A method for decelerating and / or capturing a single molecule using dielectrophoretic forces based on a nanopore device

Abstract

The application discloses a method for decelerating and / or capturing single molecules by dielectrophoresis force based on a nanopore device. The method comprises the following steps: preparing a solid-state nanopore film; taking the solid-state nanopore film as a diaphragm for separating an anode and a cathode of an electrolytic cell to obtain a nanopore sequencing device; detecting by using the nanopore sequencing device; adding a single molecule to be detected into a negative electrode grounding chamber of the electrolytic cell; during the detection, applying a direct current voltage at two ends of the diaphragm, introducing an alternating current voltage through a conductive layer, and respectively providing an electric field force and a dielectrophoresis force for the single molecule, so as to respectively serve as a driving external field for the single molecule to pass through a nanopore in the solid-state nanopore film and a capturing force for the single molecule to stay in the nanopore; and by controlling the dielectrophoresis force to be greater than or approximately equal to the electric field force, the single molecule is decelerated or captured. The application expands the measurement range of the nanopore to-be-detected substance by using dielectrophoresis, and can realize real-time controllable capturing and deceleration perforation of the single molecule, so that the detection time resolution is greatly improved.

Description

Technical Field

[0001] The present invention relates to a method for decelerating and / or capturing a single molecule by utilizing dielectrophoretic force based on a nanopore device, and belongs to the technical field of nanopore single molecule detection. Background Art

[0002] Single-molecule DNA detection and analysis based on solid-state nanopore devices is considered one of the most promising technological approaches for achieving third-generation rapid and low-cost human genome sequencing (sequencing a single human genome in 24 hours for under $1,000), and is currently a hot topic in research and application exploration. Since Clarke et al. discovered in 2009 that helicase-modified MspA biological pores could sequence DNA (Nature Nanotech. 4, 265–270, 2009), and by the IPO of Oxford Nanopore in 2021, biological nanopores have essentially achieved DNA sequencing. However, several challenges remain: error rates due to helicase failure for unknown reasons, limitations on analyte size due to difficulty adjusting the biological pore size, storage difficulties in relatively extreme environments, and high costs due to incompatibility with modern semiconductor processes. However, solid-state nanopores offer controllable pore size, tolerance to a wider range of temperatures and pH levels, ease of storage, and compatibility with semiconductor processes, enabling mass production. These advantages make continued advancement of solid-state nanopore sequencing technology highly promising and of great significance.

[0003] Single-molecule detection and analysis capabilities based on nanopore devices are achieved by electrophoretically driving molecules through a nanoscale pore in a solution. Within the confined space of a nanopore, large numbers of molecules can be rapidly analyzed using various methods. When polymer molecules pass through the nanopore, there is a one-to-one correspondence between the polymer's structural information and the detected signal characteristics. This property allows for direct characterization of single-stranded DNA molecules thousands of base pairs long, eliminating the need for amplification or labeling experimental preparation and enabling rapid and low-cost DNA sequencing. Currently, the greatest obstacle hindering the rapid development of this technology is that the speed of DNA permeation under electric field drive voltage exceeds the time resolution of conventional instruments, making it impossible to discern single-base differences. The ability to capture DNA molecules using solid-state nanopores is of great practical significance for studying the capture, manipulation, and control of single molecules in solution, as well as for studying biological processes such as chemical transformations and dynamic changes within the molecule.

[0004] Currently, the most commonly used experimental technique internationally involves applying a voltage across the nanopore. Using the electric field, the DNA molecule electrophoretically passes through the nanopore from one end. The ionic current collected in the external circuit experiences a sudden drop, and the magnitude of this sudden drop and the duration of the blockage can be used to correlate with the biological information of the DNA. However, using only electric field regulation, the DNA permeation speed is too fast, typically on the order of one microsecond per base, while the current minimum resolution of instruments is 4 microseconds. This means that if one wishes to distinguish between different bases, the time resolution achieved using existing methods is unattainable. To improve the time resolution, the DNA permeation time must be extended, effectively slowing the movement of the DNA within the pore. Slowing down the DNA permeation speed makes it possible to distinguish between different bases.

[0005] On the other hand, the basic principle of the existing second-generation high-throughput sequencing technology is to randomly break the whole genome of the species to be tested into small fragments of hundreds of bp, and then use a "sequencing by synthesis" method to read the sequence of each small fragment after in vitro amplification and modification and calibration. Finally, the sequences of all small fragments are integrated together through a certain algorithm to restore the sequence of the whole genome. It should be pointed out that each small fragment of hundreds of bp is relatively small compared to the whole human genome (about 3×10 9 bp), so the computational complexity and algorithm complexity of the small fragment alignment and splicing are extremely high. In addition, due to the large number of repeated sequences in the human genome, the whole genome obtained by the existing algorithm still has an uncertainty of 5% to 10%. Therefore, for medical diagnosis, drug screening and high-end scientific research fields with very high accuracy requirements, the entire sequence needs to be sequenced dozens or even hundreds of times, which undoubtedly greatly increases the cost of sequencing and reduces time efficiency. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for real-time controllable capture of DNA using dielectrophoresis technology based on nanopore devices, which can realize real-time controllable capture and deceleration of perforation of single molecules (such as DNA, peptide chains, etc.), greatly improving the detection time resolution.

[0007] The dielectrophoresis involved in the present invention is the phenomenon in which dielectrics are subjected to forces in a non-uniform electric field. All objects are dielectrics, and this non-uniform electric field can induce a dipole moment in objects of finite size, thereby subjecting them to forces in the non-uniform electric field. The existence of this force does not require the object itself to be charged, and the magnitude of the force depends on the dielectric properties, shape, size, and frequency of change of the field strength of the substance. Dielectrophoresis has many applications in separating biomolecules of different sizes and molecular weights, and this force does not denature the biomolecules. If a suitable dielectrophoretic force is introduced into the nanopore device to balance the electric field force, the penetration speed of the DNA molecules can be greatly reduced, the detection time resolution can be improved, and the capture and manipulation of single molecules can also be achieved.

[0008] The method provided by the present invention for decelerating and / or capturing a single molecule by utilizing dielectrophoretic force based on a nanopore device comprises the following steps:

[0009] S1, preparing a solid nanoporous film; the solid nanoporous film comprises an insulating layer I, a conductive layer and an insulating layer II which are compounded in sequence;

[0010] S2, making a nanopore sequencing device; using the solid-state nanopore film as a diaphragm separating the positive electrode and the negative electrode of the electrolytic cell to obtain the nanopore sequencing device;

[0011] S3. Performing detection using the nanopore sequencing device; adding a single molecule to be tested to the negatively grounded chamber of the electrolytic cell; during testing, applying a DC voltage across the diaphragm and introducing an AC voltage through the conductive layer to provide an electric field force and a dielectrophoretic force to the single molecule, respectively serving as a driving external field for the single molecule to pass through the nanopore in the solid-state nanopore film and a capture force for the single molecule to remain within the nanopore; deceleration or capture of the single molecule is achieved by controlling the dielectrophoretic force to be greater than or approximately equal to the electric field force;

[0012] The electric field force can cause a single molecule to electrophoretically pass through the nanopore from one end of the pore, serving as a driving external field for DNA transfection; the dielectrophoretic force can cause DNA molecules to be captured in the solid-state nanopore;

[0013] By detecting the ionic current in the positive and negative external circuits, it can be determined whether a single molecule such as a DNA molecule is captured in the nanopore.

[0014] In the above method, in the solid nanoporous film, the insulating layer I is a silicon nitride film with a thickness of 20 to 50 nm; the conductive layer is a titanium film with a thickness of 5 to 10 nm; and the insulating layer II is an aluminum oxide film with a thickness of 1 to 20 nm.

[0015] In the above method, the diameter of the nanopores in the solid-state nanoporous film is 5 to 30 nm, and the pore depth is 50 to 80 nm.

[0016] In the above method, during the test, the electrolytic cell contains an electrolyte, which is a NaCl solution, a KCl solution or a LiCl solution;

[0017] The concentration of the electrolyte is 0.1-3.2 mol / L, and the pH value is 7-10.

[0018] In the above method, the DC voltage is 0 to 1V;

[0019] The peak value of the AC voltage is 0-1V and the frequency is 0.5-2MHz;

[0020] The optimal voltage and frequency applied to reduce the DNA pore velocity can be determined through limited experimentation.

[0021] The above method can also address the problem of low temporal resolution in DNA sequencing, providing a method for DNA sequencing. By using the method of the present invention to detect DNA and determine the optimal applied voltage and frequency for reducing the DNA pore speed, the DC voltage is appropriately increased to control the DNA pore speed, thereby addressing the problem of low temporal resolution in solid-state pore sequencing.

[0022] This invention breaks through traditional DNA sequencing methods based on solid-state nanopore devices by introducing dielectrophoresis as an external field for capturing DNA. The relative magnitudes of the electric field and dielectrophoretic forces can be adjusted to control the DNA penetration rate. Using a SiN solid-state nanopore with a diameter of approximately 10 nm, the speed at which DNA molecules pass through the nanopore device is effectively reduced without compromising the signal-to-noise ratio of the measured signal. This significantly improves the temporal resolution of existing approaches, maintaining a high current signal-to-noise ratio while avoiding the uncontrolled interactions between DNA molecules and the nanopore walls that can arise from using very small nanopores (less than 5 nm in diameter). Furthermore, the experiment is simple and easy to perform, with significantly improved repeatability and controllability compared to existing techniques.

[0023] Compared with existing reports on solid-state nanopore-based DNA molecule detection, the advantages of the present invention are as follows:

[0024] 1. Existing technologies for DNA pore detection are still far from achieving single-base resolution. However, the present invention can directly capture DNA, significantly improving the previously reported temporal resolution. However, low temporal resolution is one of the biggest obstacles plaguing solid-state nanopore DNA sequencing. Many conventional methods are unable to achieve this goal.

[0025] 2. The fabrication process of this three-layer nanopore structure is fully compatible with the production process of modern silicon-based chips and is simple and fast, enabling large-scale mass production and significantly reducing sequencing costs.

[0026] 3. The method is simple and easy to operate. This method has good repeatability and is very conducive to promotion. It does not require complex circuit design and program design, does not require the introduction of complex systems and manufacturing processes, has low technical requirements, and has a high experimental success rate, which greatly improves experimental efficiency. The measurement process only requires adding the analyte and then applying specific DC voltage and AC voltage to measure the analyte. In theory, it can be used to capture and detect any molecule. This greatly improves the detection range of solid-state nanopores to the analyte and expands its application range, such as in single-molecule in vitro diagnosis and other fields.

[0027] 4. Expanding the scope of solid-state nanopore analytes. For single molecules such as peptides and proteins that have little or no charge, driving them through the pore using conventional DC voltage is difficult. However, dielectrophoresis, which does not require the object to be charged, can use the dielectrophoretic force to drive these single molecules into the nanopore, enabling detection. This greatly expands the scope of solid-state nanopore analytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a partial view of the pattern of the solid-state nanopore lithography mask used in the present invention. The entire mask is 5 inches in size, and the pattern of the mask is arranged periodically. The detailed data of the pattern is listed in the figure; a single pattern is 3mm×3mm, corresponding to the size of a single solid-state nanopore chip of 3mm×3mm; each 3mm×3mm pattern has a circular light-transmitting area with a diameter of 443μm in the center; to ensure the accuracy of the knife during the separation and dicing of each small substrate on the 4-inch silicon wafer later, and to prevent the silicon wafer from being damaged into random small pieces, some light-transmitting grooves are added to the edge of each small substrate pattern: the width of the light-transmitting strip is 282μm and the length is 2000μm.

[0029] Figure 2 Schematic diagram of the production process of nanopore device (3mm×3mm chip); double-sided spin coating of photoresist; A side with Figure 1 The mask shown is exposed, developed, and stripped; the silicon nitride on the A side is removed using reactive ion beam etching; the silicon dioxide on the A side is etched using BOE solution; the double-sided photoresist is cleaned with acetone; the silicon is anisotropically etched using potassium hydroxide solution, exposing the silicon dioxide and silicon nitride on the B side; photoresist is spin-coated on the B side to protect the silicon nitride; the silicon dioxide on the B side is etched using BOE solution; the photoresist on the B side is cleaned with acetone; metallic titanium is evaporated using a high-voltage electron beam; aluminum oxide is plated using atomic layer deposition technology; and holes are punched using a projection electron microscope.

[0030] Figure 3 A photo of a specially designed electrophoresis cell for combining dielectrophoresis technology with a solid-state nanopore chip. The cell consists of two parts, between which the chip must be placed and then joined together for normal use. Figure 3 (a) is a top view of the two electrophoretic cells; the two circular tanks below the top view are the tanks for adding ion solutions. Figure 3 (b) is a front view of the two pools; the two protrusions on the top of the front view are electrodes that apply DC voltage and measure the ion current passing through; there are four thin electrode columns in the middle of the right electrochemical pool, which are used to connect to the conductive layer of the chip and apply AC voltage (dielectrophoretic force), and in the middle is a rubber O-ring used to prevent leakage of the ion solution; in the middle of the left electrochemical pool is the B-side of the nanopore chip.

[0031] Figure 4 (a) Schematic diagram of the experimental principle. The upper chamber is the Cis chamber with the analyte (DNA) added, and the lower chamber is the Trans chamber with normal ionic solution. The two chambers are connected only by three layers of solid-state nanopores. The upper and lower chambers are connected to DC electrodes and V is applied. DC The DC voltage is applied and the ion current is recorded; the conductive layer (titanium) is connected to the AC electrode and AC is applied to generate dielectrophoresis to capture the analyte; Figure 4 (b) Actual diagram of the device for applying alternating current, a function generator.

[0032] Figure 5 (a) Schematic diagram of the ion current signal during normal DNA perforation; Figure 5 (b) The blue solid line shows the signal of DNA trapped in the pore when dielectrophoresis is activated after sample addition; the red dashed line shows the signal of ionic current when dielectrophoresis is activated without sample addition. The activation of dielectrophoresis causes a rapid redistribution of ions in the pore, thereby increasing the ionic current signal measured between the two chambers. The difference between the red dashed line and the blue solid line represents the decrease in ionic current caused by DNA in the pore. Figure 5 (c) Ionic current signal encountered during actual testing. As long as dielectrophoresis (DEP) is turned on, DNA will remain trapped in the nanopore. DETAILED DESCRIPTION

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] 1. Preparation of chip devices

[0036] The team fabricated a solid-state nanopore device that can be manipulated within a transmission electron microscope, thereby utilizing the high-energy focused electron beam within the transmission electron microscope to drill holes in the device's suspended membrane, thereby realizing the nanopore device. This involves a range of micro-nanofabrication processes involved in traditional semiconductor processing, as well as a range of modern cutting-edge nanoscale processing technologies.

[0037] The specific method is as follows:

[0038] 1. First, a 4-inch 300μm silicon wafer with (100) surface was grown with 2μm silicon oxide on both sides, and 50nm low-stress silicon nitride was deposited using low-pressure chemical vapor deposition.

[0039] 2. Then make a photolithography mask ( Figure 1 ) aims to create a periodic distribution of numerous 3mm×3mm wafers on a 4-inch silicon wafer. Each 3mm×3mm wafer has a circular, 443μm-diameter, transparent area at its center. To ensure accurate scribing when separating each 3mm×3mm wafer from the 4-inch wafer, and to prevent the wafer from breaking into random pieces, short, transparent dashes are added around the edges of each wafer pattern: 282μm wide and 2000μm long.

[0040] 3. Then, photolithography and reactive ion beam etching are used to etch through the silicon nitride on one side of the silicon wafer (side A) according to the pattern of the photolithography mask, exposing the silicon oxide surface. The other side (side B) is protected with photoresist. The protected 4-inch silicon wafer is immersed in a 10:1 BOE solution for 40 minutes to remove the silicon oxide on side A, exposing 300μm of silicon. Then, KOH anisotropic etching (40% KOH, 80℃, 6 hours) is used to etch the silicon, and the etching is carried out along the (111) surface. The etching through standard is: after the small window pane is transparent to light, it is etched for a period of time. Under an optical microscope, the silicon oxide surface is very flat and has no island structure. In this way, a small window pane (20μm) with only silicon dioxide and silicon nitride on one side is realized, with a silicon substrate as support underneath. Generally speaking, a 4-inch silicon wafer can be dissociated to obtain 800 3mm×3mm small substrates.

[0041] The chip produced by the above operation is a conventional silicon nitride solid-state nanopore operation step. It is also necessary to make a conductive layer and a second insulating layer using the high-voltage electron beam evaporation method (instrument model). At a speed of 2 nm, 2 nm titanium and 5 nm gold are evaporated. At this rate, the conductive layer evaporated has good conductivity and is sufficiently flat. To make an insulating layer, atomic layer deposition technology (instrument model) can be used, using the reaction of trimethylaluminum (TMA) with water.

[0042] Al(CH3)3+3H2O=Al(OH)3+3CH4

[0043]

[0044] Make an insulating layer of aluminum oxide. Because trimethylaluminum will spontaneously combust in the air, high-purity N2 is used as the growth environment. At the same time, the entire system needs to be heated to the required temperature, and the sample is placed and then vacuumed. N2 is continuously filled in and out of the system, taking away impurities in the sample chamber and the remaining precursors of the reaction, keeping the sample chamber clean. During the deposition process, the precursor water is first filled in, and the residual water is pumped out after adsorbing a layer. The precursor trimethylaluminum is then filled in and reacts with water to obtain a layer of the target product aluminum oxide, and then the residual trimethylaluminum is pumped out. Continuously cycle, and calculate the required number of cycles based on the temperature, material, gas flow rate, and target thickness. The parameters currently used are to control the precursor gas pressure at 1 to 3 atm at a temperature of 150°C, and complete 187 cycles to obtain a 20nm dense aluminum oxide insulating layer ( Figure 2 ).

[0045] 2. Nanopore Fabrication Using Transmission Electron Microscopy

[0046] After fabricating the 3mm×3mm chip device, it was placed under a transmission electron microscope (FEI Tecnai F30) for drilling. The magnification was adjusted to 890k, the beam spot size was set to 1:1, and the electron beam was focused to its minimum or slightly larger focus. In about 5 minutes, a three-layer nanopore structure with a diameter of approximately 10nm and a depth of approximately 80nm was obtained. Before and after drilling, the sample rod was cleaned in a plasma cleaner (O2:Ar = 1:3, v / v) for one minute to remove organic contamination. After fabrication, the nanopore device was stored in a vacuum desiccator until needed.

[0047] 3. DNA capture using three-layer solid-state nanopores

[0048] Because of this three-layer solid-state nanopore, the conductive layer needs to be connected to an AC power source, but it cannot come into contact with the electrolyte in the two chambers. The present invention uses a series of infiltration processes to encapsulate the chip into the above-mentioned electrochemical pool using a nitrile O-ring. The pool consists of a Cis cavity and a Trans cavity. The two cavities are connected only by the nanopore, without other connecting channels ( Figure 3 Then, a 1 mol sodium chloride solution was injected into the two chambers. The solution contained 1 mM EDTA and 10 mM Tris (pH = 7.4). Two Ag / AgCl electrodes were inserted into the Cis chamber and the Trans chamber (to generate DC voltage and record ion current). At the same time, the negative electrode of the signal generator was connected to the electrode at the Cis end and grounded, and the positive electrode was connected to the probe in contact with the conductive layer ( Figure 4 ).

[0049] Then, an appropriate amount of DNA solution was added to the Cis chamber, and a DC voltage of 100 mV was applied between the Cis chamber and the Trans chamber. At this time, we could observe normal DNA perforation signals ( Figure 5 (a)). A square wave AC voltage with a frequency of 1 MHz and a peak-to-peak value of 500 mV is applied between the conductive layer and the Cis terminal using a signal generator. At this time, it can be observed that the ion current is relatively stable relative to the baseline current ( Figure 5 (b), red dotted line), there is a significant decrease and stabilizes at the current value after the decrease ( Figure 5 (b), blue solid line); when the AC voltage generated by the signal generator is turned off (dielectrophoresis), the ion current can return to the baseline ( Figure 5 (b)). The magnitude of the ion current is closely related to whether dielectrophoresis is applied or not. This shows that when the dielectrophoretic force exists, the DNA is captured in the nanopore, the ion current decreases, and stabilizes at the current value, that is, the capture of the single molecule is achieved; when the dielectrophoretic force does not exist, the DNA is driven by the DC voltage to perforate, generating a perforation signal, and the ion current returns to the baseline. This experimental phenomenon proves that the capture of a single molecule (DNA) is achieved. The premise for this phenomenon is that the electric field force (F DC ) is smaller than the dielectrophoretic force (F) provided by the alternating current. DEP ), which will produce the experimental phenomenon of capture. This is actually the competition between the electric field force and the dielectrophoretic force to dominate the movement of the object to be tested. So when F acting on the object to be tested DC >F DEP When the F DC ≈F DEP When the time is high, the object under test will be observed to decelerate and perforate, thereby improving the time resolution of the detection.

[0050] 4. Using triple-layer solid-state nanopores to slow down DNA perforation and achieve sequencing

[0051] Perform the same operation as in the fourth step to capture DNA, connect the instrument, and add the sample. Fix the DC voltage to 500mV and the AC frequency to 1MHz, and change the peak-to-peak value of the AC within the range of 1mV to 1V, and change it from large to small. Since the physical and chemical properties, size, and shape of each pore are not exactly the same, an appropriate peak-to-peak value can be found to observe that the ion current returns to the baseline, that is, DNA perforation occurs. At this time, the electric field force is comparable to the dielectrophoretic force, and the resultant force on the DNA in the pore is close to 0. At this time, the perforation speed is very slow, and a low-frequency filter can be used to process the ion current obtained during perforation (the main reason why commercial nanopore sequencers can achieve sequencing at present, the perforation is slow enough, and a low-frequency filter can be used to detect the signal), thereby reading the base information of the DNA and achieving sequencing.

Claims

1. A method for decelerating and / or capturing a single molecule using dielectrophoretic force in a nanopore device, comprising the following steps: S1, preparing a solid nanoporous film; the solid nanoporous film comprises an insulating layer I, a conductive layer and an insulating layer II which are compounded in sequence; In the solid-state nanoporous film, the insulating layer I is a silicon nitride film with a thickness of 20 to 50 nm; the conductive layer is a titanium film with a thickness of 5 to 10 nm; and the insulating layer II is an aluminum oxide film with a thickness of 1 to 20 nm. S2, making a nanopore sequencing device; using the solid-state nanopore film as a diaphragm separating the positive electrode and the negative electrode of the electrolytic cell to obtain the nanopore sequencing device; S3. Use the nanopore sequencing device to perform detection; add the single molecule to be tested into the negatively grounded chamber of the electrolytic cell; during the test, apply a DC voltage to both ends of the diaphragm, and introduce an AC voltage through the conductive layer to provide an electric field force and a dielectrophoretic force to the single molecule, respectively, which serve as the driving external field for the single molecule to pass through the nanopore in the solid-state nanopore film and the capture force for the single molecule to remain in the nanopore; by controlling the dielectrophoretic force to be greater than or approximately equal to the electric field force, the single molecule can be decelerated or captured.

2. The method according to claim 1, wherein: The diameter of the nanopores in the solid-state nanopore film is 5 to 30 nm, and the pore depth is 50 to 80 nm.

3. The method according to claim 1 or 2, characterized in that: During the test, the electrolytic cell contains an electrolyte, which is a NaCl solution, a KCl solution or a LiCl solution; The concentration of the electrolyte is 0.1-3.2 mol / L, and the pH value is 7-10.

4. The method according to claim 1 or 2, characterized in that: The DC voltage is 0-1V; The peak value of the AC voltage is 0-1V, and the frequency is 0.5-2MHz.

5. The method according to claim 1 or 2, characterized in that: The single molecule is DNA or a peptide chain.

6. A DNA testing method comprising the following steps: The method according to any one of claims 1 to 5 is used to determine the voltage and frequency applied to reduce the DNA pore speed; the DC voltage is increased to control the DNA pore speed to solve the problem of low temporal resolution of DNA in solid-state pore sequencing.

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