage signal caused by the capture of single molecules by using a current amplifier.

[0023] In the above method, the voltage of the external electric field can be 40-500 mV, and specifically can be 200 mV;

[0024] The concentration of the electrolyte can be 0.1-3.2 mol / L, and specifically can be 1 mol / L;

[0025] The pH value of the electrolyte can be 8-10, and specifically can be 8;

[0026] The electrolyte can be a salt solution commonly used in solid-state nanopore detection devices, such as an aqueous solution of potassium chloride.

[0027] In the above method, the biomolecules can be nucleic acids or proteins;

[0028] The nucleic acids can be DNA or RNA, and the DNA can be double-stranded DNA or single-stranded DNA.

[0029] In specific embodiments of the present application, the biomolecules are 200 bp DNA, 500 bp DNA, and 5 nm-sized molecular cage PCC-57.

[0030] The capture and detection of the four-pore array of the present application are significantly different from that of a single solid-state nanopore. The field strength distribution of a single solid-state nanopore can be divided into a "sub-local electric field" region outside the pore (which plays a role in capturing molecules) and a "local electric field" region inside the pore (which plays a role in detecting molecules). When a molecule is captured by a single nanopore, the pore penetration speed is fast because the molecule is subjected to force in only one direction. At this time, the change in ionic current is mainly caused by the molecule passing through the central local electric field.

[0031] For a four-pore array, when the distance between the pores is less than the range of the "sub-local electric field" of a single pore, the range of the sub-local electric field for capturing will be expanded. In addition, the four-pore array will generate traction in four directions on a molecule captured in the center of the four pores, thereby prolonging the time for which the molecule stays in the "sub-local electric field" and improving the detection sensitivity. At this time, the change in ionic current is mainly caused by the molecule staying in the "sub-local electric field" region.

[0032] Compared with existing reports on protein resolution, especially solid-state nanopore resolution technology, the present application has the following advantages:

[0033] 1. More convenient operation: Compared with existing and relatively mature single-cell proteome analysis technologies, such as fluorescence Edman degradation technology and single-cell mass spectrometry technology, the present application does not require harsh acid-base conditions and complex instruments and equipment, can study the size and charge of a single molecule without labeling, greatly simplifies the experimental process, reduces the operation difficulty, and is expected to become a favorable tool for analyzing single-cell proteomics in the future.

[0034] 2. Lower cost: The preparation process of the nanopore array structure is fully compatible with the production process of modern silicon-based chips and is simple and fast, can realize mass production, is conducive to popularization, can greatly reduce the detection cost of biomolecules such as proteins, and greatly improves the resolution efficiency.

[0035] 3. Expanded range of test objects: The range of proteins that can be detected by existing solid-state nanopore detection technology is limited by the size of the pore itself. The present application adopts a different "capture" and "detection" strategy and can resolve molecules of different sizes with the same resolution accuracy.

[0036] 4. Higher sensitivity: current nanopore single molecule detection technology, mostly uses single hole detection, but due to the speed of a single protein through the nanopore is too fast, resulting in signal distortion or even unable to read. But the present application uses four-hole detection, innovatively changes the distribution of electric field, the pulling force of four holes on the molecule will play a "clamping" effect, prolong the residence time of the molecule in this area, ultimately improve the resolution of the technology, realize the high-precision resolution of various proteins. It has important promoting effect on early realization of the development of single-cell proteomics technology by using solid-state nanopore. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The pattern of the photolithography mask plate used in the present application for solid-state nanopore; wherein, Figure 1 a is the photolithography mask plate pattern designed for a 4-inch silicon wafer, Figure 1 b is a partial enlarged view of Figure 1 a, the white circular hole area corresponds to the circular light transmission area in each 3mm*3mm chip. Figure 1 c is a further enlarged view of 1b, which can clearly see the corresponding circular light transmission area in each 3mm*3mm chip. Figure 1 d is a special designed scribe line for easy cleaving single small chip from a 4-inch large silicon wafer, represented by white dotted line.

[0038] Figure 2 The flow chart for the preparation of nanopore device (3mm*3mm chip); a) glue spinning, exposure, development, glue removal, pattern b) reactive ion beam etching through one side of silicon oxide and silicon nitride; c) removing photoresist d) KOH solution anisotropic etching away silicon, exposing silicon oxide and silicon nitride suspended membrane; e) using focused ion beam to etch a small window with a depth of 1.5um in silicon oxide; f) using hydrofluoric acid buffer to remove the remaining 0.5um silicon oxide layer, only exposing 2um*2um small window size, 50nm thickness of silicon nitride suspended membrane. Figure 2 The marks in the figure are as follows: 1, 7-photoresist; 2, 6-silicon nitride; 3, 5-silicon dioxide; 4-single crystal silicon.

[0039] Figure 3 The schematic diagram of controllable preparation of solid-state nanopore array by helium ion etching technology, wherein: a-b) the size change diagram of nanopore array under the influence of different irradiation doses characterized by transmission electron microscope. c) the change diagram of nanopore measurement diameter with etching diameter. It can be seen that when the nanopore is opened, the diameter of the nanopore does not change obviously with the increase of etching dose, which shows the repeatability of the etching technology. d) helium ion scanning electron microscope image of four nanopore array prepared by helium ion etching technology. The diameter of the hole is 10nm, and the interval between the holes is 50nm.

[0040] Figure 4Fig. 1 is a schematic diagram of the present application, wherein: a) preparation of four-pore array; b) optimization of capture and detection conditions; c) expected limit current signal of two proteins A and B; d) expected current depth distribution of two proteins A and B, by which high-precision resolution of different proteins can be achieved.

[0041] Figure 5 Time resolution is insufficient when a single nanopore is used to detect small molecules, which leads to a decrease in sensitivity. a) Protein is about to pass through a solid nanopore under the action of an electric field; b) probability distribution diagram of pore passing time of different proteins calculated by one-dimensional diffusion model, the most probable pore passing time of the four proteins is much smaller than the resolution (20 microseconds) of the existing commercial current amplifier; c) blocking current signal (black solid line) caused by ideal molecular pore passing; d) blocking current signal (black solid line) caused by molecules passing too fast.

[0042] Figure 6 Comparison of four-pore array detection method used in the present application with single-pore detection method: a) cross-sectional view and electric field distribution of a single nanopore, describing the process of molecular capture and detection by the nanopore; b) three-dimensional schematic diagram of capture and detection regions of a single pore; c) cross-sectional view of a four-pore array, describing the process of molecular capture and detection by the array; d) schematic diagram of capture and detection regions of a four-pore array. The blue hemispherical dome near the array is an equipotential surface, and its field strength value is about 10 5 V / m, which can generate a capture force on the molecule.

[0043] Figure 7 Comparison of detection results of 200bp DNA by single pore and four-pore array and resolution results of different substances by four-pore array. When a single pore is used for detection, the scatter plot under an applied voltage of a) 75mV and b) 100mV. When a four-pore array is used for detection, the scatter plot under an applied voltage of c) 50mV and d) 60mV. It can be seen that the four-pore array significantly increases the detection time of short-chain DNA (200bp DNA) by the nanopore detector and improves the resolution of the nanopore, i.e. the distribution range of the signal on the ordinate is greatly reduced. In this experiment, the diameter of the nanopore is 10nm, and the spacing between the nanopore arrays is 50nm.

[0044] Figure 8 Resolution results of a) nanocage PCC-57, b) 500bp double-stranded DNA and c) 200bp double-stranded DNA by the nanopore four-pore array. The depth distribution of the current blocking signal caused by molecular pore passing is shown, and it can be seen that the three molecules are in different distribution intervals, which confirms the resolution ability of the technology for different molecules. DETAILED DESCRIPTION

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0047] Example 1: Fabrication of a solid-state nanoporous four-hole array device

[0048] 1) Fabrication of chip devices

[0049] The purpose of this step is to fabricate a solid-state nanopore chip that can be manipulated using a helium ion microscope. This allows for the use of a high-energy converging ion beam in the helium ion microscope to drill holes in the suspended thin film of the device, thereby obtaining a nanopore array chip. This involves a series of micro-nano fabrication processes used in traditional semiconductor processing, as well as a series of cutting-edge modern nanoscale fabrication technologies.

[0050] The specific method is as follows: First, a 4-inch silicon wafer with a (100) facet is used, and 2-micron silicon oxide is grown on both sides. Then, low-stress silicon nitride of about 50 nanometers is deposited using low-pressure chemical vapor deposition. Next, a photomask is fabricated to create a periodic distribution of many 3mm × 3mm small substrates on the 4-inch silicon wafer. Figure 1 a) Each 3mm×3mm small substrate pattern has a circular light-transmitting area with a diameter of 584μm at its center. Figure 1 (b, c) To ensure accurate dicing and separation of each 3mm x 3mm substrate on the 4-inch silicon wafer, and to prevent the wafer from breaking into random fragments, short, translucent scribe lines were added to the boundary of each substrate pattern: the width of the translucent strips was 5μm, and the length was 20μm. Figure 1 d). The specific fabrication parameters for the photolithography mask are: mask size: 5 inches; pattern distribution range: Ф100mm circular distribution; crystal orientation stripes: 49mm from the center, 50mm long, and 0.8mm wide. Then, photolithography is used... Figure 2 a) and reactive ion beam etching ( Figure 2 b) Erase the silicon dioxide and silicon nitride on one side of the Si wafer according to the pattern of the photomask to expose the Si surface. Then remove the photoresist. Figure 2c) Etching of silicon using KOH anisotropic etching (40% KOH, 80°C, 6 hours) which etches along (111) planes. The etching is stopped when the small windows are transparent and etching is continued for a short time. The silicon oxide surface is smooth and island free under optical microscope. This results in a suspended membrane of small windows (20-80 μm) of silicon oxide and silicon nitride on one side and silicon substrate on the other side. Figure 2 d) Next, in order to separate the 3 mm x 3 mm small chips from the 4 inch large silicon wafer, dicing is needed. A strong layer of blue tape is needed to protect the suspended membrane during dicing. The dicing depth is about 200-250 μm. After dicing, the blue tape is removed. The silicon wafer is heated to melt the wax and separate the wafer. The wax is removed by acetone. This step ensures that a large number of 3 mm x 3 mm small chips can be obtained. Typically, one 4 inch silicon wafer can be diced into 800 3 mm x 3 mm small chips. In order to reduce the area of the silicon nitride membrane exposed to the solution to reduce the capacitance noise and to reduce the possibility of breaking the silicon nitride membrane in the solution, a focused ion beam (FIB, DB 235, FEI) is used to etch 2 μm of silicon oxide with a beam current of 300 pA for about 1 minute to form a small window of 1-2 μm x 1-2 μm x 1.5 μm under the silicon nitride membrane. The 1.5 μm is the depth. The remaining 500 nm of silicon oxide is left. Figure 2 e) RCA reaction is then used to etch away the remaining 500 nm of silicon oxide to leave a suspended membrane of silicon nitride of 1-2 μm x 1-2 μm x 50 nm. Figure 2 f) The RCA reaction is NH3-H2O:H2O2 = 1:1:5 (v / v) at 70°C for 10 minutes to remove organic contamination on the wafer and to improve the wetting for the next step of BOE etching. BOE (HF:NH4F:H2O = 1:2:3) etching for 6 minutes (etching rate is 100 nm / min) to remove the remaining 500 nm of silicon oxide to leave a suspended membrane of silicon nitride. Finally, the chip is rinsed with a large amount of water to obtain a solid-state nanopore chip for the next step.

[0051] 2) Helium ion microscope for nanopore fabrication

[0052] 3mm*3mm chip device was processed and put into a helium ion microscope (Carl Zeiss, ORION NanoFab) for hole punching operation, and the best etching conditions were explored Figure 3 a-c), the field of view (FOV) was adjusted to 400 nm, the beam current was about 1 pA, the etching diameter was set to 5-10 nm, and the etching dose was set to 25 nC / μm 2 Then the parameters of the etching array were set: the lateral and longitudinal intervals of the nanopores and the number of holes in the lateral and longitudinal directions. The lateral and longitudinal intervals were set to 50 nm, and the number of holes in the lateral and longitudinal directions was set to 2, so that a four-hole nanopore array with a single hole diameter of about 10 nm, a depth of 50 nm, and a hole interval of 50 nm was obtained Figure 3 d). Before and after punching, the sample rod was placed in a plasma cleaner (O2: Ar = 1:3, v / v) for one minute to remove organic contamination. After the nanopore device was fabricated, it was stored in a vacuum drying box for future use.

[0053] Example 2, Nanopore Four-Hole Array Captures and Resolves Biomolecules

[0054] 1) Assembly of the chip

[0055] We encapsulated the chip into an upper and lower two half electrophoresis cell made of Teflon material by a series of infiltration processes with AB silicone gel (Ecoflex 00-35). The cell is composed of a Cis chamber and a Trans chamber, and the two chambers are connected only through the nanopore without other connection channels Figure 4 a-b). Then we injected a 1 molar per liter potassium chloride solution into the two chambers. The solution contains 1 mM EDTA and 10 mM Tris (pH = 8). We used two Ag / AgCl electrodes inserted into the Cis chamber and the Trans chamber, respectively.

[0056] 2) Measurement of ion current signals of test biomolecules and long-term capture

[0057] According to the electrical properties of the test molecules, we applied a suitable electric field on the two electrodes to make the test molecules pass through the nanopore from the Cis chamber to the Trans chamber. First, we injected a 100 nM solution of ultra-short DNA molecules with a length of 200 bp into the Cis chamber and monitored and recorded the ion current drop signal caused by the capture of the molecules using a patch clamp amplifier system Figure 4 c). After the measurement was completed, that is, after obtaining sufficient molecular capture signals, we flushed the Cis chamber with about 1 ml of 1 M potassium chloride ion solution while monitoring the ion current signal until there was no residual molecular-induced drop signal. Then we added another 100 nM sample of 500 bp DNA, and repeated the above steps. Finally, we added 100 nM of nanocage PCC-57 with a size of 5 nm, and repeated the above stepsFigure 4 c-d).

[0058] When a single solid-state nanopore is used to detect biomolecules, the direction of the electric field is single Figure 5 a, Figure 6 a-b), the speed of the biological small molecule is too fast. According to the one-dimensional diffusion model, the speed is much faster than the resolution of the existing commercial current amplifier ~ 20 microseconds Figure 5 b). Therefore, when a single solid-state nanopore is used to detect biological small molecules, the current blocking signal (ΔI) generated is often truncated and submerged in noise, and the phenomenon of being unable to detect any signal occurs Figure 5 c-d).

[0059] When the distance between the holes is less than the range of the "sub-local electric field" of a single hole, the range of the "sub-local electric field" for capture is expanded. In addition, the four-hole array will generate a pulling force in four directions on the molecule captured in the center of the four-hole array, thereby prolonging the time of the molecule in the "sub-local electric field" and improving the detection sensitivity Figure 6 c). At this time, the change of the ion current is mainly caused by the molecule staying in the "sub-local electric field" region Figure 6 d). Therefore, this technology can achieve the function of slowing down the ultra-short DNA molecule or capturing for a very long time Figure 7 a-d). Figure 7 a-b is a scatter plot obtained when a single nanopore detects 200bp DNA at different voltages, where each point represents a current blocking signal caused by the capture of a molecule by a nanopore array, where the abscissa is the duration of the current blocking signal, that is, the time of the molecule being captured by the nanopore, and the ordinate is the depth of the current blocking signal, which is positively correlated with the size of the molecule, and Figure 7 c-d is a scatter plot obtained when a four-hole array detects 200bp DNA. It can be seen that the capture time of the four-hole nanopore array for the molecule is significantly increased compared with the single hole, about 100 times of the capture time of the single hole.

[0060] 3) Resolution of different molecules

[0061] Due to the ability of the technology to capture biological molecules for a very long time, the resolution of the technology is greatly improved. Under an external electric field of 200mV, the four-hole nanopore array successfully distinguishes 200bp DNA, 500bp DNA and 5nm-sized molecular cage PCC-57, see Figure 8 a-c. Figure 8 The depth distribution of the current blocking signal caused by the molecule perforation is shown, and it can be seen that the three molecules are in different distribution intervals, which confirms the resolution of the technology for different molecules Figure 8 ).

[0062] The application has been described in detail. Those skilled in the art will understand that they can make modifications and alterations to this application without departing from the spirit and scope of the application. Although this application presents specific examples, it is to be understood that further modifications can be made. In general, the application is intended to cover any adaptations or variations of the present application including modifications based on the generic principles of this application as well as the present disclosure. Certain features of the application are presented in terms of examples. It is contemplated that these features can be combined with other features in the application to produce additional desirable characteristics.

Claims

1. A solid-state nanopore four-well array chip comprising nanopores through an insulating membrane, characterized in that: The nanoholes are arranged in an array, and the number of lateral holes and longitudinal holes is both 2; The distance between the centers of the two lateral holes and the two longitudinal holes is both 30-200 nm; The diameter of each nanohole is 5-10 nm, and the depth is 20-50 nm.

2. The solid-state nanopore four-well array chip of claim 1, wherein: The nanoholes are arranged in an array, and the number of lateral holes and longitudinal holes is both 2; The distance between the centers of the two lateral holes and the two longitudinal holes is both 50 nm; The diameter of each nanohole is 10 nm, and the depth is 50 nm.

3. The preparation method of the solid-state nanopore four-hole array chip according to claim 1 or 2, comprising the following steps: placing an insulating film in a helium ion microscope to etch a four-hole array, thereby obtaining the solid-state nanopore four-hole array chip.

4. The method of claim 3, wherein: The etching conditions are as follows: beam current size is 1-1.5 pA, etching dose is 10-25 nC / μm 2 .

5. The application of the solid-state nanopore four-hole array chip according to claim 1 or 2 in at least one of the following 1)-3): 1) prolonging the time of capturing biomolecules; 2) improving the resolution of biomolecules; 3) distinguishing biomolecules of different sizes.

6. Use according to claim 5, characterized in that: The biomolecules are nucleic acids or proteins; The nucleic acids are DNA or RNA, and the DNA is double-stranded DNA or single-stranded DNA.

7. A solid-state nanopore array detection device comprising an electrolytic cell provided with a positive electrode and a negative electrode, and a solid-state nanopore chip separating the positive electrode and the negative electrode of the electrolytic cell, characterized in that: The solid-state nanopore chip is the solid-state nanopore four-hole array chip according to claim 1 or 2.

8. A method for single-molecule resolution of biomolecules, comprising the following steps: placing the biomolecules to be tested in the positive electrode chamber of the solid-state nanopore array detection device containing an electrolyte according to claim 7, introducing an applied electric field to the positive electrode chamber and the negative electrode chamber during measurement, as the driving field for capturing biomolecules by the solid-state nanopore four-hole array chip, and simultaneously monitoring the current blockage signal caused by the capture of single molecules by a current amplifier.

9. The method of claim 8, wherein: The voltage of the applied electric field is 40-500 mV; The concentration of the electrolyte is 0.1-3.2 mol / L; The pH value of the electrolyte is 8-10.

10. The method according to claim 8 or 9, characterized in that: The biomolecules are nucleic acids or proteins; The nucleic acids are DNA or RNA, and the DNA is double-stranded DNA or single-stranded DNA.

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