Biosensor based on heterostructure double holes and preparation method and application thereof

Abstract

The invention provides a biosensor based on heterostructure double holes and a preparation method and application thereof. The device comprises a silicon nitride chip fixed between two electrolyte-filled liquid storage tanks, a layer of graphene film is transferred on the silicon nitride chip in advance, one nanopore is etched on a graphene interface, and the other nanopore is etched on a silicon nitride interface. The two side liquid pools are respectively connected with the positive electrode and the negative electrode of the patch clamp amplifier. When a certain potential difference is applied to the two electrodes, the biological macromolecules in the liquid pool can be captured into the nanometer channel. When two ends of a molecule are captured by two different nanopores at the same time, due to different interaction between a silicon nitride-graphene heterogeneous interface and the molecule and the action of electric field force, the molecule can form a dragsaw phenomenon between the two pores, and the time of the molecule passing through the nanopores is prolonged. According to the invention, the rate of chain molecules such as DNA, protein and the like passing through the nanopore can be regulated and controlled, and accurate control of single molecules and even gene sequencing can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanodevices, in particular to a biosensor based on a heterostructure double pore and a preparation method and application thereof. BACKGROUND

[0002] Nanopore sensing technology, as a powerful single-molecule detection platform, has shown great potential in the fields of biomacromolecule sequencing, component analysis, interaction research, etc., and has broad application prospects. The core principle is that when a biomacromolecule passes through a nanoscale pore under the driving of an electric field, it will cause a transient change in the ion current in the pore. By analyzing the characteristics (amplitude, duration, shape, etc.) of these current signals, information about the structure, sequence, conformation, etc. of the molecule can be obtained. The nanopore sensors focused on by the current third-generation sequencing technology can mainly be divided into two categories: biological nanopores and solid-state nanopores. Among them, solid-state nanopores based on materials such as silicon nitride and graphene have become an important research direction in this field due to their excellent mechanical stability, chemical inertness, size controllability, and compatibility with standard micro-nano processing technology.

[0003] However, the traditional solid-state nanopore technology still faces the challenge of too fast pore passing speed in practical applications, especially in high-throughput and high-precision sequencing: under the action of the applied transmembrane voltage, the speed of DNA and other biomacromolecules passing through the nanopore is very fast, basically in the range of 103-105 kb / s (thousand base pairs per second), far exceeding the sampling frequency (<250 kHz) of existing commercial patch clamp detection systems. This results in a very short duration of the current blockage signal generated by a single base or structural unit, which brings great pressure to the electronic measurement system with high signal-to-noise ratio and high bandwidth, and limits the signal resolution and sequencing accuracy. Secondly, the film thickness of solid-state nanopores (especially thicker SiNx pores) is usually several nanometers to several tens of nanometers, much larger than the size of a single base (about 0.34 nm). When DNA passes through, multiple bases are contained in the pore at the same time, and the current signal generated is the "average" effect of multiple bases, which significantly reduces the single-base resolution. In addition, factors such as fast pore passing, random swinging of the molecule in the pore, and non-specific adsorption of the pore wall will affect the quality and stability of the current signal, reducing the reliability and accuracy of the detection.

[0004] Therefore, there is an urgent need in the art to develop a new nanopore sensing structure that can effectively solve the problem of too fast pore passing speed of biomacromolecules, realize precise, stable and diversified manipulation of the movement of single molecules in the pore, and at the same time improve the spatial resolution and signal quality of the sensing, in order to meet the strict requirements of high-precision nanopore sequencing and single-molecule dynamics research. SUMMARY

[0005] In order to solve the problem of the third generation sequencing technology that the biological macromolecule over-pore speed is too fast, the application discloses a biological sensor based on a heterogeneous structure double-pore, so as to realize the manipulation control of the biological macromolecule over-pore speed and state.

[0006] The application discloses a biological sensor based on a heterogeneous structure double-pore, wherein the main body part of the sensor is a silicon nitride chip, a graphene film is laid on one side of the silicon nitride chip, and a circular nano-pore with similar size is etched on the graphene interface and the silicon nitride interface respectively.

[0007] The processing and manufacturing method of the silicon nitride chip is as follows: First, polish a silicon wafer with a thickness of 300 microns on both sides to obtain a silicon substrate; A 20-100 nm thick silicon nitride film is deposited on the surface of the silicon substrate by a low-pressure chemical vapor deposition method; A square window with a side length of 100-200 microns is etched on the surface of the silicon nitride by a photoetching and reactive ion etching (RIE) process.

[0008] The silicon substrate exposed by the window is etched and released by using a potassium hydroxide (KOH) solution; Finally, the buffer oxide solution (BOE) obtained by mixing hydrogen fluoride (49%) solution and ammonium fluoride (40%) solution according to a volume ratio of 1:6 is used to etch the silicon dioxide and the silicon nitride, so that the silicon nitride film which can support its own weight is finally obtained.

[0009] The processed and treated silicon nitride chip is clamped in the middle of two liquid pool channels and is sealed by a silica gel gasket. The positions of the two liquid pools and the chip are fixed by a clamp; electrolyte solution and biological macromolecules are added into the liquid cavities of the two liquid pools, and two electrodes of a patch clamp amplifier are connected; an electric field can be generated between the patch clamp electrodes to drive the biological macromolecules to pass through the nano-pores, in addition, the patch clamp amplifier is used to collect the ion current between the two electrodes, and the collected ion current data is transmitted to a computer, and the computer is used to receive the ion current data from the patch clamp amplifier and record the detection process of the biological macromolecules.

[0010] Further, the two nano-pores are through holes with a diameter of 5nm to 100nm and a hole distance of 200-1000nm.

[0011] Further, the thickness of the graphene film is 0.34-3.4nm, and the number of layers is 1-10.

[0012] Further, the biological macromolecule should be selected from DNA, protein and other biological macromolecules with ductility, the material of the gasket should be selected from natural rubber or silica gel, the material of the liquid pool should be selected from PMMA, and the electrolyte solution added in the liquid pool should be potassium chloride salt solution or sodium chloride salt solution.

[0013] The application further discloses an operating method of the biological sensor based on the heterostructure double-hole. 1, place the silicon nitride chip with graphene film on one side and etched double-hole in the middle of two liquid pools and fix it; 2, add electrolyte solution in the two liquid pools, the electrolyte solution is 1mol / L KCl and 10mM Tris-HCl buffer solution, and a certain concentration of biological macromolecule (eg. 30pM lambda DNA) is added in one liquid pool.

[0014] 3, place two electrodes of the patch clamp amplifier in the two liquid pools, and set a detection voltage, generally in the range of 100-600mV.

[0015] 4, under the action of an electric field, the biological macromolecule to be measured gradually passes through and enters the nanometer hole, and part of the biological macromolecule is captured by one of the nanometer holes in the double-hole system under the action of electrophoretic force, and then the other end gradually approaches the surface of the silicon nitride chip and is captured by the other nanometer hole in the process of molecular movement. Therefore, the two ends of the biological macromolecule are captured by the silicon nitride nanometer hole and the graphene-silicon nitride nanometer hole respectively to form a double-hole co-capture phenomenon. Under a certain voltage, the biological macromolecule will be repeatedly pulled back and forth between the two holes due to the electrophoretic force.

[0016] 5, changing the transmembrane voltage can control the movement state of the biological macromolecule, and the movement state and structure information of the biological macromolecule can be obtained through the current data measured by the patch clamp amplifier.

[0017] 6, stop the measurement of the patch clamp amplifier.

[0018] In addition, the application discloses a preparation method of the biological sensor based on the heterostructure double-hole, mainly including processing and manufacturing of the chip and post-processing, comprising: 1, deposit a layer of 50-200nm thick silicon nitride film on the upper surface of the silicon substrate, preferably 50nm thick, and etch a square window with a side length of 100-200um on the silicon substrate; 2, transfer a layer of graphene film above the silicon nitride chip by using a dry transfer method; 3. Using a focused ion beam (FIB) method to etch two circular nanoholes with similar sizes on the graphene interface and the silicon nitride interface, respectively; the diameter of the nanoholes ranges from 5 nm to 100 nm, and the hole distance ranges from 200 nm to 1000 nm; 4. Hydrophilic treatment of the chip. A plasma cleaning machine is used to perform vacuum plasma treatment on the chip. Generally, low-power vacuum plasma treatment is performed on both sides of the two-dimensional material for 30 seconds each.

[0019] Advantages of the present application: 1. The present application innovatively combines double-hole and graphene-silicon nitride heterostructures. When the two ends of a molecule are captured by two different nanoholes, due to the different interactions between the silicon nitride-graphene hetero-interface and the molecule and the electric field force, the molecule will form a tug-of-war phenomenon between the two holes and prolong the time of the molecule passing through the nanohole. By controlling the different interactions of biological macromolecules in the heterostructure, the movement state of the biological macromolecule in the nanohole is influenced to some extent during the process of co-capturing by double holes, the pore passing time of the biological macromolecule is prolonged, and even the movement of the DNA molecule in the hole can be controlled. The present application can improve the accuracy of the third-generation sequencing technology for biological macromolecule detection and make up for the shortcomings of existing facilities and technologies.

[0020] 2. The present application uses silicon nitride chips and graphene as the main materials. These materials are all non-biological materials with high stability and wear resistance, corrosion resistance. In addition, in terms of structure, the stability of the device of the present application is more stable than that of the traditional graphene nanohole, and it is less likely to deform and denature. The graphene surface is also less likely to have problems such as wrinkles, damage and collapse.

[0021] 3. The present application uses dry method throughout the preparation process, and does not involve any liquid reagent. Therefore, no additional chemical contamination impurities and cleaning steps are introduced in the entire process, reducing the interference of external factors on the quality of the device, improving the success rate of sensor preparation, and the preparation cost is relatively low.

[0022] 4. There is a certain degree of interaction between DNA and other biological molecules and the graphene film, which makes it more likely to adhere to the surface of the graphene layer when passing through the graphene nanohole, thereby prolonging the pore passing time of the biological molecule. Therefore, the present design increases a graphene film on one side of the double-hole, which will further prolong the pore passing time of the biological molecule and the pulling time of the molecule between the double holes compared to the double silicon nitride hole or single silicon nitride hole system.

[0023] 5. The two nanopores in this patent are independent of each other and there is no channel connecting them. During the experiment, some biomolecules will enter the silicon nitride nanopore at one end and the graphene-silicon nitride nanopore at the other end. The long biomolecules are repeatedly pulled between the two pores under the action of electrophoretic force, forming a unique dual-pore co-capture. This capture phenomenon will further extend the pore passage time.

[0024] 6. The graphene transfer methods used are different. Existing graphene transfer technologies mostly use wet copper-based graphene transfer, which results in a relatively thick number of graphene layers, and the thickness and size cannot be flexibly controlled, thus having certain limitations. This patent uses dry thermal transfer, which can obtain fewer graphene layers (as low as a single layer), and the number of layers can be flexibly changed to obtain different experimental results. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the biosensor based on a heterogeneous dual-pore structure in this invention; Figure 2 This is a top view of the biosensor based on a heterogeneous dual-pore structure in this invention.

[0026] List of reference numerals in the attached diagram: 1-Silicon nitride chip; 2-Graphene film; 3-Nanopore; 4-Biomacromolecule; 5-Patch clamp amplifier; 6-Electrode. Detailed Implementation

[0027] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. It should be noted that the terms "front," "rear," "left," "right," "up," and "down" used in the following description refer to directions in the accompanying drawings, and the terms "inner" and "outer" refer to directions toward or away from the geometric center of a specific component, respectively.

[0028] As attached Figure 1 and attached Figure 2 As shown, this embodiment provides a biosensor based on a heterogeneous dual-pore structure. It includes 1. a silicon nitride chip, 2. a graphene film, 3. nanopores, 4. biomolecules, 5. a patch-clamp amplifier, and 6. electrodes. The silicon nitride chip 1 consists of layers of different materials from top to bottom: silicon nitride, silicon oxide, a silicon substrate, silicon oxide, and silicon nitride. The graphene film 2 is placed on one side above the silicon nitride chip, and can be one or more layers. Two nanopores 3 are spaced apart on the silicon nitride chip, one on the graphene interface and the other on the silicon nitride interface. The electrodes 6, connected to the patch-clamp amplifier 5, are inserted into electrolyte solutions in two liquid pools.

[0029] In this invention, to ensure that the substrate material of the nanopore is non-conductive, has sufficient mechanical strength, and meets the requirements of nanopore sequencing, a silicon-based material, comprising silicon nitride, silicon oxide, and silicon, is selected as the chip material used as the substrate.

[0030] In one specific implementation of this embodiment, after the silicon nitride chip is fabricated, a graphene film 2 of appropriate size and number of layers is transferred onto the silicon nitride chip 1 using a dry transfer technique, ensuring that the graphene film is well-laid out on the chip. The thickness of the graphene film is 0.34-3.4 nm, and the number of layers is between 1 and 10. Depending on the size of the biomolecule to be tested, two circular nanopores are etched on both the silicon nitride film and the graphene film using a focused ion beam (FIB). The diameter of the two circular nanopores ranges from 5 nm to 100 nm, and the distance between the two pores is between 200-1000 nm. The specific pore diameter and pore spacing data are further determined according to the specific analyte.

[0031] To ensure the normal operation and mechanical strength of the heterostructure-based dual-pore biosensor, the thickness of the silicon nitride layer on the silicon nitride chip 1 must not be less than 20 nm. To ensure that the substrate material of the nanopore is non-conductive, possesses sufficient mechanical strength, and meets the requirements of nanopore sequencing, the chip material used as the substrate is selected as a silicon-based material, comprising silicon nitride, silicon oxide, and silicon.

[0032] As attached Figure 1 and attached Figure 2 As shown in this embodiment, a method for fabricating a biosensor based on a heterogeneous dual-pore structure includes the following steps: 1. Deposit a silicon nitride film 50-200 nm thick, preferably 50 nm thick, on the surface of a silicon substrate. Etch a square window with a side length of 100 μm-200 μm on the silicon substrate; 2. A graphene film is transferred over a silicon nitride chip using a dry transfer method; 3. Two circular nanopores of similar size were etched on the graphene interface and the silicon nitride interface respectively using the focused ion beam (FIB) method; the diameter of the nanopores ranged from 5 nm to 100 nm, and the pore spacing was between 200 and 1000 nm. 4. Perform hydrophilic treatment on the chip. Use a plasma cleaner to perform vacuum plasma treatment on the chip. Generally, for two-dimensional materials, perform low-power vacuum plasma treatment on both sides for 30 seconds each.

[0033] In one specific embodiment of this invention, the method for manufacturing the silicon nitride chip 1 is as follows: 1. First, a silicon wafer with a thickness of 300μm is polished on both sides to obtain a silicon substrate; 2. A silicon nitride film with a thickness of 20-100 nm is deposited on the surface of a silicon substrate by low-pressure chemical vapor deposition. 3. A square window with a side length of 100-200μm is etched on the surface of silicon nitride using photolithography and reactive ion etching (RIE) processes.

[0034] 4. Use potassium hydroxide (KOH) solution to etch the exposed silicon substrate through the release window; 5. Finally, a buffer oxide solution (BOE) obtained by mixing hydrogen fluoride (49%) solution and ammonia fluoride (40%) solution at a volume ratio of 1:6 is used to etch silicon dioxide and silicon nitride, ultimately obtaining a silicon nitride film that can support its own weight.

[0035] In one specific implementation of this embodiment, the graphene film 2 transfer process adopts a dry transfer method, and the process is as follows: 1. Use blue glue to pick up a small amount of graphite ore, and repeatedly fold it to obtain a few layers of graphene.

[0036] 2. Place the material obtained in the previous step under a microscope for observation, and observe and record the appropriate number of layers, size, and clean surface of graphene.

[0037] 3. On the transfer stage, align the position of the graphene on the PDMS film with the chip and make them fully bonded.

[0038] 4. Heat and cure at 80℃ for 20 minutes.

[0039] 5. Separate the PDMS film from the glass slide, and then separate the chip from the PDMS to obtain the chip after the graphene film has been transferred.

[0040] This embodiment includes an operation method for a biosensor based on a heterostructure dual-pore structure. This method applies a specific voltage to both sides of the nanopore through the two electrodes of a patch-clamp amplifier, thereby controlling and manipulating the movement of biomolecules within the pore. Compared with normal sequencing, this method can extend the pore passage time of biomolecules and improve the detection accuracy of biomolecules.

[0041] Specifically, the operation method includes the following steps: 1. Place a silicon nitride chip with a graphene film on one side and two etched holes at the connection point of the channels in the two liquid pools; 2. Use clamps to secure the liquid pool and the chip; 3. Add electrolyte solutions to both liquid tanks. The electrolyte solutions are 1 mol / L KCl and 10 mM Tris-HCl buffer solution. Add a certain concentration of biological macromolecules (e.g., 30 pM λ-DNA) to one liquid tank.

[0042] 4. Place the two electrodes of the patch clamp amplifier in the liquid pools on both sides, and set the detection voltage. The typical voltage range is 100-600mV.

[0043] 5. Under the influence of an electric field, the biomolecules to be tested gradually pass through and enter the interior of the nanopores. Under the influence of electrophoretic force, one end of some biomolecules is initially captured by one of the nanopores in the dual-pore system, and then, during molecular motion, the other end gradually approaches the surface of the silicon nitride chip and is captured by the other nanopore. Therefore, the two ends of the biomolecules are captured by both silicon nitride nanopores and graphene-silicon nitride nanopores, forming a dual-pore co-capture phenomenon. Under a certain voltage, the biomolecules will be repeatedly pulled back and forth between the two pores due to the electrophoretic force.

[0044] 6. Changing the transmembrane voltage can control the motion state of biological macromolecules. The motion state and structural information of biological macromolecules can be obtained by measuring the current data through a patch-clamp amplifier.

[0045] The operating method of this invention utilizes the principle that charged objects experience forces in an electric field. Most biological macromolecules in nature carry an electric charge, and under the influence of an electric field, they can move rapidly. Simultaneously, there are interaction forces between molecules; when the distance is too close, these forces manifest as repulsion. Using this principle, biological macromolecules can exhibit different motion states under different interactions with heterogeneous material interfaces (silicon nitride and graphene), thereby achieving manipulation and control of the movement of biological macromolecules within the dual-pore structure.

[0046] The technical means disclosed in this invention are not limited to those disclosed in the above embodiments, but also include technical solutions composed of any combination of the above technical features.

Claims

1. A biosensor based on a heterogeneous dual-pore structure, comprising a silicon nitride chip (1), a graphene film (2), a nanopore (3), a biomacromolecule (4), a patch-clamp amplifier (5), and patch-clamp electrodes (6); the patch-clamp electrodes (6) are connected to both sides of a liquid pool, and the patch-clamp amplifier (5) is communicatively connected to a computer. Its features are: A graphene film (2) is also provided, which is laid flat on top of the silicon nitride chip (1); two nanopores (3) are provided at a certain distance on the silicon nitride chip (1), one of the nanopores (3) is on the interface of the graphene film (2), and the other nanopore (3) is on the interface of the silicon nitride chip (1); an electric field is generated between the patch clamp electrodes (6) to drive biomacromolecules (4) through the nanopores (3).

2. The biosensor based on a heterogeneous dual-pore structure according to claim 1, characterized in that: Both nanopores (3) are through pores with a diameter between 5 nm and 100 nm and a pore spacing between 200 and 1000 nm.

3. The biosensor based on a heterogeneous dual-pore structure according to claim 1, characterized in that: The graphene film has a thickness of 0.34-3.4 nm and has 1-10 layers.

4. The biosensor based on a heterogeneous dual-pore structure according to claim 1, characterized in that: The biological macromolecules (4) should be selected from chain-like biological macromolecules with extensibility, such as DNA and proteins. The electrolyte solutions added to the liquid pools on both sides of the chip should be potassium chloride salt solutions or sodium chloride salt solutions.

5. A method for operating a biosensor based on a heterogeneous dual-pore structure as described in any one of claims 1-4, comprising the following specific operating steps: Step 1: Place the silicon nitride chip with a graphene film on one side and two holes etched on it between the two liquid pools and fix it in place; Step 2: Add electrolyte solutions to both liquid tanks. The electrolyte solutions are 1 mol / L KCl and 10 mM Tris-HCl buffer solution. Add a certain concentration of biomacromolecules (e.g., 30 pM λ DNA) to one liquid tank. Step 3: Place the two electrodes of the patch-clamp amplifier into the liquid pools on both sides, and set the detection voltage. The typical voltage range is 100-600mV. Step 4: Under the influence of an electric field, the biomolecules to be tested gradually pass through and enter the interior of the nanopores. Under the influence of electrophoretic force, one end of some biomolecules is initially captured by one of the nanopores in the dual-pore system, and then, during molecular motion, the other end gradually approaches the surface of the silicon nitride chip and is captured by the other nanopore. Therefore, the two ends of the biomolecules are captured by both the silicon nitride nanopores and the graphene-silicon nitride nanopores, forming a dual-pore co-capture phenomenon. Under a certain voltage, the biomolecules will be repeatedly pulled back and forth between the two pores due to the electrophoretic force. Step 5: Changing the transmembrane voltage can control the motion state of biological macromolecules. The motion state and structural information of biological macromolecules can be obtained by measuring the current data through a patch-clamp amplifier. Step 6: Stop the measurement using the patch clamp amplifier.

6. A method for fabricating a biosensor based on a heterostructure dual-pore structure according to any one of claims 1-5, mainly comprising chip fabrication and post-processing, including the following steps; Step 1: Deposit a silicon nitride film 50-200 nm thick, preferably 50 nm thick, on the surface of a silicon substrate. Etch a square window with sides of 100 μm-200 μm on the silicon substrate; Step 2: A graphene film is transferred over the silicon nitride chip using a dry transfer method; Step 3: Use focused ion beam (FIB) to etch two circular nanopores of similar size on the graphene interface and the silicon nitride interface respectively; the diameter of the nanopores ranges from 5 nm to 100 nm, and the pore spacing is between 200 and 1000 nm. Step 4: Perform hydrophilic treatment on the chip. Use a plasma cleaner to perform vacuum plasma treatment on the chip. Generally, for two-dimensional materials, perform low-power vacuum plasma treatment on both sides for 30 seconds each.

7. An application of a biosensor based on a heterogeneous dual-pore structure according to any one of claims 1-6, characterized in that: The device is a biological macromolecule detection device, mainly used for sequencing chain-like biological macromolecules such as nucleic acids and proteins.

Images