Flexible nanopore channel, preparation method thereof and mechanical regulation and control device

Abstract

The invention relates to the technical field of micro-nano machining, and discloses a flexible nanopore channel, a preparation method thereof and a mechanical regulation and control device. The nanopore channel is a through hole and is formed in an organic film; the organic film is arranged on the silicon substrate, a window is formed in the silicon substrate, and the nano pore channel is formed in the window area. The nanopore channel has good elasticity and can bear repeated deformation, and the signal resolution and the detection precision can be remarkably improved by adjusting the aperture of the nanopore channel in a variable-diameter mode.

Description

Technical Field

[0001] The present invention relates to the field of micro-nano processing technology, and specifically relates to a flexible nanochannel, a preparation method thereof, and a mechanical regulation device. Background Art

[0002] Since the rise of nanopore detection technology at the end of the 20th century, it has gradually developed from biological nanopores to solid-state nanopores (such as silicon nitride and silicon oxide). Due to its high stability, repeatable processability, and ability to withstand extreme environments, it has become a core tool in the fields of gene sequencing, single-molecule analysis, etc. Solid-state nanopore technology represented by silicon nitride has achieved nanoscale control of pore diameters through precision processing (such as focused ion beam etching), and has significantly improved detection sensitivity in composite structure designs (such as combining graphene films and silicon nitride films). However, the limitations of solid-state nanopores are as follows:

[0003] (1) The mechanical strength of the material is insufficient. Most solid pores of rigid materials, such as the brittleness of silicon oxide and silicon nitride films, make them prone to breakage during use, which to a certain extent increases the use cost.

[0004] (2) Rigid materials are difficult to meet the requirements of multi-functional applications. For example, the physical rigidity of silicon nitride films results in their pore diameters being unable to be dynamically adjusted with external conditions (such as pressure and electric field), and solid pores of rigid materials are prone to breakage under pressure. And there are certain limitations in mechanical regulation applications. For example, when driving biomolecules through pores under pressure, the fixed pore diameter cannot adjust the fluid resistance through deformation, resulting in it being difficult to balance the molecular pore passing speed and signal resolution. Reducing the driving voltage can extend the pore passing time, but sacrifices the signal-to-noise ratio.

[0005] There are also certain limitations in the mechanical force application method. Existing mechanical regulation means (such as piezoelectric actuators and mechanical stretching devices) have problems such as complex systems, high costs, or large volumes. For example, piezoelectric driving requires precise circuit control, while mechanical stretching devices are difficult to achieve micron-level precise regulation and are difficult to integrate with liquid pools. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides a flexible nanochannel, a preparation method thereof, and a mechanical regulation device. By using a PDMS nanochannel, it has good elasticity and can withstand repeated deformation. By adjusting the variable diameter of the pore diameter of the nanochannel, the signal resolution and detection accuracy can be significantly improved.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] Combined with the first aspect, a flexible nanochannel, the nanochannel is a through-hole and is opened on an organic membrane; the organic membrane is arranged on a silicon substrate, a window is opened on the silicon substrate, and the nanochannel is opened within the window area.

[0009] In combination with the first aspect, further, the window is square with a side length of 30 μm to 200 μm; the diameter of the nanochannel is 10 nm to 100 nm; the thickness of the organic film is 50 nm to 1000 nm.

[0010] The geometric size of the nanochannel needs to be determined according to specific applications. For example, the pore size of the nanochannel is determined according to the size of the analyte molecules to be passed; the size of the square window of the silicon substrate needs to be determined according to the elastic properties of the organic film, and the specific determination method is the prior art.

[0011] In combination with the first aspect, further, the organic film is a PDMS film, or a polyethylene terephthalate film (PET), or a polymethyl methacrylate film (PMMA), or other elastic organic films. Preferably, the organic film is a PDMS film, that is, a polydimethylsiloxane film.

[0012] The pore diameter of the nanochannel of the present invention can be regulated by an external mechanical force. When a mechanical force is applied, the PDMS flexible film undergoes elastic deformation, resulting in a change in the pore diameter of the nanochannel. The regulation range is ±10% to ±70% of the initial pore diameter.

[0013] In a second aspect, further, a method for preparing a flexible nanochannel for preparing the above nanochannel includes the following steps:

[0014] Double-side polish a silicon wafer to obtain a silicon substrate, and perform directional etching on the silicon substrate with tetramethylammonium hydroxide to etch a window on the silicon substrate;

[0015] Spin-coat a PDMS solution on a copper sheet by spin coating, and form a PDMS film after curing;

[0016] Transfer the cured PDMS film to the surface of the silicon substrate by wet transfer, so that the PDMS film covers the window area to form a self-supporting structure;

[0017] Use a focused ion beam etching technique to etch nanochannels in the PDMS film corresponding to the window area.

[0018] In combination with the second aspect, further, perform directional etching with a tetramethylammonium hydroxide (TMAH) solution, the etching temperature is 80 °C to 95 °C, and the etching time is 2 to 8 hours.

[0019] In combination with the second aspect, further, the spin-coating of the PDMS solution on the copper sheet by spin coating and forming a PDMS film after curing includes:

[0020] The mass ratio of the PDMS solution to the curing agent is 10:1 to 8:1. The specific process of spin coating is as follows: Place the tabletop spin coater in a fume hood, turn on the power supply, stick the prepared clean copper sheet on the ITO glass sheet and place it on the vacuum suction seat of the spin coater with the front side of the copper sheet facing up. Start the vacuum pump to firmly suck the glass sheet, and use a dropper to suck a few drops of the prepared PMMA solution and drop it on the copper sheet. Start the motor to rotate the sample and spin coat it at a speed of 100 - 500 rpm for 10 - 15 seconds first, and then spin coat it at a speed of 3000 - 4000 rpm for 40 - 60 seconds to initially obtain a uniform thin film sample. Then cure it. The curing temperature is 80°C - 100°C, and the curing time is 2 - 5 hours. After curing, a copper sheet with a PDMS film is obtained.

[0021] Combined with the second aspect, further, the process of wet transferring the cured PDMS film to the surface of the silicon substrate so that the PDMS film covers the window area includes:

[0022] The copper sheet with the PDMS film is immersed in the ferric chloride solution with the front side of the PDMS film facing up and the back side contacting the ferric chloride solution to etch the copper sheet and place it for 2 - 4 h until the copper sheet is completely dissolved; after the copper sheet is completely dissolved, the PDMS film floats on the surface of the ferric chloride solution; then wash the PDMS film with deionized water, transfer the washed PDMS film to the surface of the silicon substrate, and bake it after natural air drying to make the PDMS film and the silicon substrate fit well.

[0023] Combined with the second aspect, further, the process of etching nanopores in the PDMS film corresponding to the window area by using the focused ion beam etching technology includes: Using the focused ion beam to sputter nanopores 3 on the PDMS film 2, and the etching parameters are an ion beam current of 1.1 pA - 40 pA and an etching time of 500 ms - 20 s.

[0024] Combined with the third aspect, a mechanical regulation device for flexible nanopores is used to mechanically regulate and detect the nanopores of the present invention, and includes a patch clamp amplifier, a microfluidic pump, a computer, a pair of electrodes, a first liquid pool, a second liquid pool, and a chip; the chip includes the above-mentioned nanopores; the chip is arranged between the first liquid pool and the second liquid pool; the second liquid pool is a closed liquid pool and is integrated with one of the motors and a liquid injection hose; another motor is arranged in the first liquid pool, and one ends of the pair of motors are respectively inserted into the first liquid pool and the second liquid pool, and the other ends are both connected to the patch clamp amplifier; the patch clamp amplifier is communicatively connected to the computer and is used to collect the ion current between the two electrodes and transmit the collected ion current data to the computer, and the computer is used to receive the ion current data from the patch clamp amplifier to record the pore size change process of the nanopores.

[0025] Apply an axial mechanical force to the flexible nanopore of the present invention to cause elastic deformation of the PDMS flexible film, thereby dynamically adjusting the pore diameter of the nanopore. The axial mechanical force is achieved by injecting liquid into the sealed second liquid pool through a microfluidic pump to generate axial pressure.

[0026] Inject liquid into the sealed second liquid pool through a microfluidic pump, and at the same time apply a bias voltage using a patch clamp amplifier. Under the action of pressure, the pore diameter structure of the nanopores on the PDMS membrane changes, and further the ion transport also changes, which is reflected by the change in the magnitude of the ion current of the patch clamp amplifier.

[0027] Combined with the third aspect, further, the device of the present invention further includes a catheter, and the syringe of the microfluidic pump is connected to the air hole seal on the second liquid pool through the catheter. Preferably, the catheter is a polytetrafluoroethylene (PTFE) catheter. Preferably, both the first liquid pool and the second liquid pool are plexiglass liquid pools.

[0028] Combined with the third aspect, further, the second liquid pool further includes an electrode seal and an air hole seal. The electrode seal is used to seal the inner electrode therein, and the air hole seal is used to seal the air hole matching the catheter.

[0029] Combined with the third aspect, further, one of the pair of electrodes is a cathode electrode and the other is an anode electrode, and a silver / silver chloride electrode is optional.

[0030] Combined with the third aspect, still further, potassium chloride (KCl) solution or sodium chloride (NaCl) solution or other salt solutions are placed in the first liquid pool and the second liquid pool. When potassium chloride salt solution is selected, the potassium chloride salt solution is a KCl solution with a concentration of 0.001 mol / L to 1 mol / L.

[0031] Combined with the third aspect, further, the device of the present invention further includes rubber gaskets. Rubber gaskets are respectively arranged in the counterbores of the first liquid pool and the second liquid pool, and the rubber gaskets in the counterbores of the first liquid pool and the second liquid pool clamp the chip in the middle.

[0032] Combined with the third aspect, further, the window on the chip is arranged close to the first liquid pool and far from the second liquid pool.

[0033] During the mechanical regulation process, the microfluidic pump connects the injection hole of the syringe integrated with the sealed second liquid pool through a soft catheter. The syringe contains KCl solution with the same concentration. The single injection volume is controlled to be 5 μL - 50 μL by setting the microfluidic pump program (the specific pressure can be converted according to the internal volume of the sealed second liquid pool). Under the action of pressure, the PDMS membrane on the silicon substrate will undergo a certain degree of elastic deformation, thereby driving the change of the effective aperture of the flexible nanopore. The patch clamp amplifier records the ionic current signal during the deformation process of the nanopore in the PDMS membrane.

[0034] The present invention uses a PDMS flexible thin film, which effectively improves the toughness of the nanopore compared with the rigid solid pore. By applying mechanical force to the flexible PDMS nanopore through the method of pressurizing the microfluidic pump, the pore undergoes elastic deformation, realizing the mechanical regulation of the structural morphology of the flexible nanopore, thereby affecting the ion transport in the pore, and the regulation range is ±10% - ±70% of the initial aperture.

[0035] Compared with the prior art, the present invention provides a flexible nanopore and its preparation method and a mechanical regulation device, having the following beneficial effects:

[0036] (1) The present invention uses a PDMS thin film, which has stronger toughness, good elasticity, can withstand repeated deformation, and avoids permanent damage caused by pressure; the combination of the rigid support of the silicon substrate and the flexible PDMS thin film not only avoids collapse but also retains the elastic regulation ability, realizing stable operation under high-pressure environments.

[0037] (2) The present invention can perform dynamic aperture adjustment. Through external pressure or mechanical force, the aperture of the flexible nanopore of the present invention can be regulated in real time and reversibly, adapting to the usage requirements in different environments, and combining with the observation of the ionic current by the patch clamp amplifier to judge the change of the effective aperture.

[0038] (3) The present invention adopts high-precision processing and integration. The focused ion beam (FIB) etching process is used to process nanopores in the window area of the PDMS thin film, and the PDMS thin film is seamlessly combined with the silicon substrate through the wet transfer technology, avoiding interface delamination; the design of the rubber gasket further optimizes the liquid pool sealing performance and reduces pressure leakage.

[0039] (4) The present invention has the characteristics of flexible processing and cost advantages. The thin film is efficiently prepared through processes such as spin coating, etching, and wet transfer, without the need for complex equipment.

[0040] (5) The present invention integrates a dynamic regulation system. By using a microfluidic pump device to inject liquid into a sealed liquid pool, pressure is applied to the nanopores of the PDMS thin film. By controlling the amount of liquid injected and withdrawn, the magnitude of the applied force can be effectively controlled, realizing real-time and reversible regulation of the pore size (±10% to ±70%). Combining with the pressure-driven technology, the change in the pore size can be accurately controlled, solving the limitations of existing mechanical force application methods, as well as the problems of complex systems, high costs, and large volumes in existing mechanical regulation means.

[0041] (6) The present invention adopts the coordinated regulation of pressure and elasticity. By injecting liquid into a sealed plexiglass liquid pool using a microfluidic pump to generate controllable pressure, and utilizing the elastic characteristics of the PDMS thin film, the nanopores undergo uniform deformation under the action of liquid pressure (the pore size regulation range is ±10% to ±70%). Description of the Drawings

[0042] Figure 1 It is a flow chart of the preparation method of the flexible nanopores in the present invention.

[0043] Figure 2 It is a schematic diagram of the flexible nanopores being pressurized in the present invention.

[0044] Figure 3 It is a light microscope photo of a PDMS film covering the window of a silicon substrate in the present invention.

[0045] Figure 4 It is a scanning electron microscope photo after processing the flexible nanopores of the present invention on the PDMS film.

[0046] Figure 5 It is an exploded schematic diagram of the mechanical regulation device of the flexible nanopores in the present invention.

[0047] Figure 6 It is a volt-ampere characteristic curve (I-V) diagram measured in KCl solutions with different concentrations from 0.001 mol / L to 1 mol / L in Example 1 of the present invention.

[0048] Figure 7 It is a diagram of the changes in the I-V curve, conductance, and pore size measured when the flexible nanopores in Example 1 of the present invention are under pressure regulation; among them, Figure 7 a is the I-V curve diagram of the first three pressurizations, with obvious changes. Figure 7 b is the I-V curve diagram after the subsequent 4 to 10 pressurizations, with a smaller change amplitude, which should be caused by the PDMS thin film reaching the elastic limit. Figure 7 c is the conductance change diagram during the pressurization process. Figure 7 d is the diagram of the change in pore size with pressure.

[0049] Figure 8I-V curve and conductance change diagram measured during pressure release regulation of the flexible nanopore channel in Embodiment 1 of the present invention; among them, Figure 8 a is the measured I-V curve diagram after pressure release, Figure 8 b is the corresponding conductance change diagram. A large rebound phenomenon appears during a single pressure release. The subsequent continuous reduction of the current reference should be caused by reverse pressure application. The fact that the current does not rebound to the original reference after pressure release is presumably due to plastic deformation caused by continuous pressure application beyond the elastic limit.

[0050] Figure 9 Scanning electron microscope photograph of Chip 10 after processing the flexible nanopore channel of the present invention on the PDMS film in Comparative Example 1 of the present invention.

[0051] Figure 10 Volt-ampere characteristic curve (I-V) diagram measured in KCl solutions with different concentrations from 0.0001 mol / L to 1 mol / L in Comparative Example 1 of the present invention.

[0052] The meanings of the reference numerals in the figure are as follows:

[0053] 1 - silicon substrate; 2 - PDMS film; 3 - nanopore channel; 4 - microfluidic pump; 5 - catheter; 6 - first liquid pool; 7, 9 - rubber gaskets; 8 - chip; 10 - second liquid pool; 13 - air hole sealing plug; 14 - electrode sealing plug; 13 - electrode; 14 - patch clamp amplifier; 15 - computer. Specific Embodiments

[0054] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0055] Unless otherwise specifically stated, the relative arrangements, numerical expressions, and numerical values of the components and steps described in these embodiments do not limit the scope of the present invention. At the same time, it should be understood that for the convenience of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship. Technologies, methods, and devices known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods, and devices should be regarded as part of the specification. In all the examples shown and discussed here, any specific value should be interpreted as merely exemplary, rather than as a limitation. Therefore, other examples of the exemplary embodiments may also include different values. It should be noted that similar reference numerals and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0056] In the description of the present application, it should be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "upper", "lower", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus cannot be construed as a limitation on the protected content of the present invention.

[0057] Example 1

[0058] As Figure 2 shown, the nanopore 3 of this embodiment is opened on the PDMS film 2, the PDMS film 2 is disposed on the stacked silicon substrate 1, a window is opened on the silicon substrate 1, and the nanopore 3 communicates with the window.

[0059] In a specific implementation manner of this embodiment, the pore diameter of the nanopore 3 in this embodiment is 10 - 100 nm; the thickness of the PDMS film 2 is 50 nm - 1000 nm; the side length of the square window of the silicon substrate 1 is 30 μm - 200 μm.

[0060] The chip 8 includes the silicon substrate 1, the PDMS film 2, and the nanopore 3 of this embodiment.

[0061] Example 2

[0062] As Figure 1 shown, this embodiment proposes a preparation method for a flexible nanopore, for preparing the nanopore in Example 1, including the following steps:

[0063] Step S1, perform double-sided polishing on a silicon wafer to obtain the silicon substrate 1, and then use tetramethylammonium hydroxide (TMAH) for directional etching to etch a square window with a side length of 30 μm - 200 μm on the silicon substrate, thus obtaining a rigid substrate for supporting the elastic film, as Figure 2 and Figure 3 shown, Figure 2 the trapezoidal opening at the center of the silicon substrate 1 in Figure 3 is the light microscope photograph of the square window of the silicon substrate 1.

[0064] Step S2, transfer the PDMS film 2 above the silicon substrate 1.

[0065] Step S3, use a focused ion beam to etch a through hole, namely the nanopore 3, on the PDMS film 2, as Figure 4 shown, the pore diameter of the nanopore 3 is 10 nm - 100 nm; preferably, the measured Figure 4 pore diameter in

[0066] In a specific implementation manner of this embodiment, the wet transfer technology is adopted in the transfer process of the PDMS film 2, and the transfer process is as follows:

[0067] First, prepare the PDMS film 2. Mix the PDMS solution and the curing agent evenly at a ratio of 10:1 to 8:1, then drop the mixture on the copper sheet. Next, use a spin coater for spin coating. First, spin coat at a speed of 100 - 500 rpm for 10 - 15 seconds, and then spin coat at a speed of 3000 - 4000 rpm for 40 - 60 seconds to form a uniform PDMS film 2. The spin coating and the ratio of the solution to the curing agent can be adjusted according to the required thickness. Place the spin-coated copper sheet in an oven at 80 - 100 °C for baking for 2 - 5 hours for curing, and ensure that the PDMS film 2 is closely attached to the copper sheet. After curing, a copper sheet with a PDMS film is obtained.

[0068] Next, transfer. The specific transfer method of the polydimethylsiloxane (PDMS) film is as follows: Cut the baked sample into an appropriate size and put it into the ferric chloride (FeCl3) solution with the PDMS-coated side facing up and the back contacting the solution to etch the copper sheet. Place it for 2 - 4 h until the copper sheet is completely dissolved and the PDMS film 2 floats on the surface of the ferric chloride solution. Use a clean silicon wafer to gently drag out the PDMS film 2 floating on the surface of the ferric chloride solution and transfer it to deionized water for cleaning. Repeat the cleaning 3 times, about 10 minutes each time, to remove the residual ferric chloride on the PDMS film 2. Then fish out the PDMS film 2 from the water with a hydrophilized silicon substrate, so that the PDMS film 2 covers the square window in the center of the silicon substrate, and then air-dry it naturally and put it into an oven at 90 °C for baking for 1 - 3 h to make the PDMS film 2 and the silicon substrate fit closely, that is, the transfer process of the PDMS film 2 is completed.

[0069] In a specific implementation manner of this embodiment, the processing method of the nanopores 3 is to use a focused ion beam to sputter nanopores 3 on the PDMS film 2. The etching parameters are an ion beam current of 1.1 pA - 40 pA and an etching time of 500 ms - 20 s. The etching time is specifically determined by the thickness of the PDMS film 2. The thicker the PDMS film 2, the longer the required etching time. The pore diameter accuracy is controlled within ±5 nm, and damage to the mechanical properties of the PDMS film 2 should be avoided during the etching process. The specific pore size can be controlled by the focused ion beam sputtering time and the beam current size.

[0070] Example 3

[0071] The difference between this embodiment and Embodiment 2 is as follows: This embodiment proposes specific parameters for the preparation of the flexible PDMS film 2. After uniformly mixing the PDMS solution and the curing agent at a ratio of 8:1, it is dropped on a copper sheet. Then, a spin coater is used for spin coating. First, spin coat at a speed of 300 rpm for 15 seconds, and then spin coat at a speed of 4000 rpm for 50 seconds to form a uniform PDMS film 2. The spin-coated copper sheet is placed in an oven at 90 °C and baked for 4 hours for curing, obtaining a PDMS film 2 with good toughness and easy processing. Good nanopores can be etched using the focused ion beam sputtering process, such as Figure 4 as shown, and has good penetration, such as Figure 6 shown, Figure 6 is the I-V curve diagram at different concentrations.

[0072] Comparative Example 1

[0073] This comparative example presents a comparison of the preparation of the flexible PDMS film 2 in Embodiment 3. After uniformly mixing the PDMS solution and the curing agent at a ratio of 5:1, it is dropped on a copper sheet. Then, a spin coater is used for spin coating. First, spin coat at a speed of 200 rpm for 15 seconds, and then spin coat at a speed of 2000 rpm for 50 seconds to form a uniform PDMS film 2. The spin-coated copper sheet is placed in an oven at 90 °C and baked for 4 hours for curing. The obtained PDMS film 2 has a very high thickness and strong viscosity, making it difficult to process. Through scanning electron microscope observation, the area after focused ion beam sputtering shows strong fluidity, and the processed holes are difficult to form, such as Figure 9 shown, and may close after a period of time, such as Figure 10 shown, Figure 10 is the I-V curve diagram of this comparative example at different concentrations. The current reference hardly changes at different concentrations, and the current reference is very small, only at the picoampere level. It is speculated that this is due to the poor formability of the thin film and the closure of the pores after processing.

[0074] Embodiment 4

[0075] Such as Figure 3As shown in the figure, this embodiment proposes a mechanical regulation device for flexible nanopores. The device includes a microfluidic pump 4, a catheter 5, a first liquid pool 6, a chip 8 in the present invention, a second liquid pool 10, a gas hole sealing plug 11, an electrode sealing plug 12, electrodes 13 (arranged in pairs), a patch clamp amplifier 14, and a computer 15. The chip 8 of the present invention is clamped between two liquid pools 6 and 10. The catheter 5 is used to connect the syringe on the microfluidic pump 4 and the reserved channel on the gas hole sealing plug 11. Two electrodes 13 are respectively arranged in the two liquid pools 6 and 10. One of the electrodes 13 is placed in the reserved hole of the electrode sealing plug 12 and sealed. The two electrodes 13 are respectively connected to the patch clamp amplifier 14, and the patch clamp amplifier 14 is also communicatively connected to the computer 15. The patch clamp amplifier 14 is used to collect the ion current between the two electrodes 13 and transmit the collected ion current data to the computer 15. The computer 15 is used to receive the ion current data from the patch clamp amplifier 14, so as to record the mechanical regulation process of the flexible nanopore 3. By using the microfluidic pump 4 to apply pressure to the liquid pumped into and out of the closed second liquid pool 10 and applying a bias voltage through the patch clamp amplifier 14, the ion current when the membrane deforms is recorded.

[0076] The two liquid pools 6 and 10 clamp the chip 8 of the present invention in the middle, forming two independent cavities (the two cavities refer to the two liquid pools 6 and 10). The middle nanopore 3 is the only flow channel between the two cavities, and the ion current can only pass through this pore, so the size change of the pore can be directly reflected.

[0077] In a specific implementation manner of this embodiment, the mechanical regulation device of this embodiment further includes rubber gaskets 7 and 9. The rubber gasket 7 is arranged between the nanopore sample 8 and the first liquid pool 6, and the rubber gasket 9 is arranged between the nanopore sample 8 and the second liquid pool 10. The rubber gaskets 7 and 9 play a sealing role on the one hand and can slow down the force between the clamp holding the two liquid pools 6 and 10 and the nanopore sample 8 on the other hand.

[0078] In a specific implementation manner of this embodiment, the two liquid pools 6 and 10 are both plexiglass liquid pools, and the second liquid pool 10 is a closed liquid pool.

[0079] The experimental steps for mechanically regulating the nanopore sample 8 by using the mechanical regulation device of the present invention are as follows:

[0080] First, place a rubber washer 7 in the counterbore of the first liquid cell 6, and at the same time place a rubber washer 9 in the counterbore of the second liquid cell 10. Clamp the nanopore sample 8 of the present invention in the middle, and then clamp the two liquid cells 6 and 10 using a fixture. Inject 1 mol / L KCl solution into both liquid cells 6 and 10. The microfluidic pump 4 is connected to the second liquid cell 10 through a conduit 5. Apply pressure to the liquid cell by controlling the single injection volume of 5 μL to 50 μL through a program. Insert silver / silver chloride electrodes 13 into the two liquid cells 6 and 10 respectively. The other end of the electrode 13 is connected to a patch clamp amplifier 14. Collect the ion current signal of the nanopore sample 8 of the present invention during the membrane deformation process through a computer 15, as Figure 5 shown.

[0081] First, apply a voltage of -1000 mV to 1000 mV using the patch clamp amplifier 14, measure one point every 200 mV, and measure the volt-ampere characteristic curves (I-V curves) under KCl solutions with concentrations of 0.001 mol / L, 0.01 mol / L, 0.1 mol / L, 0.5 mol / L, and 1 mol / L respectively, as Figure 6 shown. And take this as the reference ion current, and calculate the membrane thickness in combination with the pore size of 87 nm of the nanopore 3 measured by a scanning electron microscope. The specific calculation is obtained from the formula where G is the measured conductance, I is the current, V is the voltage, σ is the conductivity of the KCl solution, l is the membrane thickness, and d is the diameter of the nanopore 3. Finally, the membrane thickness is approximately 530 nm. Taking this as the calibrated membrane thickness, the pore size change of the nanopore 3 after subsequent pressurization can be calculated from the above formula.

[0082] Inject liquid into the closed second liquid cell 10 through the microfluidic pump 4. The injection direction is away from the silicon substrate 1. Increase the pressure by continuously injecting liquid (from an increase of 5.84×10 - ³ N to 29.2×10 - ³ N). The single injection volume is 10 μL (i.e., corresponding to a pressure of 5.84×10 - ³ N). The specific pressure is calculated from the injection volume in combination with the internal volume of the closed second liquid cell 10. The injection volume can be adjusted as needed, and record the ion current changes under different pressures. As Figure 7 shown in a, Figure 7a is the volt-ampere characteristic curve (I-V curve) of the first three liquid injections. The numbers in the legend represent the number of liquid injections. The current reference has obvious and uniform changes. The amplitude of the current decreases by about 20 nA each time, and the single-time decrease is about 20% of the unpressurized state. That is, the pore diameter of nanopore 3 decreases linearly with the increase of pressure (according to Poisson's effect, when the film is in a tensile state, the material around the pore undergoes lateral contraction, and the pore diameter shrinks). At the same time, according to the above pore diameter calculation formula, the single-time pore diameter reduction is about 15 - 20 nm, and the single-time shrinkage accounts for 17% - 23% of the initial pore diameter. As Figure 7 shown in Figure 7 b. b is the volt-ampere characteristic curve (I-V curve) corresponding to the subsequent 4th to 10th pressurizations. The influence of pressurization on deformation becomes smaller, and the change of the current reference is smaller. The amplitude decreases by about 0.2 nA each time, and the single-time decrease only accounts for about 0.2% of the unpressurized state. The single-time pore diameter reduction is about 0.1 nm - 0.5 nm, accounting for 0.1% - 0.6% of the initial pore diameter, indicating that the PDMS film reaches the elastic limit. As Figure 7 shown in Figure 7 c. c is the curve graph of the conductance of nanopore 3 of the present invention changing with the applied pressure. Its change is synchronous with the current change. The first three times show a significant downward trend in the single-time change rate, and the single-time decrease rate is about 23% - 30%. The change in the latter 7 times is smaller, and the single-time decrease rate is only 0.3% - 0.6%. As Figure 7 shown in Figure 7 d. d is the curve graph of the effective pore diameter of nanopore 3 of the present invention changing with the increase of pressure, which basically corresponds to the conductance change.

[0083] As Figure 8 shown in Figure 8 a. a is the I-V curve of the liquid extraction process, that is, the I-V curve corresponding after pressure release. Liquid extraction is carried out after the liquid injection is completed. A large rebound phenomenon appears during a single pressure release, and the amplitude rebounds by about 10 nA, indicating that partial elastic recovery of the PDMS film 2 occurs, resulting in an increase in the pore diameter of nanopore 3, accounting for 15% of the initial current amplitude. After pressure release, the current does not rebound to the original reference. It is speculated that due to the irreversible deformation of the PDMS film 2 beyond the elastic limit during the previous pressurization process, the subsequent continuous reduction of the current reference should be caused by the start of reverse pressurization. The change in the single-time pressurization amplitude is basically the same as that of Figure 7 b, and the amplitude decreases by about 0.2 nA each time. Figure 8 b is the curve graph of the conductance of nanopore 3 under pressure release changing with the number of liquid injections. The conductance value rebounds by about 15% (rising from the lowest value of 8 nS after pressurization to 20 nS), which is consistent with the amplitude rebound of the I-V curve. After continuous pressurization, it enters the reverse regulation stage. At this time, the pore diameter of nanopore 3 shrinks due to compression. Due to the possible irreversible deformation after the previous pressurization beyond the elastic limit, the elastic performance of the PDMS film 2 decreases, and the single-time decrease rate is about 0.4% - 0.6%.

[0084] It should be noted that in this application, relative terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprise", "include" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the phrase "comprising an..." does not exclude the presence of additional identical elements in the process, method, article or device comprising said element.

[0085] Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A flexible nanopore channel, characterized in that: The nanopore is a through-hole formed in an organic film; the organic film is disposed on a silicon substrate, a window is formed in the silicon substrate, and the nanopore is formed within the window area.

2. The flexible nanopore according to claim 1, characterized in that: The window is square with a side length of 30 μm to 200 μm; the diameter of the nanopore is 10 nm to 100 nm; the thickness of the organic film is 50 nm to 1000 nm.

3. The flexible nanopore according to claim 1, wherein: The organic film is a PDMS film.

4. A preparation method of a flexible nanopore, characterized in that, The method for preparing the nanopore according to claim 3 includes the following steps: Double-side polish a silicon wafer to obtain a silicon substrate, and directionally etch the silicon substrate with tetramethylammonium hydroxide to etch a window in the silicon substrate. Spin coat a PDMS solution onto a copper sheet, and cure it to form a PDMS film. Transfer the cured PDMS film onto the surface of the silicon substrate by wet transfer method, such that the PDMS film covers the window area. Use a focused ion beam etching technique to etch nanopores in the PDMS film corresponding to the window area.

5. The preparation method of the flexible nanopore channel according to claim 4, wherein: The step of spin coating the PDMS solution onto the copper sheet and curing it to form a PDMS film includes: Mix the PDMS solution and the curing agent in a ratio of 10:1 to 8:1, and then drop the mixture onto the copper sheet. Next, use a spin coater to spin coat it. First, spin coat at a speed of 100 to 500 rpm for 10 to 15 seconds, and then spin coat at a speed of 3000 to 4000 rpm for 40 to 60 seconds to form a preliminary sample of a uniform PDMS film. Place the spin-coated copper sheet in an oven at 80 to 100 °C and bake for 2 to 5 hours for curing, and ensure that the PDMS film is closely attached to the copper sheet. After curing, a copper sheet with a PDMS film is obtained.

6. The preparation method of the flexible nanopore channel according to claim 4, wherein: The step of transferring the cured PDMS film onto the surface of the silicon substrate by wet transfer method, such that the PDMS film covers the window area, includes: Immerse the copper sheet with the PDMS film in a ferric chloride solution, with the side of the PDMS film facing up and the back in contact with the ferric chloride solution, etch the copper sheet, and place it for 2 to 4 hours until the copper sheet is completely dissolved. After the copper sheet is completely dissolved, the PDMS film floats on the surface of the ferric chloride solution. Subsequently, wash the PDMS film with deionized water, transfer the washed PDMS film onto the surface of the silicon substrate, and bake it after natural drying to make the PDMS film fully adhere to the silicon substrate.

7. The preparation method of the flexible nanopore according to claim 4, characterized in that: The step of using a focused ion beam etching technique to etch nanopores in the PDMS film corresponding to the window area includes: Use a focused ion beam to sputter nanopores in the PDMS film 2, and the etching parameters are an ion beam current of 1.1 pA to 40 pA and an etching time of 500 ms to 20 s.

8. A mechanical regulation device for flexible nanopores, characterized in that It includes a patch clamp amplifier, a microfluidic pump, a computer, a pair of electrodes, a first liquid cell, a second liquid cell, and a chip; the chip includes the nanopore channels described in any one of claims 1 to 4; the chip is disposed between the first liquid cell and the second liquid cell; the second liquid cell is a sealed liquid cell and integrates one of the motors and a liquid injection hose; the other motor is disposed in the first liquid cell, and one ends of the pair of motors are respectively inserted into the first liquid cell and the second liquid cell, and the other ends are both connected to the patch clamp amplifier; the patch clamp amplifier is communicatively connected to the computer, and is used to collect the ionic current between the two electrodes and transmit the collected ionic current data to the computer, and the computer is used to receive the ionic current data from the patch clamp amplifier, so as to record the pore size change process of the nanopore channels.

9. The mechanical regulation device of the flexible nanopore channel according to claim 8, wherein: It further includes rubber gaskets, and rubber gaskets are respectively disposed in the counterbores of the first liquid cell and the second liquid cell, and the rubber gaskets in the counterbores of the first liquid cell and the second liquid cell clamp the chip in the middle.

10. The mechanical regulation device of the flexible nanopore channel according to claim 8, characterized in that: The window on the chip is arranged close to the first liquid cell and far from the second liquid cell.

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