Silicon-based high-flux single-molecule detection nanopore chip and preparation method thereof

Abstract

The invention belongs to the technical field of high-throughput sequencing sensors, and relates to a silicon-based high-throughput single-molecule detection nanopore chip and a preparation method thereof, and the silicon-based high-throughput single-molecule detection nanopore chip comprises a silicon substrate, a nano through hole array and a sample cell array; the nano through hole array and the sample cell array are respectively arranged on the upper surface and the lower surface of the silicon substrate; the nano through hole array comprises M * N nano through holes; the sample cell array comprises a plurality of non-intercommunicated sample cells; the number of the sample pools corresponds to that of the nanometer through holes; through holes with the number corresponding to that of the nano through holes are formed in the through silicon substrate; and the nano through hole is communicated with the sample cell through the through hole. According to the invention, low cost, portable use, multiple application scenes, high sensitivity and high accuracy of the nanopore can be realized, a sequencer is developed by using the technology, and the development of a fourth-generation sequencing technology is promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of high-throughput sequencing sensors, and relates to a nanopore chip and a preparation method thereof, and in particular to a silicon-based high-throughput single-molecule detection nanopore chip and a preparation method thereof. Background Art

[0002] The third generation sequencing technology is single molecule sequencing, which is characterized by the fact that it does not require PCR amplification of samples and can achieve high-throughput sequencing. It is the main direction of the development of sequencing technology in the future. There are two main core technologies for the third generation sequencing: single molecule fluorescence technology and single molecule nanopore technology. The more mature nanopore technology currently uses biological pores, which use motor proteins to pull single strands of DNA through nanopore proteins and detect the current through the pores. The structure of biological pores can be artificially designed and prepared by artificial synthesis. The advantage of biological pores is that the pore size is small, it is more sensitive to molecular pore signals, and the pore current can be directly recorded for the bases of single-stranded DNA. However, biological pores also have many disadvantages. The production cost of biological pores is high, the pore size is difficult to control, the storage of biological pore chips requires low-temperature storage, and the number of reuses is extremely limited. Another nanopore technology that is still under development is solid-state pores, which use electric field forces to drive charged DNA molecules through nanopores and detect the current through the pores. Solid-state pores use semiconductor components to open nanopores on them through physical and chemical processing. Due to the controllability of the pore size, chips can be designed for different biological molecules, not only limited to DNA and RNA, but also for proteins and other biological macromolecules.

[0003] However, the current limitations of solid-state pores are also obvious. First of all, the design and application of single-channel solid-state pores are becoming mature, but with the expansion of sequencing scale, the speed of a single channel is limited and cannot meet the demand. The design of multiple channels can effectively solve this demand, but affected by the design scheme, use process and processing conditions, a series of problems have arisen in the application, including but not limited to: 1) During the manufacturing process, the size and shape of the nanopores in each channel are different; 2) There is signal interference between channels; 3) As the number of channels increases, the heat dissipation problem becomes more prominent, affecting the accuracy and shortening the service life. Secondly, due to the limitations of integration, heat dissipation treatment and process level, most solid-state pores adopt a single electrode structure, which has limitations in signal detection and detection of changes in electrical properties when molecules pass through nanopores. Since there is only one electrode, it is impossible to provide sufficient electric field to drive molecules through nanopores, and the sensitivity is low when detecting small molecules. In addition, the traditional solid-state pore array does not have a dedicated external pad designed for the electrode, resulting in signal crosstalk between different channels, and cannot complete the independent control of a specific number of apertures, nor can it accurately troubleshoot when the molecule passes through the pore. Summary of the invention

[0004] Purpose of the invention: In view of the limitations and shortcomings of the above-mentioned prior art, the present invention proposes a silicon-based high-throughput single-molecule detection nanopore chip to solve the existing technical defects of solid-state pores; at the same time, a method for preparing a silicon-based high-throughput single-molecule detection nanopore chip is proposed to achieve low cost, portable use, multiple application scenarios, high sensitivity and high accuracy of nanopores, and use this technology to develop sequencers to promote the development of fourth-generation sequencing technology.

[0005] Technical solution: In order to achieve the above-mentioned purpose, the present invention adopts the following technical solution: a silicon-based high-throughput single-molecule detection nanopore chip, the silicon-based high-throughput single-molecule detection nanopore chip includes a silicon substrate, a nanopore array and a sample pool array; the nanopore array and the sample pool array are respectively arranged on the upper surface and the lower surface of the silicon substrate; the nanopore array includes M×N nanopores; the sample pool array includes multiple non-interconnected sample pools; the number of the sample pools corresponds to the number of nanopores; the through-holes corresponding to the number of nanopores are arranged on the through silicon substrate; the nanopores are connected to the sample pools through the through-holes.

[0006] Wherein, M represents a number consistent with the number of matrix rows; N represents a number consistent with the number of matrix columns; M×N, that is, represents M×N nano-through holes.

[0007] Preferably, the silicon-based high-throughput single-molecule detection nanopore chip adopted in the present invention includes a nanopore forming unit group, which includes a plurality of nanopore forming units, and the plurality of nanopore forming units are arranged in a matrix manner on the upper surface of the silicon substrate; each nanopore forming unit is provided with nanopores with exactly the same structure, and the nanopores on the plurality of nanopore forming units together form a nanopore array.

[0008] Preferably, the structures of the nano-through-hole forming units used in the present invention are completely the same.

[0009] Preferably, the nano-via forming unit used in the present invention includes a Si3N4 passivation layer, an upper Pt electrode layer, a Si3N4 insulating layer and a lower Pt electrode layer arranged on a silicon substrate from top to bottom; the nano-via sequentially penetrates the Si3N4 passivation layer, the upper Pt electrode layer, the Si3N4 insulating layer and the lower Pt electrode layer.

[0010] Preferably, the lower Pt electrode layer used in the present invention includes a lower Pt electrode, a lower Pt wire and a lower Pt electrode interface; the lower Pt electrode interface is connected to the lower Pt electrode through the lower Pt wire; the lower Pt electrode is disc-shaped as a whole; and the nano-through hole is arranged along the center of the lower Pt electrode.

[0011] Preferably, the lower Pt electrode interface of each nano-through hole forming unit in the nano-through hole forming unit group used in the present invention is placed on the same side of the lower Pt electrode, or is relatively arranged on both sides of the lower Pt electrode.

[0012] Preferably, the structure of the upper Pt electrode layer used in the present invention is completely the same as that of the lower Pt electrode layer.

[0013] Preferably, the nano-through hole array used in the present invention includes 2×2 nano-through holes.

[0014] Preferably, the pore diameter of the nano-through hole used in the present invention is not greater than 50 nm.

[0015] A method for preparing the aforementioned silicon-based high-throughput single-molecule detection nanopore chip, the method comprising the following steps:

[0016] 1) sputtering and depositing 100-300 nm thick Pt on a silicon substrate, coating with photoresist, exposing, developing, and etching Pt to form a lower Pt electrode layer;

[0017] 2) growing a 50-100 nm thick Si3N4 layer on the substrate obtained in step 1) by chemical vapor deposition, and photolithography Si3N4 to form a Si3N4 insulating layer;

[0018] 3) On the substrate obtained in step 2), a 100-300 nm thick Pt layer is deposited by sputtering, a photoresist is coated, the Pt layer is exposed, developed, and the Pt layer is etched to form an upper Pt electrode layer;

[0019] 4) growing a 50-100 nm thick Si3N4 layer on the substrate obtained in step 3) by chemical vapor deposition, and photolithography Si3N4 to form a Si3N4 passivation layer;

[0020] 5) On the substrate obtained in step 4), using electron beam lithography, a nano-through hole is etched at the center of the upper Pt electrode layer, and the nano-through hole penetrates the Si3N4 passivation layer, the upper Pt electrode layer, the Si3N4 insulating layer and the lower Pt electrode layer from top to bottom, wherein the aperture of the nano-through hole is not greater than 50 nm;

[0021] 6) coating a photoresist on the back side of the substrate obtained in step 5), exposing, developing, and etching the silicon substrate by deep reactive ion etching process to retain a silicon substrate with a thickness of about 50±5 μm;

[0022] 7) Perform isotropic etching in HNA solution to etch away the silicon substrate with a thickness of about 50±5 μm retained in step 6) to form a silicon-based high-throughput single-molecule detection nanopore chip.

[0023] The beneficial effects of the present invention are:

[0024] The nanopore chip of the present invention is prepared based on silicon-based semiconductor technology and has a low production cost. Compared with traditional biological pore chips, it can be stored at room temperature and can be reused many times. The pore size and number of the nanopore chip of the present invention are controllable, and the requirements of high-throughput sequencing can be met through strict control of the design scheme and processing technology. The nanopore chip of the present invention adopts a dual-electrode structure, and realizes selective response to specific ions by accurately controlling the electrode potential, effectively controlling and adjusting the electrochemical environment of the electrode interface; at the same time, it provides a stable and uniform electric field, reduces noise, and can also freely adjust the voltage value as needed to select a suitable macromolecule through-hole speed. The dual-electrode system can balance the charge transfer between the electrodes, reduce the potential fluctuation caused by the change of the electrode surface state, and ensure the stability and reproducibility of the sensor. This stability is particularly important for repeated use and high-precision applications. The nanopore chip of the present invention is designed with efficient metal routing and exclusive external pads for each metal electrode. Through the exclusive pad, voltage can be applied to each hole and the signal can be read separately, thereby achieving more precise control and operation. At the same time, it helps to isolate the signal paths of each channel and reduce electromagnetic interference between different channels. By optimizing the pad layout and the distance between the holes, crosstalk can be further reduced and the signal-to-noise ratio of the system can be improved. The design of a specific voltage waveform and current detection strategy can help provide better control when molecules pass through the nanopore and reduce the possibility of molecular blockage. When blockage occurs, the voltage on the dedicated pad can be adjusted to try to clear the blocked molecules and restore the function of the nanopore. The design of the dedicated pad allows each channel to be independently connected to the external circuit, which facilitates the integration and expansion of the system. This design provides greater flexibility when more channels need to be added or the system needs to be upgraded in the future. When a nanopore in the system fails, the problem channel can be quickly located and repaired or replaced separately without affecting the operation of the entire system. The present invention has the characteristics of high sequencing stability, high sequencing throughput, miniaturization and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a longitudinal cross-sectional view of the silicon-based high-throughput single-molecule detection nanopore chip provided by the present invention;

[0026] Figure 2 It is a structural diagram of the lower Pt electrode layer in the single-side layout method adopted by the present invention;

[0027] Figure 3 It is a structural diagram of the upper Pt electrode layer in the single-side layout adopted by the present invention;

[0028] Figure 4 It is a top view of the overall structure of the single-side layout adopted by the present invention;

[0029] Figure 5 It is a structural diagram of the upper Pt electrode layer and the lower Pt electrode layer in the double-sided layout adopted by the present invention;

[0030] Figure 6 It is a top view of the overall structure of the double-sided layout adopted by the present invention;

[0031] Figure 7 is a schematic diagram of the nanopore structure used in the present invention;

[0032] in:

[0033] 1-silicon substrate; 2-lower Pt electrode layer; 3-Si3N4 insulating layer; 4-upper Pt electrode layer, 5-sample cell array; 6-nano-through hole array; 7-Si3N4 passivation layer. DETAILED DESCRIPTION

[0034] The present invention provides a silicon-based high-throughput single-molecule detection nanopore chip, comprising a silicon substrate 1, a nanopore array 6 and a sample pool array 5; the nanopore array 6 and the sample pool array 5 are respectively arranged on the upper surface and the lower surface of the silicon substrate 1; the nanopore array 6 includes M×N nanopores; the sample pool array 5 includes a plurality of non-interconnected sample pools; the number of sample pools corresponds to the number of nanopores; through holes corresponding to the number of nanopores are arranged on the silicon substrate 1; the nanopores are connected to the sample pools through the through holes. M rows*N columns

[0035] The silicon-based high-throughput single-molecule detection nanopore chip includes a nanopore forming unit group, which includes a plurality of nanopore forming units, and the plurality of nanopore forming units are arranged in a matrix on the upper surface of a silicon substrate 1; each nanopore forming unit is provided with a nanopore with a completely identical structure, and the nanopores on the plurality of nanopore forming units together form a nanopore array 6. The nanopore forming units used in the present invention have completely identical structures, and illustratively, the nanopore forming units include a Si3N4 passivation layer 7, an upper Pt electrode layer 4, a Si3N4 insulating layer 3, and a lower Pt electrode layer 2, which are sequentially arranged on the silicon substrate 1 from top to bottom; the nanopores sequentially penetrate the Si3N4 passivation layer 7, the upper Pt electrode layer 4, the Si3N4 insulating layer 3, and the lower Pt electrode layer 2. The lower Pt electrode layer 2 includes a lower Pt electrode, a lower Pt wire and a lower Pt electrode interface; the lower Pt electrode interface is connected to the lower Pt electrode through the lower Pt wire; the lower Pt electrode is disc-shaped as a whole; and the nano-through hole is arranged along the center of the lower Pt electrode. The lower Pt electrode interface of each nano-through hole forming unit in the nano-through hole forming unit group is placed on the same side of the lower Pt electrode, or is relatively arranged on both sides of the lower Pt electrode. The structure of the upper Pt electrode layer 4 is exactly the same as that of the lower Pt electrode layer 2.

[0036] The working principle of the high-throughput single-molecule detection nanopore chip is introduced by taking the single-sided layout as an example. When the biomacromolecule passes through the hole in the horizontal direction, the chip adopts a single-sided layout, and the chip is placed vertically in the sample liquid. The lower Pt electrode interface and the upper Pt electrode interface are designed to be located on one side of the chip, and are located on the side above the liquid surface when in use. The through-hole current in the nanopore can be detected by the upper Pt electrode and the lower Pt electrode on both sides of the nanopore, so as to analyze the through-hole electrical signal generated when the molecule to be tested passes through the hole. The single-sided layout places the lower Pt electrode interface and the upper Pt electrode interface on the upper side of the sample liquid, avoiding problems such as short circuit and excessive structural consumption caused by liquid corrosion penetration. At the same time, the upper Pt electrode interface and the lower Pt electrode interface are placed in a similar area, which is convenient for integrated design and maintenance of functional partitions. The double-sided layout is applied to the case of vertical through-holes of biomolecules, and the principle is similar to that of the single-sided layout. By increasing the number of nanopores and improving the chip integration, the chip can be applied to larger-scale sequencing.

[0037] Below, the technical solution provided by the present invention will be described in detail by taking a 2×2 nano-through hole array 6 as an example and combining with the corresponding drawings:

[0038] like Figure 1 As shown, the present invention provides a silicon-based high-throughput single-molecule detection nanopore chip, which includes, from bottom to top, a silicon substrate 1, a lower Pt electrode layer 2, a Si3N4 insulating layer 3, an upper Pt electrode layer 4, and a Si3N4 passivation layer 7. The nano-through hole array 6 includes 4 identical nano-holes, which are located at the center of the upper Pt electrode layer 4, have a pore size of less than 50nm, and penetrate the Si3N4 passivation layer 7, the upper Pt electrode layer 4, the Si3N4 insulating layer 3, and the lower Pt electrode layer 2. The sample pool array 5 includes 4 identical and non-interconnected sample pools, which are located on the back of the silicon substrate 1, penetrate to the lower surface of the lower Pt electrode layer 2, and are located directly below the nano-through hole array 6.

[0039] See also Figure 2 , the lower Pt electrode layer 2 includes 4 lower Pt electrodes, lower Pt wires and 4 lower Pt electrode interfaces. Among them, each lower Pt electrode is connected to the corresponding lower Pt electrode interface through a lower Pt wire. The 4 lower Pt electrodes are equal in area and thickness and are not connected to each other. The overall distribution is in a 2×2 square array, and the distances between adjacent lower Pt electrodes in the longitudinal and transverse directions are equal. The lower Pt electrodes are in the shape of regular disks, so as to effectively read the via signals and reduce the signal errors caused by the irregularity of the electrodes themselves. The lower Pt wires are arranged in parallel, with a routing corner of 90 degrees, and no two wires cross each other. The 4 lower Pt electrode interfaces are of the same size and isolated from each other. According to the vertical or horizontal placement direction of the chip in the sample liquid, the layout of the lower Pt wires and the lower Pt electrode interfaces can be single-sided or double-sided.

[0040] See also Figure 5 When the chip is placed vertically, a single-side layout scheme is adopted. The lower Pt electrode interface is located on the vertical upper side of the lower Pt electrode and is on the same horizontal line. The four lower Pt electrode interfaces are connected to the lower left electrode, upper left electrode, upper right electrode and lower right electrode of the lower Pt electrode from left to right. Figure 6 ,When the chip is placed horizontally, a double-sided layout scheme is adopted, and the lower Pt electrode interface is located on the upper and lower Pt electrode.

[0041] See also Figure 1 The Si3N4 insulating layer 3 uniformly covers the upper and side surfaces of the lower Pt electrode layer 2 and the surface of the silicon substrate 1 where the lower Pt electrode layer 2 is not formed, completely isolating the lower Pt electrode layer 2 from the air.

[0042] like Figure 3 As shown, the upper Pt electrode layer 4 includes 4 upper Pt electrodes, an upper Pt wire and 4 upper Pt electrode interfaces. Among them, each upper Pt electrode is connected to the corresponding upper Pt electrode interface through an upper Pt wire. The size and thickness of the upper Pt electrode are exactly the same as those of the lower Pt electrode. The duality of the circuit is guaranteed by the manufacturing process to improve the accuracy of sequencing. The upper Pt wires are arranged in parallel, with a routing angle of 90 degrees, and no two wires cross each other. The four upper Pt electrode interfaces are the same size and isolated from each other. According to the vertical or horizontal placement direction of the chip in the sample liquid, the layout of the upper Pt wire and the upper Pt electrode interface also has two layout modes: single-sided layout and double-sided layout, which matches the layout of the lower Pt electrode interface and the lower Pt wire.

[0043] like Figure 4 , Figure 5 as well as Figure 6 As shown, when the chip is placed vertically, a single-sided layout scheme is adopted. The upper Pt electrode interfaces are all located on the vertical upper side of the upper Pt electrode, and are connected to the lower left electrode, upper left electrode, upper right electrode, and lower right electrode of the upper Pt electrode from left to right. The upper Pt electrode interface and the lower Pt electrode interface are located on the same straight line, and are symmetrically distributed on the left and right sides. When the chip is placed horizontally, a double-sided layout scheme is adopted. The upper Pt electrode interface is located on the upper and lower sides of the upper Pt electrode. The upper Pt electrode interface and the lower Pt electrode interface are overall symmetrically distributed up and down.

[0044] The method for preparing the above-mentioned silicon-based high-throughput single-molecule detection nanopore chip comprises:

[0045] Step 1: deposit a Pt layer with a thickness of 100-300 nm on a silicon substrate 1 by sputtering or the like, apply photoresist, expose, develop, and etch the Pt to form a lower Pt electrode layer 2.

[0046] Step 2: On the substrate obtained in step 1, grow a 50-100 nm thick Si3N4 layer by chemical vapor deposition or other processes, and photolithography Si3N4 to form a Si3N4 insulating layer 3.

[0047] Step 3: On the substrate obtained in step 2, a layer of Pt with a thickness of 100-300 nm is deposited by sputtering or the like, photoresist is coated, exposed, developed, and Pt is etched to form an upper Pt electrode layer 4.

[0048] Step 4: On the substrate obtained in step 3, a 50-100 nm thick Si3N4 layer is grown by chemical vapor deposition or other processes, and Si3N4 is photolithographically processed to form a Si3N4 passivation layer 7.

[0049] Step 5: On the substrate obtained in step 4, use electron beam lithography technology to etch through holes at the centers of the four upper Pt electrodes, penetrating the Si3N4 passivation layer 7, the upper Pt electrode layer 4, the Si3N4 insulating layer 3 and the lower Pt electrode layer 2 from top to bottom, and the through hole diameter is less than 50nm.

[0050] Step 6: coating photoresist on the back of the substrate obtained in step 5, exposing, developing, and etching the silicon substrate 1 by deep reactive ion etching process, etching away the silicon substrate 1 of about 350 μm thickness, and retaining the silicon substrate 1 of about 50 μm thickness;

[0051] Step 7: Perform isotropic etching in the HNA solution to etch away the silicon substrate 1 with a thickness of about 50 μm retained in step 6, thereby forming a silicon-based high-throughput single-molecule detection nanopore chip.

Claims

1. A silicon-based high-throughput single-molecule detection nanopore chip, characterized in that: The silicon-based high-throughput single-molecule detection nanopore chip comprises a silicon substrate (1), a nanopore array (6) and a sample pool array (5); the nanopore array (6) and the sample pool array (5) are respectively arranged on the upper surface and the lower surface of the silicon substrate (1); the nanopore array (6) comprises M×N nanopores; the sample pool array (5) comprises a plurality of non-interconnected sample pools; the number of the sample pools corresponds to the number of the nanopores; the through-holes corresponding to the number of the nanopores are arranged on the through silicon substrate (1); and the nanopores are connected to the sample pools via the through-holes.

2. The silicon-based high-throughput single-molecule detection nanopore chip according to claim 1, characterized in that: The silicon-based high-throughput single-molecule detection nanopore chip comprises a nanopore forming unit group, wherein the nanopore forming unit group comprises a plurality of nanopore forming units, and the plurality of nanopore forming units are arranged in a matrix manner on the upper surface of a silicon substrate (1); each nanopore forming unit is provided with a nanopore having a completely identical structure, and the nanopores on the plurality of nanopore forming units together form a nanopore array (6).

3. The silicon-based high-throughput single-molecule detection nanopore chip according to claim 2, characterized in that: The structures of the nano-through-hole forming units are completely the same.

4. The silicon-based high-throughput single-molecule detection nanopore chip according to claim 3, characterized in that: The nano-through hole forming unit comprises a Si3N4 passivation layer (7), an upper Pt electrode layer (4), a Si3N4 insulating layer (3) and a lower Pt electrode layer (2) which are arranged on a silicon substrate (1) in sequence from top to bottom; the nano-through hole sequentially penetrates the Si3N4 passivation layer (7), the upper Pt electrode layer (4), the Si3N4 insulating layer (3) and the lower Pt electrode layer (2).

5. The silicon-based high-throughput single-molecule detection nanopore chip according to claim 4, characterized in that: The lower Pt electrode layer (2) comprises a lower Pt electrode, a lower Pt wire and a lower Pt electrode interface; the lower Pt electrode interface is connected to the lower Pt electrode via the lower Pt wire; the lower Pt electrode is disc-shaped as a whole; and the nano-through hole is arranged along the center of the lower Pt electrode.

6. The silicon-based high-throughput single-molecule detection nanopore chip according to claim 5, characterized in that: The lower Pt electrode interface of each nano-through hole forming unit in the nano-through hole forming unit group is placed on the same side of the lower Pt electrode, or is relatively arranged on both sides of the lower Pt electrode.

7. The silicon-based high-throughput single-molecule detection nanopore chip according to claim 6, characterized in that: The structure of the upper Pt electrode layer (4) is completely identical to that of the lower Pt electrode layer (2).

8. The silicon-based high-throughput single-molecule detection nanopore chip according to any one of claims 1 to 7, characterized in that: The nano-through hole array (6) includes 2×2 nano-through holes.

9. The silicon-based high-throughput single-molecule detection nanopore chip according to claim 8, characterized in that: The pore diameter of the nano-through hole is no greater than 50 nm.

10. A method for preparing a silicon-based high-throughput single-molecule detection nanopore chip as claimed in claim 9, characterized in that: The preparation method comprises the following steps: 1) sputtering and depositing a 100-300 nm thick Pt layer on a silicon substrate (1), coating with a photoresist, exposing, developing, and etching the Pt to form a lower Pt electrode layer (2); 2) On the substrate obtained in step 1), a 50-100 nm thick Si3N4 layer is grown by chemical vapor deposition, and Si3N4 is photolithographically formed into a Si3N4 insulating layer (3); 3) On the substrate obtained in step 2), a 100-300 nm thick Pt layer is deposited by sputtering, a photoresist is coated, the Pt layer is exposed, developed, and the Pt layer is etched to form an upper Pt electrode layer (4); 4) growing a 50-100 nm thick Si3N4 layer on the substrate obtained in step 3) by chemical vapor deposition, and photolithography Si3N4 to form a Si3N4 passivation layer (7); 5) On the substrate obtained in step 4), using electron beam lithography, a nano-through hole is etched at the center of the upper Pt electrode layer (4), and the nano-through hole penetrates the Si3N4 passivation layer (7), the upper Pt electrode layer (4), the Si3N4 insulating layer (3) and the lower Pt electrode layer (2) from top to bottom, wherein the aperture of the nano-through hole is not greater than 50 nm; 6) coating a photoresist on the back side of the substrate obtained in step 5), exposing, developing, and etching the silicon substrate (1) by a deep reactive ion etching process, leaving a 50±5 μm thick silicon substrate (1); 7) Perform isotropic etching in HNA solution to etch away the 50±5 μm thick silicon substrate (1) retained in step 6) to form a silicon-based high-throughput single-molecule detection nanopore chip.

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