A method for the preparation of low-noise solid-state nanopores

Abstract

The application provides a low-noise solid-state nanopore preparation method, and belongs to the technical field of molecular detection and sequencing, which comprises the following steps: step S1, nanometer film chip preparation, a double-layer material solid-state nanometer film chip is constructed on a wafer; step S2, microfluidic chip assembly, according to the appearance size of the double-layer material solid-state film chip, a chip with a multilayer structure and a micro flow channel is designed and processed; step S3, processing parameter setting, according to the thickness and material dielectric constant of the double-layer material solid-state film, the output current and output voltage of a constant current source system are set, and are used for preparing a solid-state nanopore with a small aperture; and step S4, solid-state nanopore preparation, according to the processing parameters set in step S3, a solid-state nanopore is prepared by using an ultrahigh electric field. Based on the microfluidic microdroplet contact method, the application precisely controls the time and position of microdroplet contact above the nanometer film, and realizes the control of the processing position and speed of the solid-state nanopore.

Description

Technical Field

[0001] This invention belongs to the field of molecular detection and sequencing technology, and specifically relates to a method for preparing low-noise solid nanopores. Background Technology

[0002] With the development of nanoscience and technology, nanopore-based sensors have become outstanding tools for single-molecule analysis of key components of living organisms, including nucleic acids, proteins, glycans, and a large number of biomolecules that play vital roles in life and medicine. Solid-state nanopores are considered an effective alternative to biological nanopores due to their controllable pore size, good environmental adaptability, chemical stability, and scalable fabrication and integration capabilities. However, high noise interference remains the most serious problem restricting their application in DNA sequencing. Exploring methods to reduce solid-state nanopore noise and researching low-noise solid-state nanopore fabrication methods are of great significance for breakthroughs in solid-state nanopore technology.

[0003] Currently, transmission electron microscopy (TEM) electron beam, focused ion beam, and dielectric breakdown methods are three commonly used fabrication techniques for small-aperture thin-film nanopores. However, the extremely high cost of electron beam / ion beam equipment and the stringent requirements of laboratory conditions limit the applicability of these methods. Existing noise reduction schemes for solid-state nanopores all employ a photolithography-transfer process for the film capping layer. This involves first photolithographically creating windows on a polymer film, then transferring the capping layer onto a silicon nitride film, and finally fabricating silicon nitride nanopores at the polymer window locations. The thickness and opening area of ​​this capping layer are affected by the photolithography and transfer processes, and it cannot completely cover the surface of the silicon nitride film.

[0004] If a polymer capping layer is pre-coated onto a silicon nitride film before direct fabrication of solid-state nanopores in bilayer materials, the unbroken portions of the silicon nitride film will be completely covered by the polymer. This minimizes the contact between the electrolyte solution and the silicon nitride film surface, significantly reducing the overall capacitance and detection noise of the nanopores. Currently used fabrication techniques for small-aperture thin-film nanopores (transmission electron microscopy, focused ion beam, and dielectric breakdown method) cannot meet the requirements for fabricating solid-state bilayer materials. Furthermore, the fabrication of solid-state bilayer materials places ultra-high electric field requirements on the preparation of solid-state nanopores using the dielectric breakdown method—the applied electric field must reach the breakdown requirements of the silicon nitride film to achieve nanopore fabrication. This results in the polymer capping layer being simultaneously placed under an ultra-high electric field much higher than its own breakdown electric field, which can lead to the severe consequence of the polymer capping layer itself being broken down. Summary of the Invention

[0005] Addressing the limitation that the electric field applied by the dielectric breakdown method in existing technologies cannot exceed the breakdown electric field of solid thin films, this invention provides a method for preparing low-noise solid nanopores. This method can prepare solid nanopore devices with various pore sizes on various types of thin film substrates, especially multilayer thin film substrates, in a single operation. In particular, it can realize the preparation and single-molecule testing of low-noise nanopores on the same platform, and can be widely used in fields such as nanopore single-molecule detection and nanopore sequencing.

[0006] According to a first aspect of the present invention, a method for preparing low-noise solid-state nanopores is provided, comprising a low-noise nanopore fabrication step and a nanopore testing step, wherein the low-noise nanopore fabrication step includes the following steps:

[0007] Step S1, Nanofilm chip fabrication: Constructing a bilayer material solid nanofilm chip on a thin film;

[0008] Step S2: Microfluidic chip assembly. Based on the external dimensions of the bilayer material solid thin film chip, design and fabricate a multilayer chip with microchannels.

[0009] Step S3, setting processing parameters: Based on the thickness of the bilayer solid film and the dielectric constant of the material, set the output current and output voltage of the constant current source system to prepare small-aperture solid nanopores;

[0010] Step S4: Solid-state nanopore preparation. Based on the processing parameters set in step S3, solid-state nanopores are prepared using an ultra-high electric field.

[0011] The in-situ nanopore testing step involves performing in-situ nanopore testing on the low-noise nanopores prepared above, which includes the following steps:

[0012] Step W1: Replace the power meter with a multi-channel nanopore current testing system to test the nanopore pore size and ensure that the testing performance and indicators of the nanopore meet the requirements.

[0013] Step W2: Replace the buffer solution at the front end of the multi-channel peristaltic pump with the sample solution to be tested;

[0014] Step W3, Single molecule detection: The test solution is injected into one end of a solid nanopore under the drive of a multi-channel peristaltic pump. Under the action of a bias electric field, it passes through the solid nanopore in the form of single molecules, forming an ion current signal that can be recorded by patch clamp.

[0015] Step W4 involves storing and analyzing the sample test signal based on the nanopore through-pore current recorded in step W3.

[0016] Furthermore, in step S1, a PMMA capping layer with a thickness of less than 80 nm is directly suspended on a silicon nitride thin film with a TEM window to form a bilayer material solid thin film chip composed of a PMMA capping layer and a silicon nitride thin film.

[0017] Further, in step S1, a bilayer material solid nanofilm is constructed.

[0018] Preferably, a three-layer chip with microchannels is designed and fabricated based on the external dimensions of the two-layer material solid thin film chip.

[0019] Preferably, the bilayer solid film chip is installed in the middle layer of the microfluidic chip and fixedly bonded; after installation, a solution pipeline is connected to control the buffer solution to enter the lower common flow channel layer of the solid film, and the buffer solution is in complete contact with the lower part of the bilayer solid film until the buffer solution flows out of the microfluidic outlet and connects to the common electrode.

[0020] Preferably, the solution tubing and electrodes at the upper end of the microfluidic chip are connected to the solution inlet at the upper end of the microfluidic chip, but no buffer solution is injected. The buffer solution is injected according to the set processing parameters after the DC power is turned on.

[0021] Preferably, based on the thickness of the bilayer solid film and the dielectric constant of the material, the output current of the constant current source system is set to 5nA, and the output voltage is 1 to 5 times the breakdown electric field corresponding to the nanofilm layer, for the preparation of small-aperture solid nanopores.

[0022] More preferably, a pre-set low-noise nanopore controllable preparation process is used to apply an ultra-high electric field (1.5*E) on the microfluidic chip. bd Preparation of solid nanopores.

[0023] According to a second aspect of the present invention, a method for preparing low-noise solid-state nanopores employs a microfluidic-based locally controllable solid-state nanopore preparation system. This system precisely controls the contact position and contact time between the solution at the tip of the nanoneedle and the solid film via a microfluidic system and nanoneedles in a microfluidic chip, achieving locally controllable preparation of solid-state nanopores. The microfluidic locally controllable solid-state nanopore preparation system includes a microfluidic chip, a multi-channel peristaltic pump, a solution tubing, a multilayer solid nanofilm, electrodes, a power meter, and a processing system; wherein...

[0024] Microfluidic chips are used to mount and fix solid nanopore chips, precisely control the solutions on both sides of the solid nanopores, and add samples to be tested.

[0025] Multichannel peristaltic pumps are used to control the flow rate of the solution within the microfluidic chip and to add buffer solutions and the types of samples to be tested;

[0026] The solution tubing is used to connect the buffer solution, the sample solution bottle to the microfluidic chip, and the T-connector for accessing the electrodes. The flow rate and volume of the solution in the tubing are precisely controlled by a multi-channel peristaltic pump.

[0027] Multilayer solid nanofilms are used to prepare solid nanopores. The multilayer solid nanofilm is the original substrate of the solid nanopore. When stimulated by the local electric field of nanoneedles, it is broken down to form solid nanopores of multilayer material.

[0028] The electrode is used to connect the buffer ion solution to the power meter or nanopore test circuit to ensure smooth ion exchange between the ion solution and the electrode interface.

[0029] The power meter is used to provide an adjustable regulated power supply or a constant current source; when performing nanopore through-hole current testing, the power meter is replaced by a multi-channel patch clamp detection system to record the through-hole current of solid nanopores in real time.

[0030] The processing system is used for programmable power meters or nanopore testing circuits, providing a graphical interface for user interaction, and enabling functions such as real-time control of the current and voltage applied by the power meter, testing of nanopore pore size, and detection of single-molecule through-pore signals.

[0031] Compared with existing technologies, the method for preparing low-noise solid-state nanopores of the present invention has the following technical or beneficial effects:

[0032] 1. This invention is based on the microfluidic microdroplet contact method, which precisely controls the time and position of microdroplet contact above the nanofilm, thereby achieving control over the processing position and speed of solid nanopores.

[0033] 2. This invention employs a locally controllable dielectric breakdown method, which can process solid nanopores using an electric field several times higher than the material breakdown electric field. It has the ability to process thin films of different materials simultaneously, and provides a new method for directly processing bilayer or multilayer solid nanopores.

[0034] 3. The present invention controls the micro-contact between the solution and the nanofilm using nanoneedles, resulting in a small contact area and improving the signal-to-noise ratio and throughput of solid-state nanopore detection. This means that it can realize the individual preparation and detection of nanopores for each nanoneedle channel.

[0035] 4. The present invention enables the in-situ preparation of solid nanopores by simply changing the reagent at the front end of the buffer solution, thereby changing the sample to be tested. This solves the problem of cumbersome solution changing steps and large amounts of solution used in common nanopore detection systems. It is a high-throughput nanopore preparation and testing system that allows for simultaneous preparation and testing. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the low-noise nanopore preparation system according to the present invention.

[0037] Figure 2 for Figure 1 A schematic diagram of the internal structure of the microfluidic chip 1.

[0038] Figure 3 This is a schematic diagram of the preparation process of the low-noise solid nanopore preparation method according to the present invention.

[0039] Figure 4 for Figure 3 The flowchart shows the process control of the preparation method for low-noise solid nanopores.

[0040] Labels in the attached diagram:

[0041] Figure 1 1: Microfluidic chip, 2: Multichannel peristaltic pump, 3: Tube, 4: Multilayer solid nanofilm, 5: Electrode, 6: Power meter, 7: Processing system;

[0042] Figure 2 101: Solution inlet, 102: Solution outlet, 103: Nanoneedles, 104: Microchannel, 401: Polymer layer, 402: Nanofilm layer, 601: Current source, 602: Voltmeter;

[0043] Figure 3 103: Nano needles, 401: Polymer layer, 402: Nano film layer, 403: Low-noise nanopores of bilayer material, 5: Electrode, 601: Current source, 602: Voltmeter;

[0044] Figure 4 In this diagram, Z represents the distance between the solution in the nanoneedles and the upper surface of the nanofilm. When a high voltage of 15V is applied, the program controls a peristaltic pump to inject a buffer solution at a rate of 15µl / s, causing the distance between the solution in the nanoneedles and the upper surface of the nanofilm to gradually approach, until contact Z = 0. At this point, due to the ultra-high electric field of 1.5*Ebd applied across the solid film, the solid film will momentarily break down, creating solid nanopores. The sharp drop in the feedback voltage of the applied constant current source can be detected immediately upon the breakdown. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Furthermore, the scope of protection of the present invention should not be limited to the specific structures, components, or parameters described below.

[0046] It should be noted that when a component is referred to as "fixed to" or "set on" another component, it can be directly or indirectly attached to that other component. When a component is referred to as "connected to" another component, it can be directly or indirectly connected to that other component. Unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0047] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or component referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention.

[0048] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0049] This invention proposes a method for fabricating low-noise solid-state nanopores, which is a method for controllably fabricating low-noise nanopores under localized ultra-high electric fields. It enables the fabrication of nanopores under electric fields several times higher than the breakdown electric field, providing the possibility for ultra-high electric field fabrication of nanopores in bilayer thin films of different materials, and realizing the in-situ fabrication of low-noise solid-state nanopores. Furthermore, this invention's method for fabricating low-noise solid-state nanopores constructs a multilayer solid thin film substrate and uses a cross-electric field fabrication method to fabricate and completely cover low-noise nanopores.

[0050] Based on the preparation and research of solid nanopores in this invention, in the solid nanopore detection system, the interaction between the ionic solution and the nanopore wall and the surface of the nanofilm is the main cause of nanopore noise. The nanopore noise is directly determined by the nanofilm material and structure (electron mobility, dielectric constant, area and thickness).

[0051] The equipment used in the low-noise solid-state nanopore fabrication method of this invention is a microfluidic-based locally controllable solid-state nanopore fabrication system. This system precisely controls the contact position and contact time between the solution at the tip of the microfluidic needle and the solid film through a microfluidic system and nanoneedles in a microfluidic chip, achieving locally controllable fabrication of solid-state nanopores. The microfluidic-based locally controllable solid-state nanopore fabrication system includes a microfluidic chip 1, a multi-channel peristaltic pump 2, a solution tubing 3, a multilayer solid nanofilm 4, electrodes 5, a power meter 6, and a processing system 7.

[0052] Microfluidic chip 1 is used to mount and fix a solid nanoporous chip, precisely control the solution on both sides of the solid nanoporous pore, and add the sample to be tested; microfluidic chip 1 includes a solution inlet 101, a solution outlet 102, nanoneedles 103, and a microchannel structure 104.

[0053] Multichannel peristaltic pump 2 is used to control the flow rate of the solution in the microfluidic chip and the type of buffer solution and sample to be added;

[0054] Solution tubing 3 is used to connect the buffer solution, the sample solution bottle to the microfluidic chip, and provides a 3-way connector for accessing electrode 5. The flow rate and volume of the solution in the tubing are precisely controlled by the multi-channel peristaltic pump 2.

[0055] Multilayer solid nanofilm 4 is used to prepare solid nanopores. The multilayer solid nanofilm serves as the original substrate for the solid nanopores. When stimulated by a localized electric field from nanoneedles, it is broken down, forming a multilayer solid nanopore. Multilayer solid nanofilm 4 includes a polymer layer 401 and a nanofilm layer 402. The polymer layer is typically a high-molecular-weight material, such as PMMA, PDMS, SU8 photoresist, etc., with a thickness ranging from 1 nm to 1 μm. The nanofilm layer uses commonly used solid nanopore processing materials, such as silicon dioxide, silicon nitride, molybdenum disulfide, graphene, hafnium oxide, etc., with a thickness ranging from 0.35 nm to 30 nm.

[0056] Electrode 5 is used to connect the buffer ion solution to the power meter 6 or the nanopore test circuit. This electrode is usually an Ag / AgCl electrode, but Au and Pt electrodes can also be used to ensure smooth ion exchange between the ion solution and the electrode interface.

[0057] Power meter 6 provides an adjustable regulated power supply or a constant current source. This power meter can operate in voltage source mode and current source mode. In voltage source mode, it provides a specified voltage to the system and can detect the current in the circuit in real time (two-wire operation mode); in current source mode, it provides a constant current source to the system and can detect the voltage in the circuit in real time, with the maximum output voltage controlled by a specified voltage. In the figure, power meter 6 includes a current source 601 and a voltmeter 602, i.e., it operates in constant current source mode. When performing nanopore through-hole current testing, power meter 6 is replaced by a multi-channel patch-clamp detection system or other nanopore testing circuitry to record the through-hole current of solid-state nanopores in real time.

[0058] The processing system 7 is used to program-control the power meter 6 or the nanopore test circuit, providing a graphical interface for user interaction, and realizing functions such as real-time control of the current and voltage applied by the power meter, testing of nanopore pore size, and detection of single-molecule through-pore signals.

[0059] The microfluidic chip 1 is equipped with hollow nanoneedles 103. Each nanoneedle 103 is connected to the solution inlet 101 through a microchannel 104. The multi-channel peristaltic pump 2 controls the solution flow rate in the microchannel 104, which is the key to realizing the instantaneous processing of low-noise nanopores with ultra-high electric fields.

[0060] The microfluidic chip 1 has a three-layer structure. The top layer includes a solution inlet 101 (solution injection interface) and a microchannel 104 connecting the solution inlet 101 and the nanoneedles 103. The function of the top layer is to input the solution from the outside to the tip of the nanoneedles 103 through the microchannel 104. The solution flow rate of each microchannel is precisely controlled by the multi-channel peristaltic pump 2. The middle layer includes a multilayer solid nanofilm 4 (solid nanofilm chip) and its fixing structure. The multilayer solid nanofilm chip is constructed using a low-noise polymer layer 401 (preferably a polymer film layer) and a solid nanofilm layer 402. The purpose is to create solid film nanopores whose upper and lower surfaces are completely covered by polymer, while possessing the advantages of low noise and high spatial resolution. The bottom layer of the microfluidic chip 1 includes a structure that communicates with the solution injection port 101 and allows the solution to flow into the lower part of the multilayer solid nanofilm 4, and then connects to the electrode 5 after flowing out.

[0061] Furthermore, after the multilayer solid nanofilm 4 is assembled, a micrometer-level gap is formed between the upper surface of the multilayer solid nanofilm 4 and the nanoneedles 103. During the fabrication of nanopores, after applying an electric field, the solution in each microchannel is controlled by a multi-channel peristaltic pump 2 to contact the upper surface of the multilayer solid nanofilm 4. The solution between the lower surface of the multilayer solid nanofilm 4 and the lower layer structure is in contact, ultimately achieving communication with the electrode 5.

[0062] The multi-channel peristaltic pump system 2 is connected to the processing system via a serial bus, enabling program control of the processing system and precise control of the solution volume and injection rate within the nanoneedles 103. Preferably, the multi-channel peristaltic pump system 2 can be a structure with multiple pump heads driving multiple flow channels, simultaneously controlling the solution within all nanoneedle structures; alternatively, the multi-channel peristaltic pump system 2 can be a structure with a single peristaltic pump and an electric valve to control the solution within a single channel.

[0063] The nanoneedles 103 are fabricated microfluidic devices with a tip radius of less than 2 μm. In a preferred embodiment, the nanoneedles 103 are fabricated by using glass material to create capillaries with a tip radius of less than 2 μm, and then embedding the capillaries into the microfluidic channels 104 to form a microfluidic system in which the solution at the tip of the nanoneedles can be controlled by a peristaltic pump. Another fabrication process for the nanoneedles 103 is to use the same process as the piezoelectric spraying process of inkjet printers to directly injection mold a microfluidic structure with an outlet hole of less than 2 μm at the lower end, thereby achieving control of the droplets at the outlet position of the channel.

[0064] The tubing 3 is a commonly used tubing in microfluidic systems, used for conveying lithium chloride buffer solution and detection sample solution in the low-noise nanopore preparation system. The tubing 3 is connected to the microfluidic chip 1 through the solution inlet 101 of the microchannel. The solution inlet end is connected to the lithium chloride buffer solution container and the sample solution container to be detected, and the solution outlet is connected to the waste liquid pool.

[0065] The multilayer solid nanofilm chip 4 is a solid nanofilm designed according to the requirements of nanopore preparation. The overall thickness of the nanofilm is within 100nm. The main material 402 of the nanofilm can be graphene, silicon nitride, molybdenum disulfide, tungsten disulfide, silicon dioxide, and boron nitride, etc. The polymer layer material can be PMMA, PDMS, and other organic polymer materials.

[0066] In this invention, electrode 5 is preferably a silver chloride / silver electrode / gold / platinum electrode, wherein the silver electrode needs to be chlorinated before use. The electrode is inserted into the solution through a flexible tube interface and connected to the solution in each microchannel respectively. The electrode in the solution at the lower end of the multilayer nanofilm chip forms an electrode pair with each electrode in the microneedle at the upper end.

[0067] The power meter 6 of this invention is a current source 601 that can stably provide a constant current source from pA to mA level, and a voltage meter 602 that can synchronously detect voltage signals via a two-wire system. The voltage detection range is 0 to 200V. The current source can limit the output power during use, and the power limit can be flexibly adjusted.

[0068] The processing system 7 of this invention provides a runtime environment such as Python, MATLAB, C++, or LabVIEW, enabling automated data processing. The processing system 7 connects to a power meter and a multi-channel peristaltic pump via an Ethernet cable or USB communication bus. By controlling the power applied to the power meter and the position of the solution in the nanoneedles through software control, the system determines the formation status of the nanopores and the pore size of the nanopores after preparation.

[0069] Based on the microfluidic solid-state nanopore locally controllable fabrication system provided above, this invention provides a method for fabricating low-noise solid-state nanopores, comprising two main steps: low-noise nanopore fabrication and nanopore testing. The low-noise nanopore fabrication step includes the following steps:

[0070] Step S1: Fabrication of nanofilm chips. A bilayer material solid nanofilm chip is constructed on a thin film.

[0071] In a preferred embodiment, a construction is performed as follows: Figure 2 The bilayer material solid nanofilm shown is, for example, a PMMA capping layer with a thickness of less than 80 nm is directly suspended on a silicon nitride film with a TEM window to form a bilayer material solid film chip composed of a PMMA capping layer and a silicon nitride film.

[0072] Step S2: Microfluidic chip assembly. Based on the appearance and dimensions of the double-layer material solid thin film chip, a multi-layer chip with microchannels is designed and fabricated.

[0073] In a preferred embodiment, a three-layer chip with microchannels is designed and fabricated based on the external dimensions of the bilayer solid-film chip. Furthermore, the bilayer solid-film chip is mounted on the middle layer of the microfluidic chip for fixed bonding; after mounting, a solution pipeline is connected, controlling the buffer solution to enter the lower common flow channel layer of the solid film. The buffer solution is in complete contact with the lower part of the bilayer solid-film chip until it flows out of the microchannel outlet and connects to the common electrode 5.

[0074] The solution pipeline and electrode 5 at the upper end of the microfluidic chip are connected to the solution inlet at the upper end of the microfluidic chip, but the buffer solution is not injected. After the processing parameters are set and the DC power is turned on, the buffer solution is injected according to the set processing parameters.

[0075] This step uses the following method: Figure 2 The internal structure of the microfluidic chip is shown.

[0076] Step S3: Processing parameter setting. Based on the thickness of the bilayer solid film and the dielectric constant of the material, set the output current and output voltage of the constant current source system to prepare small-aperture solid nanopores.

[0077] In a preferred embodiment, based on the thickness of the bilayer solid film and the dielectric constant of the material, the output current of the constant current source system is set to 5nA, and the output voltage is 1 to 5 times the breakdown electric field corresponding to the nanofilm layer 402, for the preparation of small-aperture solid nanopores; for example, the thickness of the silicon nitride film is 10nm and the thickness of the PMMA film is 80nm. At this time, the breakdown voltage of the silicon nitride film and the PMMA film is 10V, so the limited output voltage of the constant current source system is set to 15V, which is 1.5 times the breakdown voltage (10V).

[0078] Furthermore, parameters such as flow rate, film thickness, and solution conductivity of the microfluidic chip's microfluidic system are set to provide a basis for nanopore fabrication and real-time pore size testing. The flow rate of the microfluidic system affects the speed at which the solution inside the nanoneedles contacts the solid film surface. Optimization is performed during actual debugging based on the designed microchannel volume and nanoneedle size. In this example, the overall volume of the microchannel is 200 μL, and the inner diameter of the nanoneedle tip is 1 μm. Setting the flow rate to 15 μL / s allows control over the contact between the solution at the nanoneedle tip and the solid film surface within 15 seconds. The film thickness refers to the thickness of the solid film nanofilm layer. This thickness affects the breakdown electric field strength and the thickness of the formed nanopores. In this example, the PMMA+silicon nitride film thickness is 80 nm + 10 nm, so the film thickness is set to 10 nm (silicon nitride film thickness), as this thickness determines the breakdown electric field strength. The solution conductivity is the actual conductivity of the buffer solution used in nanopore fabrication. In this example, the conductivity of the 1M LiCl buffer solution used is 7 S / m.

[0079] Step S4, solid-state nanopore preparation: Based on the processing parameters set in step S3, solid-state nanopores are prepared using an ultra-high electric field. For example... Figure 3 The method illustrates the fabrication of low-noise solid-state nanopores. First, a multilayer solid film chip 4 is fixed onto a microfluidic chip 1, and a multi-channel peristaltic pump system 2 is controlled to inject a buffer solution, such that... Figure 3 In the left image, the lower end of the multilayer solid film is in contact with the solution, while the upper end of the multilayer solid film is not in contact with the solution at the tip of the nanoneedle. Then, power meter 6 is turned on to complete step S3. A constant current source (1nA-50nA) is applied according to the film thickness parameters. After the feedback voltage of the constant current source reaches the limit value, the multichannel peristaltic pump is controlled to continue injecting solution into the nanoneedle, so that the solution at the tip comes into contact with the surface of the multilayer solid film, forming an ultra-high electric field. This causes the multilayer solid film to break down instantaneously, forming a solid nanopore, thus realizing the preparation of low-noise solid nanopores.

[0080] In a preferred embodiment, a pre-set low-noise nanopore controllable preparation process is used to apply an ultra-high electric field (1.5*E) on the microfluidic chip. bd This involves the rapid fabrication of solid-state nanopores, specifically silicon nitride nanopores. Furthermore, it utilizes... Figure 4 The low-noise nanopore controllable fabrication process shown can be achieved by applying an ultra-high electric field (1.5*E). bd Preparation of solid-state nanopores of silicon nitride. Among them, E... bd The breakdown electric field corresponding to the material can be calculated by IV testing of the thin film material. It is an inherent property of the material. Different materials have different breakdown electric fields. For example, silicon nitride has a breakdown electric field of about 1 V / nm, silicon dioxide has a breakdown electric field of about 1.5 V / nm, and PMMA has a breakdown electric field of 0.02 V / nm.

[0081] Preparatory work before program operation: (1) Processing preparation. Fix the multilayer solid thin film chip into the microfluidic chip, connect the electrodes and solution interface, and turn on the power supply 6 and the multichannel peristaltic pump 2;

[0082] (2) Initial Positioning. The multi-channel peristaltic pump 2 is controlled to inject solution into the microfluidic chip. The solution at the lower end of the solid film is fully injected and flows to the outlet end, ensuring contact between the lower end of the film and the solution. The solution volume at the upper end of the solid film is precisely controlled to be 150 μL, ensuring that the solution does not reach the tip of the nanoneedle. Testing shows that at this point, the distance from the solution surface inside the glass microtube to the tip and the distance from the tip to the upper surface of the solid film are 5 μm.

[0083] The fabrication process of low-noise solid nanopores is as follows: Figure 4 As shown:

[0084] (1) Set parameters and start applying pressure. Set software parameters, run the power meter program, output current with a constant current source of 5nA and a limited output voltage of 15V, and monitor voltage changes in real time.

[0085] (2) Solution injection. Since the solution is not in contact with the upper part of the solid film at this stage, the circuit resistance is infinite (R0). L =R air +R PMMA +R SiNx →∞, V=V set The power supply output will gradually increase until the output voltage reaches the 15V limit. Control the multi-channel peristaltic pump 2 to inject the upper buffer solution at a rate of 15uL / s.

[0086] As more solution is injected, the liquid surface will approach the top of the solid film until contact occurs at Z=0.

[0087] (3) Dielectric breakdown forms solid nanopores. At the moment of contact, because the voltage of 15V is much higher than the voltage of 10V corresponding to the breakdown electric field, an ultra-high electric field is formed, which will instantaneously break down the solid film and form solid nanopores.

[0088] (4) Constant current source pore expansion stage. After the solid nanopores are formed, R L =Rnanopore As the load resistance decreases, the power supply output voltage, which is the product of the constant current source and the nanopore resistance, is much lower than the set voltage Vset, preventing further breakdown of the solid film. During this stage, the interaction between the nA-level ion current and the nanopore walls leads to a slow pore-expanding process. The pore-expanding rate is related to the actual current magnitude (1nA-50nA). The pore-expanding rate and nanopore diameter accuracy can be adjusted by changing this current value.

[0089] Because this ultra-high electric field is much higher than the breakdown electric field of PMMA thin film, a nanoporous structure with large PMMA nanopores and small silicon nitride nanopores was fabricated 403. Figure 3 This does not affect the spatial resolution of the silicon nitride nanopores. In another embodiment, during the actual fabrication process, the pore size and pore shape of the nanopores can be adjusted by regulating the magnitude, duration, and polarity of the pore-expanding current, thereby achieving the fabrication of low-noise, high-resolution bilayer solid-state thin film nanopores.

[0090] The in-situ nanopore testing procedure only requires replacing the power meter 6 of the nanopore preparation system with a multi-channel patch-clamp testing system. Connect the electrodes of each channel to the input interface of the patch-clamp system, and connect the lower common electrode to the input ground of the patch-clamp system. The in-situ nanopore testing can then be performed, and it includes the following steps:

[0091] Step W1 involves testing the nanopore size to ensure that the testing performance and various indicators of the nanopore meet the requirements. Furthermore, the power meter 6 is replaced with a multi-channel patch clamp system or other nanopore current testing circuit to test the nanopore size and ensure that the testing performance and indicators of the nanopore meet the requirements.

[0092] A gradient voltage of -0.1V to 0.1V is output between the electrode pairs of each channel nanopore, and the current value generated by the gradient voltage is detected to obtain the IV scan curve of the nanopore chip. The nanopore pore size is calculated by fitting the IV curve and the nanopore conductance formula.

[0093] The nanopore size testing is divided into three stages. The first stage involves controlling the moment when microdroplets contact the solid film to create initial nanopores, at which point the first nanopore size test is performed. The test result is compared with the target pore size to determine whether to proceed to the nanopore expansion stage. The second stage is the expansion stage, where nanopore size testing is performed again after each expansion cycle. The test result is compared with the target pore size, and high-precision processing of the target pore size is achieved through a gradual approximation method. The third stage involves using a patch-clamp system or nanopore detection circuit to test the nanopore size before adding the sample, ensuring that the nanopore testing performance and various indicators meet the requirements.

[0094] Step W2: Add the sample to be tested into the nanopore that meets the performance and specification requirements. Specifically, replace the buffer solution at the front end of the multichannel peristaltic pump 2 with the sample solution to be tested.

[0095] In this invention, the nanopores are prepared in situ. Therefore, only the buffer solution at the upper / lower solution inlet needs to be replaced with the sample to be tested. When the sample to be tested is added to the upper solution inlet, the solution in each channel's nanoneedle needs to be first exported by reverse flow using a multi-channel peristaltic pump, and then replaced with the sample to be tested before introduction. This allows for the detection of different solution molecules in each channel. When the sample to be tested is added to the lower solution inlet, only the reagent at the solution inlet needs to be directly replaced and introduced, allowing for simultaneous testing of multiple channels. The sample to be tested is a sample solution prepared using a buffer solution according to the testing requirements.

[0096] Step W3, single-molecule detection: The test solution is injected into one end of the solid nanopore under the drive of the multi-channel peristaltic pump 2. Under the action of the bias electric field, it passes through the solid nanopore in the form of single molecules, forming an ion current signal that can be recorded by patch clamp.

[0097] After the test solution is replaced, the multi-channel peristaltic pump 2 is started to inject the test solution into one end of the solid nanopore (the common end below). Then, the patch-clamp detection system, which replaced the power meter 6, is started to record the ion current information of each channel of the solid nanopore. Next, a bias voltage (0-1V) is applied to each channel, forming a bias electric field around the solid nanopore. Molecules in the test solution are subjected to Brownian motion and electric field forces, are captured by the electric field force of the solid nanopore, and pass through the solid nanopore as individual molecules, causing changes in ion current, thereby achieving real-time detection of individual molecules passing through the solid nanopore in each channel.

[0098] Step W4: Based on the nanopore through-pore current recorded in step W3, perform signal storage and analysis for sample testing.

[0099] After the required sample test signal of the sample to be tested is detected, the processing system stores the test signal and analyzes information such as the amplitude, time and frequency of the through-hole signal, providing data and feedback such as the concentration, size and electrical properties of the analyte molecule for experimental testing, thus completing the nanopore detection work.

[0100] Compared with existing technologies, this invention proposes a method for preparing low-noise solid-state nanopores. Based on a microfluidic locally controllable solid-state nanopore preparation system, it constructs a multilayer solid film substrate and fabricates fully covered low-noise nanopores using a cross-electric field processing method. This system enables the fabrication of nanopores at electric fields several times higher than the breakdown electric field, providing a new method and equipment for ultra-high electric field processing of nanopores in bilayer or multilayer films of different materials, achieving in-situ preparation of low-noise solid-state nanopores. It has the following beneficial effects:

[0101] 1. This invention is based on the microfluidic microdroplet contact method, which precisely controls the time and position of microdroplet contact above the nanofilm, thereby achieving control over the processing position and speed of solid nanopores.

[0102] 2. This invention employs a locally controllable dielectric breakdown method, which can process solid nanopores using an electric field several times higher than the material breakdown electric field. It has the ability to process thin films of different materials simultaneously, and provides a new method for directly processing bilayer or multilayer solid nanopores.

[0103] 3. The present invention controls the micro-contact between the solution and the nanofilm using nanoneedles, resulting in a small contact area and improving the signal-to-noise ratio and throughput of solid-state nanopore detection. This means that it can realize the individual preparation and detection of nanopores for each nanoneedle channel.

[0104] 4. The present invention enables the in-situ preparation of solid nanopores by simply changing the reagent at the front end of the buffer solution, thereby changing the sample to be tested. This solves the problem of cumbersome solution changing steps and large amounts of solution used in common nanopore detection systems. It is a high-throughput nanopore preparation and testing system that allows for simultaneous preparation and testing.

[0105] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A method of preparing a low-noise solid-state nanopore, characterized in that, It includes a low-noise nanopore fabrication step and a nanopore testing step. The low-noise nanopore fabrication step includes the following steps: Step S1, Nanofilm preparation: Constructing a bilayer material solid nanofilm on a thin film; Step S2: Microfluidic chip assembly. Based on the appearance and dimensions of the bilayer material solid nanofilm, a multilayer chip with microchannels is designed and fabricated. Step S3, setting processing parameters: Based on the thickness of the bilayer solid nanofilm and the dielectric constant of the material, set the output current and output voltage of the constant current source system to prepare small-aperture solid nanopores; Step S4, solid nanopore preparation: solid nanopores are prepared using an ultra-high electric field according to the processing parameters set in step S3. The ultra-high electric field is 1.5*E. bd ; The in-situ nanopore testing procedure involves performing in-situ nanopore testing on low-noise nanopores, and includes the following steps: Step W1: Replace the power meter with a multi-channel patch clamp system to test the nanopore pore size and ensure that the test performance and indicators of the nanopore meet the requirements. Step W2: Replace the buffer solution at the front end of the multi-channel peristaltic pump with the sample solution to be tested; Step W3, Single molecule detection: The test solution is injected into one end of a solid nanopore under the drive of a multi-channel peristaltic pump. Under the action of a bias electric field, it passes through the solid nanopore in the form of single molecules, forming an ion current signal that can be recorded by patch clamp. Step W4: Based on the ion current signal recorded in step W3, perform signal storage and analysis for sample testing; After solid nanopores are formed, the load resistance decreases, and the power supply output voltage is the product of the constant current source and the nanopore resistance. The power supply output voltage is less than the set voltage, and the expansion speed and nanopore diameter accuracy can be adjusted by changing the current value.

2. The method of claim 1, wherein the method further comprises, In step S1, a PMMA capping layer with a thickness of less than 80 nm is directly suspended on a silicon nitride thin film with a TEM window to form a bilayer material solid nanofilm composed of a PMMA capping layer and a silicon nitride thin film.

3. The method of claim 2, wherein the method further comprises, Based on the external dimensions of the bilayer solid nanofilm, a three-layer chip with microchannels was designed and fabricated.

4. The method of claim 2, wherein the solid-state nanopore is prepared with low noise. A bilayer solid nanofilm is installed on the middle layer of a microfluidic chip and fixedly bonded. After installation, a solution pipeline is connected to control the buffer solution to enter the lower common flow channel layer of the solid film. The buffer solution is in complete contact with the bottom of the bilayer solid nanofilm until the buffer solution flows out of the microchannel outlet and connects to the common electrode.

5. The method of claim 4, wherein the solid-state nanopore is prepared with low noise, and The solution tubing and electrodes at the top of the microfluidic chip are connected to the solution inlet at the top of the microfluidic chip, but the buffer solution is not injected. After the processing parameters are set and the DC power is turned on, the buffer solution is injected according to the set processing parameters.

6. The method of claim 4, wherein the solid-state nanopore is prepared with low noise, and Based on the thickness and dielectric constant of the bilayer solid nanofilm, the output current of the constant current source system is set to 5nA, and the output voltage is 1 to 5 times the breakdown electric field corresponding to the nanofilm layer, which is used to prepare solid nanopores with small apertures.

7. The method of claim 1, wherein the method further comprises: It employs a microfluidic-based locally controllable solid-state nanopore preparation system, which includes a microfluidic chip, a multi-channel peristaltic pump, a solution tubing, a bilayer solid nanofilm, electrodes, a power meter, and a processing system. The microfluidic chip is used to mount and fix the solid-state nanopore chip, precisely control the solutions on both sides of the solid-state nanopore, and add the sample to be tested. Multichannel peristaltic pumps are used to control the flow rate of the solution within the microfluidic chip and to add buffer solutions and the types of samples to be tested; The solution tubing is used to connect the buffer solution, the sample solution bottle to the microfluidic chip, and the T-connector for accessing the electrodes. The flow rate and volume of the solution in the tubing are precisely controlled by a multi-channel peristaltic pump. Bilayer material solid nanofilms are used to prepare solid nanopores. When stimulated by the local electric field of nanoneedles, they are broken down to form solid nanopores of bilayer material. The electrode is used to connect the buffer ion solution to the power meter or nanopore test circuit to ensure smooth ion exchange between the ion solution and the electrode interface. The power meter is used to provide an adjustable regulated power supply or a constant current source; when performing nanopore through-hole current testing, the power meter is replaced by a multi-channel patch clamp detection system to record the through-hole current of solid nanopores in real time. The processing system is used in programmable power meters or nanopore testing circuits, providing a graphical interface for user interaction, enabling real-time control of the applied current and voltage of the power meter, testing of nanopore pore size, and detection of single-molecule through-pore signals.

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