Preparation method and application of nanopore sensor based on specific antibody

Abstract

The invention discloses a preparation method and application of a nanopore sensor based on a specific antibody, and relates to the field of heavy metal monitoring. The method comprises the following steps: preparing Si3N4 nanopores in a Si3N4 film substrate based on an electron beam exposure micro-nano processing technology to obtain a Si3N4 nanopore array; carrying out surface functionalization on the Si3N4 nanopore array so as to fix a specific antibody on the surfaces of nanopores; wherein the specific antibody can generate electric signal characteristic change under the bias voltage after reacting and combining with the target heavy metal. Compared with the prior art, the prepared nanopore sensor is low in cost and shows high selectivity and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of heavy metal monitoring, and more specifically, to a preparation method of a nanopore sensor based on specific antibodies and application thereof. Background Art

[0002] With the acceleration of industrialization, heavy metal pollution in mining areas and water bodies has become increasingly serious. Heavy metals such as lead (Pb), cadmium (Cd), copper (Cu) and zinc (Zn) have long existed in the environment and caused great harm to the ecosystem and human health, becoming a major environmental pollution problem that needs to be solved urgently. Traditional heavy metal detection methods such as atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS) have high sensitivity, but they have problems such as complex instruments, cumbersome operations, long time and high costs, and cannot achieve rapid on-site detection.

[0003] In recent years, sensors based on nanotechnology have shown great potential in environmental monitoring. In particular, solid-state nanopore sensors have gradually become an important tool for heavy metal pollutant detection due to their high sensitivity, label-free detection, and real-time analysis. Si3N4 nanopores have good stability and applicability and are currently one of the materials widely used in biological and chemical sensing. Si3N4 nanopore arrays prepared by electron beam lithography (HIM) can provide accurate channels for molecules and can effectively monitor the penetration behavior of heavy metal ions. However, the current Si3N4 nanopore sensors are easily affected by non-target metal ions when detecting heavy metal pollutants, and have poor selectivity and sensitivity to target metal ions. Summary of the invention

[0004] In order to overcome the defects of poor selectivity and sensitivity described in the above-mentioned prior art, the present invention provides a preparation method of a nanopore sensor based on specific antibodies and its application.

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0006] In a first aspect, a method for preparing a nanopore sensor based on a specific antibody comprises:

[0007] Based on the electron beam exposure micro-nano processing technology, Si3N4 nanopores were prepared on the Si3N4 film substrate to obtain Si3N4 nanopore arrays;

[0008] The Si3N4 nanopore array is surface functionalized to fix specific antibodies on the nanopore surface; wherein the specific antibodies can react and bind with the target heavy metal to generate characteristic changes in electrical signals under a bias voltage.

[0009] In a second aspect, a nanopore sensor based on specific antibodies comprises a Si3N4 nanopore array, wherein the nanopore surface of the Si3N4 nanopore array has specific antibodies fixed thereon;

[0010] The specific antibody is used to react and bind with the target heavy metal to generate characteristic changes in the electrical signal under a bias voltage.

[0011] In a third aspect, a method for detecting heavy metal pollutants comprises:

[0012] Fixing the nanopore sensor as described in the second aspect in a fluid pool whose cross section is adapted to the size of the nanopore sensor, so that the nanopore on the nanopore sensor becomes the only channel in the fluid pool, and the fluid pool is divided into two independent fluid pools by the nanopore sensor;

[0013] Injecting electrolyte solution into the two independent fluid pools, and placing the entire fluid pool and the nanopore sensor in a Faraday shielding box;

[0014] Inserting Ag / AgCl electrodes into the electrolyte solution, applying voltage across the nanopore sensor and recording a reference current based on a patch clamp amplifier;

[0015] The electrolyte solution at the cis end of the fluid cell is replaced with the sample solution to be tested, a bias voltage is applied and a time-current diagram is recorded;

[0016] According to the time-current diagram and the reference current, it is determined whether molecular translocation occurs, and the qualitative / quantitative detection result of the target heavy metal in the sample to be detected is determined according to the molecular translocation result.

[0017] In a fourth aspect, a heavy metal pollutant detection device comprises:

[0018] A Faraday shielding box, in which a fluid pool and the nanopore sensor as claimed in claim 8 are placed, the nanopore sensor is fixed between the fluid pools, and the nanopore on the nanopore sensor becomes the only channel in the fluid pool, and the fluid pool is divided into two independent fluid pools by the nanopore sensor; wherein the fluid pool is also used to contain an electrolyte solution and / or a sample solution to be detected;

[0019] A power source for applying an electrical signal to both sides of the nanopore sensor;

[0020] The data processing module is used to collect the electrical signal of the nanopore sensor through a patch clamp amplifier to determine the reference current and the time-current diagram; it is also used to determine whether molecular translocation occurs based on the time-current diagram and the reference current, and determine the qualitative / quantitative detection results of the target heavy metal in the sample solution to be detected based on the molecular translocation results.

[0021] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0022] The present application discloses a method for preparing a nanopore sensor based on specific antibodies and its application. Si3N4 nanopores are prepared by electron beam exposure micro-nano processing technology, which has low cost and high operability both in laboratory scale and large-scale production; the selectivity of the nanopore is optimized by specific antibody modification, and the sensitivity of the nanopore sensor for heavy metal detection is improved. In addition, the prepared nanopore sensor has high mechanical strength, chemical stability and good electrical properties, can work stably for a long time in a complex environment, and has good repeatability. It can be applied to the detection of heavy metal pollutants in mining areas, other environmental heavy metal pollutants, and even food safety detection and biomedical fields, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the process of preparing the nanopore sensor in Example 1 of the present application;

[0024] Figure 2 This is a schematic diagram of imaging of a nanopore prepared based on HIM in Example 1 of the present application;

[0025] Figure 3 This is a schematic diagram of the preparation process of the Si3N4 thin film substrate in Example 1 of the present application;

[0026] Figure 4 This is a schematic diagram of the process of surface functionalization in Example 1 of the present application;

[0027] Figure 5 This is a schematic diagram of the process of the method for detecting heavy metal pollutants in Example 2 of the present application;

[0028] Figure 6 This is a schematic diagram of the sample determination principle in Example 2 of the present application;

[0029] Figure 7 This is a schematic diagram of the standard concentration current diagram of four heavy metals in Example 2 of the present application;

[0030] Figure 8 This is a schematic diagram of the structure of the heavy metal pollutant detection device in Example 2 of the present application. DETAILED DESCRIPTION

[0031] The terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the terms used in this way can be interchanged in appropriate cases, which is only to describe the distinction mode adopted by the objects of the same attributes in the embodiments of the present application when describing. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, so that the process, method, system, product or equipment containing a series of units need not be limited to those units, but may include other units that are not clearly listed or inherent to these processes, methods, products or equipment. The term "determine" widely covers various actions, and may include acquisition, calculation, calculation, processing, derivation, investigation, search (for example, search in a table, database or other data structure), ascertainment, and similar actions, and may also include reception (for example, receiving information), access (for example, accessing data in a memory) and similar actions, and may also include generation, creation, establishment and similar actions, as well as parsing, selection, selection and similar actions, etc. The relevant definitions of other terms will be given in the following description.

[0032] It should be noted that when an element is considered to be "connected" to another element, it can be directly connected to the other element, or connected to the other element through an intermediate element. In addition, the "connection" in the following embodiments should be understood as "electrical connection", "communication connection", etc. if there is transmission of electrical signals or data between the connected objects.

[0033] The drawings are for illustrative purposes only and should not be construed as limiting the present patent;

[0034] In order to better illustrate the present embodiment, some parts in the drawings may be omitted, enlarged or reduced, and do not represent the size of the actual product;

[0035] It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0036] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0037] Example 1

[0038] This embodiment provides a method for preparing a nanopore sensor based on a specific antibody, see Figure 1 ,include:

[0039] Based on the electron beam exposure micro-nano processing technology, Si3N4 nanopores were prepared on the Si3N4 film substrate to obtain Si3N4 nanopore arrays;

[0040] The Si3N4 nanopore array is surface functionalized to fix specific antibodies on the nanopore surface; wherein the specific antibodies can react and bind with the target heavy metal to generate characteristic changes in electrical signals under a bias voltage.

[0041] It should be noted that the use of Si3N4 material as the nanopore substrate has high mechanical strength, chemical stability and good electrical properties, and can work stably for a long time in complex environments. In addition, the antibody-modified nanopore sensor has good repeatability, ensuring consistency in multiple tests.

[0042] It should also be noted that the preparation process uses simple and mature micro-nano processing technology, which has low cost and high operability both in laboratory scale and large-scale production. At the same time, the use of surface functionalization technology simplifies the assembly process of the sensor and improves work efficiency.

[0043] It should be emphasized that antibody modification can enhance the sensor's ability to recognize heavy metals such as lead, cadmium, arsenic, copper and zinc by specifically binding to the target heavy metals. In this modified nanopore sensor, heavy metal ions cause current changes when passing through the nanopore, thus providing direct electrical signal feedback for detection. The selectivity of the nanopore is optimized through specific antibody modification, and combined with the current signal analysis method, efficient, accurate and real-time detection of various heavy metal pollutants in the environment can be achieved.

[0044] It should also be emphasized that the nanopore sensor described in this embodiment can complete the detection of heavy metal pollutants in a short time, and can achieve real-time monitoring during the entire detection process. Compared with the disadvantages of traditional methods that require a long time for sample pretreatment or complex analysis processes, the detection of heavy metals (such as lead, cadmium, copper, and zinc) based on the nanopore sensor has obvious speed advantages and is suitable for real-time monitoring and rapid on-site detection.

[0045] Those skilled in the art should understand that heavy metals can be regarded as special antigens, and the specific antibodies are antibodies that can specifically recognize target heavy metals, including but not limited to monoclonal antibodies against heavy metal ions of lead, cadmium, copper, zinc, and arsenic.

[0046] More specifically, as non-limiting examples, the specific antibodies include but are not limited to heavy metal cadmium monoclonal antibody CD Ab, Anti-ZN462 Antibody, Anti-CU070 (C-TERM) antibody produced in rabbit antibody, Mouse polyclonal to Pb12 antibody.

[0047] In some preferred embodiments, the step of preparing Si3N4 nanopores on a Si3N4 thin film substrate comprises:

[0048] The Si3N4 thin film substrate was etched using a helium ion microscope to obtain nanopores with a size within a preset diameter range. The acceleration voltage during the etching process was 20 kV to 30 kV and the helium working pressure was 1.5×10 -10 Torr to 2.0×10 -10 Torr, and / or a base vacuum pressure of 3.5×10 -10 Torr to 4.5×10 -10 Torr's conditions.

[0049] It should be noted that precise processing of the Si3N4 substrate using a helium ion microscope (HIM) can achieve precise control of the nanopore diameter, thereby adjusting the sensitivity of the sensor and adapting to heavy metal pollutants in different concentration ranges, ensuring stability, high sensitivity and reliability during the detection process.

[0050] In some specific implementations, a helium ion microscope (HIM) is used to etch the Si3N4 thin film substrate to obtain nanopores with a diameter of 250 nm to 350 nm.

[0051] In some optional embodiments, before preparing Si3N4 nanopores on the Si3N4 thin film substrate, the Si3N4 thin film substrate is pretreated, including:

[0052] In a first water bath environment, the Si3N4 thin film substrate is placed in a highly corrosive oxidant solution for a first preset time, and then the residual solution on the Si3N4 thin film substrate is removed by deionized water, and the substrate is placed in an oven for drying for a second preset time;

[0053] Alternatively, after soaking the Si3N4 film substrate in acetone for a third preset time, the Si3N4 film substrate is sequentially soaked in anhydrous ethanol for a fourth preset time and in deionized water for a fifth preset time, and then placed in an oven for drying for a second preset time.

[0054] In some specific implementation processes, a piranha solution (V concentrated sulfuric acid: V hydrogen peroxide = 3:1) is prepared as the highly corrosive oxidant solution, and the solution is cleaned in a 90°C ± 5°C water bath (i.e., the first water bath environment) for 20 min to 30 min (i.e., the first preset time). The residual solution on the Si3N4 film substrate is removed with deionized water, and the solution is dried in an oven at 100°C ± 10°C for 20 min to 30 min (i.e., the second preset time).

[0055] In other specific implementation processes, the Si3N4 thin film substrate is immersed in acetone for 3h to 5h (i.e., the third preset time), and then immersed in anhydrous ethanol and deionized water for 5min to 15min respectively (i.e., the fourth preset time and the fifth preset time), and finally dried in an oven at 100℃±10℃ for 20min to 30min (i.e., the second preset time).

[0056] In some optional embodiments, when a Si3N4 thin film substrate is etched using a helium ion microscope, the processed pattern is a circle corresponding to the preset diameter range, the ion beam current is 10pA to 20pA, and the ion dose is 60nC / μm 2 Up to 100nC / μm 2 , dwell time of 800μs to 1200μs, and / or pixel size of 8nm 2 To 12nm 2 .

[0057] In some specific implementations, the helium ion microscope is focused and the Si3N4 film substrate is moved to the center of the field of view, and the processing area and processing parameters are set. The processing pattern is a circle with a diameter of 250nm to 350nm, the ion beam current is set to 10pA to 20pA, and the ion dose is 60nC / μm 2 Up to 100nC / μm 2 , dwell time 800μs to 1200μs, pixel size 8nm 2 Up to 12nm 2 , and finally form Figure 2 The nanopore is shown.

[0058] In some preferred embodiments, the preparation process of the Si3N4 thin film substrate includes: Figure 3 :

[0059] Using an n-type medium-resistance silicon wafer with a first thickness, double-sided polishing, and a crystal phase plane of (100) as a substrate, depositing a low-stress silicon nitride film with a second thickness on the upper and lower surfaces of the substrate respectively by an LPCVD (low pressure chemical vapor deposition) method;

[0060] Coating photoresist on one side of the silicon nitride film, which is referred to as the back side of the chip, and forming a square photoresist window by using photolithography technology;

[0061] Using CHF3, SiF6 and He as etching gases, using reactive ion etching (RIE) technology to remove the silicon nitride under the protection of the photoresist, expose the silicon substrate, and remove the photoresist;

[0062] Using a wet etching process, using a 30% potassium hydroxide solution, heating in a second water bath environment for a fifth preset time, etching the silicon substrate along the (111) crystal plane until another layer of silicon nitride is etched to obtain a square silicon nitride film of a preset size;

[0063] Based on the laser scribing technology, at least one Si3N4 film substrate is cut on the square silicon nitride film.

[0064] In some specific implementations, SiH 2 Cl 2 and NH 3 are used as reactive gases during the low pressure chemical vapor deposition process.

[0065] In some specific implementation processes, an n-type medium-resistance silicon wafer with a thickness of 200 μm±10 μm (i.e., the first thickness), double-sided polishing, and a crystal phase plane of (100) is selected as a substrate; a low-stress silicon nitride film with a thickness of 100 nm±10 nm (i.e., the second thickness) is deposited on the upper and lower surfaces of the silicon wafer by low-pressure chemical vapor deposition (LPCVD) using SiH2Cl2 and NH3 as reaction gases; a photoresist is coated on one side of the silicon nitride film, which side is defined as the back side of the chip, and a square photoresist window is formed by photolithography technology, and the window size is 10 μm×10 μm to 50 μm×50 μm; Reactive ion etching (RIE) technology is used with CHF3, SiF6 and He as etching gases to remove the silicon nitride under the protection of the photoresist, expose the silicon substrate, and remove the photoresist; a wet etching process is adopted, using a 30% concentration of potassium hydroxide solution, heated in a water bath at 80℃±5℃ for 5h to 10h, and the silicon substrate is etched along the (111) crystal plane until another layer of silicon nitride is etched to obtain a square silicon nitride film with a size of 5μm×5μm±0.5μm; finally, laser scribing technology is used to obtain a single Si3N4 thin film substrate with a unit of 2.4mm×2.4mm±0.1mm.

[0066] In some preferred embodiments, the Si3N4 nanopore array is surface functionalized, see Figure 4 ,include:

[0067] After cleaning the Si3N4 nanopore array, subjecting the Si3N4 nanopore array to oxygen plasma treatment within a first preset power interval to introduce oxygen-containing functional groups; wherein the oxygen-containing functional groups include carboxyl groups;

[0068] The carboxyl groups on the surface of the Si3N4 nanopore array are activated using a CDI (1-chloro-3-chloromethyl-1,3,5-triazine) solution, and the specific antibody is bound to the surface of the Si3N4 nanopore array through an EDC / NHS cross-linking reaction to form a sensing interface with high specificity.

[0069] In some optional embodiments, the step of activating the carboxyl groups on the surface of the Si3N4 nanopore array using a CDI solution and covalently binding the specific antibody to the surface of the Si3N4 nanopore array by an EDC / NHS cross-linking reaction comprises:

[0070] Prepare a 0.1M to 1M CDI solution, place the Si3N4 nanopore array in the CDI solution, and react at room temperature for a sixth preset time to activate the carboxyl groups on the surface of the Si3N4 nanopore array;

[0071] The activated carboxyl groups are converted into active esters through the EDC / NHS cross-linking reaction for a seventh preset time; wherein the solution ratio is 1 mM EDC and 1 mM NHS;

[0072] After dissolving the specific antibody in a buffer solution at a specified concentration range, adding the buffer solution containing the specific antibody to the surface-activated Si3N4 nanopore array, and performing a reaction for an eighth preset time to covalently bind the specific antibody to the surface of the Si3N4 nanopore array;

[0073] After the reaction is completed, PBS solution is used for washing to remove the unbound specific antibody to obtain the functionalized nanopore sensor.

[0074] In some specific implementations, the Si3N4 nanopore array is first cleaned in deionized water and soaked in acetone and ethanol for 5 to 15 minutes to remove surface organic matter and pollutants. Then, the cleaned Si3N4 nanopore array is placed in an oxygen plasma treatment device and subjected to plasma treatment at a power of 20W to 40W for 10 to 20 minutes to introduce oxygen-containing functional groups such as carboxyl (-COOH) and hydroxyl (-OH). Next, 1-chloro-3-chloromethyl-1,3,5-triazine (CDI) is used to activate the surface carboxyl groups, prepare a 0.1M to 1M CDI solution, and react at room temperature for 1h to 2h. Furthermore, the activated carboxyl groups are converted into active esters by an EDC / NHS cross-linking reaction, with a solution ratio of 1mM EDC and 1mM NHS, and a reaction time of 30min to 1h. Finally, the specific antibody (such as an antibody against a target molecule) is dissolved in an appropriate buffer at a concentration of 0.1 mg / mL to 1 mg / mL, and added to the surface-activated Si3N4 nanopore array for a reaction of 4 to 12 hours to complete the covalent binding of the antibody. After the reaction is completed, the unbound antibody is removed by washing with a PBS solution to obtain a functionalized Si3N4 nanopore array surface.

[0075] Example 2

[0076] This embodiment provides a nanopore sensor based on specific antibodies, including a Si3N4 nanopore array, wherein specific antibodies are fixed on the nanopore surface of the Si3N4 nanopore array;

[0077] The specific antibody is used to react and bind with the target heavy metal to generate characteristic changes in the electrical signal under a bias voltage.

[0078] This embodiment also provides a method for detecting heavy metal pollutants. Figure 5 ,include:

[0079] Fixing the aforementioned nanopore sensor in a fluid pool whose cross section is adapted to the size of the nanopore sensor, so that the nanopore on the nanopore sensor becomes the only channel in the fluid pool, and the fluid pool is divided into two independent fluid pools by the nanopore sensor;

[0080] Injecting electrolyte solution into the two independent fluid pools, and placing the entire fluid pool and the nanopore sensor in a Faraday shielding box;

[0081] Inserting Ag / AgCl electrodes into the electrolyte solution, applying voltage across the nanopore sensor and recording a reference current based on a patch clamp amplifier;

[0082] The electrolyte solution at the cis end of the fluid cell is replaced with the sample solution to be tested, a bias voltage is applied and a time-current diagram is recorded;

[0083] According to the time-current diagram and the reference current, it is determined whether molecular translocation occurs, and the qualitative / quantitative detection result of the target heavy metal in the sample to be detected is determined according to the molecular translocation result.

[0084] It should be noted that the use of solid-state nanopore sensors for label-free detection reduces the reliance on markers or reagents compared to traditional immunoassays or chromatography, and avoids potential errors and high costs in the labeling process. In addition, nanopore arrays can achieve multi-channel high-throughput detection, further improving experimental efficiency.

[0085] In some specific implementations, the Si3N4 nanopore chip and the silicone gasket are clamped between the organic glass liquid pool (i.e., the fluid pool) and fixed with bolts to ensure that the nanopore becomes the only channel, and the organic glass liquid pool is divided into two independent fluid pools (usually referred to as the cis end and the trans end). Next, use a 100μL pipette to wet both sides of the liquid pool with anhydrous ethanol and clean with deionized water, and then inject potassium chloride solution. Place the entire device in a Faraday shielding box, insert the Ag / AgCl electrode into the electrolyte solution, and cover the shielding box to isolate external interference. Use a patch clamp amplifier and a data acquisition system to record the reference current, set the sampling frequency to 250kHz, and use a low-pass Bessel filter set to 10kHz to ensure that the signal is stable and has a high signal-to-noise ratio.

[0086] In some specific implementations, a voltage ranging from 100 mV to 150 mV is applied across the Si3N4 solid-state nanopore to drive heavy metal ions (such as Pb 2+ 、Cd 2+ , Cu 2+ 、Zn 2+ In this process, the presence of heavy metal ions or their complexes will have a blocking effect on the nanopore current, resulting in a change in the current amplitude (i.e., the blocking current amplitude ΔI), see Figure 6 .

[0087] Those skilled in the art will appreciate that, based on the time-current diagram (reflecting the trend of the current amplitude over time, which may be a curve diagram or a scatter diagram) and the reference current, an ion current diagram (reflecting the trend of the ion current amplitude over time after removing the influence of the reference current) can be determined. Based on the ion current diagram, by real-time monitoring and recording the current change (ΔI) and the passing time (Δt) when passing through the nanopore, it can be determined whether molecular translocation occurs.

[0088] As a non-limiting example, the time-current diagram is drawn using Clampfit software, and the parameters such as the current amplitude and the through-hole time of the signal are counted. Then, data screening and distribution map drawing are performed by Excel and Origin. The screening criteria are: the current amplitude is greater than 3 times the through-hole current noise (based on RMS calculation), and the signal-to-noise ratio is greater than 3. After meeting this condition, the reference current and the through-hole signal can be effectively distinguished to ensure the accuracy of the signal, thereby identifying whether the molecule has been translocated.

[0089] It should be noted that, according to the ion current diagram obtained by fitting, the concentration of each heavy metal ion is further calculated, and the concentration of each heavy metal pollutant in the sample to be tested can be determined by comparing the test results obtained with the standard concentration range. Exemplarily, a heavy metal solution with a concentration range of 0.5mol / L to 1.5mol / L is prepared as a standard solution, added to the fluid cell, and fully contacted with the electrodes on both sides of the fluid cell. The standard curve of known concentration is fitted by detecting the ion current diagram under the standard solution, and compared with the ion current diagram when the sample is actually detected, the concentration of each heavy metal pollutant in the sample to be detected can be determined. This method has high sensitivity and can distinguish different heavy metal ions, and is suitable for efficient and rapid analysis of environmental monitoring and pollutant detection.

[0090] It should be understood that the concentration of the standard solution can be adjusted by those skilled in the art through experiments to ensure uniform distribution of the heavy metal solution.

[0091] In some specific implementations, a Gaussian fitting method is used to obtain the current curve.

[0092] In some specific implementations, the standard solution is a solution of lead (Pb), cadmium (Cd), arsenic (As), copper (Cu), or zinc (Zn) at a concentration of 1 mmol / L, and the corresponding ion current diagram at the standard concentration (referred to as "standard concentration current diagram") is as follows: Figure 7 shown.

[0093] This embodiment also provides a heavy metal pollutant detection device, see Figure 8 ,include:

[0094] Faraday shielding box( Figure 8 d), a fluid pool and the nanopore sensor as claimed in claim 8 are placed in the Faraday shielding box, and the nanopore sensor is fixed in the fluid pool ( Figure 8 e), and the nanopore on the nanopore sensor becomes the only channel in the fluid pool, and the fluid pool is divided into two independent fluid pools by the nanopore sensor; wherein the fluid pool is also used to contain an electrolyte solution and / or a sample solution to be detected;

[0095] power supply( Figure 8 b), used to apply electrical signals to both sides of the nanopore sensor;

[0096] Data processing module ( Figure 8 a), used for patch clamp amplifier ( Figure 8c) collecting the electrical signal of the nanopore sensor to determine the reference current and the time-current diagram; and also used to determine whether molecular translocation occurs based on the time-current diagram and the reference current, and determine the qualitative / quantitative detection results of the target heavy metal in the sample solution to be detected based on the molecular translocation results.

[0097] It can be understood that the device of this embodiment corresponds to the method of the aforementioned embodiment, and the options in the aforementioned embodiment are also applicable to this embodiment, so they will not be described repeatedly here.

[0098] The same or similar reference numerals correspond to the same or similar components;

[0099] The terms used to describe the positional relationship in the drawings are only used for illustrative purposes and should not be construed as limiting the present application;

[0100] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application may be combined with each other.

[0101] In different specific implementations, the method or system described in the present application can be implemented in software, hardware or a combination thereof. In addition, the order of the steps of the method can be changed, and various elements can be added, reordered, combined, omitted, modified, etc.

[0102] Obviously, the above-mentioned embodiments of the present application are merely examples for clearly illustrating the present application, and are not intended to limit the implementation methods of the present application, and are not intended to limit the present application. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. Each discrete structure / function module or unit can be integrated together to form an independent part, or each module can exist separately, or two or more modules can be integrated to form an independent part, and the structure and function of the discrete components can be implemented as a combined structure or component. It is not necessary and impossible to enumerate all the implementation methods here. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the scope of protection of the claims of the present application.

Claims

1. A method for preparing a nanopore sensor based on specific antibodies, characterized in that: include: Based on the electron beam exposure micro-nano processing technology, Si3N4 nanopores were prepared on the Si3N4 film substrate to obtain Si3N4 nanopore arrays; The Si3N4 nanopore array is surface functionalized to fix specific antibodies on the nanopore surface; wherein the specific antibodies can react and bind with the target heavy metal to generate characteristic changes in electrical signals under a bias voltage.

2. The method for preparing a nanopore sensor based on a specific antibody according to claim 1, characterized in that: The method of preparing Si3N4 nanopores on a Si3N4 thin film substrate comprises: The Si3N4 thin film substrate was etched using a helium ion microscope to obtain nanopores with a size within a preset diameter range. The acceleration voltage during the etching process was 20 kV to 30 kV and the helium working pressure was 1.5×10 -10 Torr to 2.0×10 -10 Torr, and / or a base vacuum pressure of 3.5×10 -10 Torr to 4.5×10 -10 Torr's conditions.

3. The method for preparing a nanopore sensor based on specific antibodies according to claim 2, characterized in that: Before preparing Si3N4 nanopores on a Si3N4 thin film substrate, the Si3N4 thin film substrate is pretreated, including: In a first water bath environment, the Si3N4 thin film substrate is placed in a highly corrosive oxidant solution for a first preset time, and then the residual solution on the Si3N4 thin film substrate is removed by deionized water, and the substrate is placed in an oven for drying for a second preset time; Alternatively, after soaking the Si3N4 film substrate in acetone for a third preset time, the Si3N4 film substrate is sequentially soaked in anhydrous ethanol for a fourth preset time and in deionized water for a fifth preset time, and then placed in an oven for drying for a second preset time.

4. The method for preparing a nanopore sensor based on a specific antibody according to claim 2, characterized in that: When etching the Si3N4 thin film substrate using a helium ion microscope, the processed pattern is a circle corresponding to the preset diameter range, the ion beam current is 10pA to 20pA, and the ion dose is 60nC / μm 2 Up to 100nC / μm 2 , dwell time of 800μs to 1200μs, and / or pixel size of 8nm 2 Up to 12nm 2 .

5. The method for preparing a nanopore sensor based on a specific antibody according to claim 1, characterized in that: The preparation process of the Si3N4 thin film substrate includes: Using an n-type medium-resistance silicon wafer with a first thickness, double-sided polishing, and a crystal phase plane of (100) as a substrate, and depositing a low-stress silicon nitride film with a second thickness on the upper and lower surfaces of the substrate respectively by an LPCVD method; Coating photoresist on one side of the silicon nitride film, which is referred to as the back side of the chip, and forming a square photoresist window by using photolithography technology; Using CHF3, SiF6 and He as etching gases, a reactive ion etching technique is used to remove the silicon nitride under the protection of the photoresist, expose the silicon substrate, and remove the photoresist; Using a wet etching process, using a 30% potassium hydroxide solution, heating in a second water bath environment for a fifth preset time, etching the silicon substrate along the (111) crystal plane until another layer of silicon nitride is etched to obtain a square silicon nitride film of a preset size; Based on the laser scribing technology, at least one Si3N4 film substrate is cut on the square silicon nitride film.

6. A method for preparing a nanopore sensor based on specific antibodies according to any one of claims 1 to 5, characterized in that: The Si3N4 nanopore array is surface functionalized, comprising: After cleaning the Si3N4 nanopore array, subjecting the Si3N4 nanopore array to oxygen plasma treatment within a first preset power interval to introduce oxygen-containing functional groups; wherein the oxygen-containing functional groups include carboxyl groups; The carboxyl groups on the surface of the Si3N4 nanopore array are activated using a CDI solution, and the specific antibody is bound to the surface of the Si3N4 nanopore array through an EDC / NHS cross-linking reaction to form a sensing interface with high specificity.

7. The method for preparing a nanopore sensor based on a specific antibody according to claim 6, characterized in that: The method of using CDI solution to activate the carboxyl groups on the surface of the Si3N4 nanopore array and covalently binding the specific antibody to the surface of the Si3N4 nanopore array through an EDC / NHS cross-linking reaction comprises: Prepare a 0.1M to 1M CDI solution, place the Si3N4 nanopore array in the CDI solution, and react at room temperature for a sixth preset time to activate the carboxyl groups on the surface of the Si3N4 nanopore array; The activated carboxyl groups are converted into active esters through the EDC / NHS cross-linking reaction for a seventh preset time; wherein the solution ratio is 1 mM EDC and 1 mM NHS; After dissolving the specific antibody in a buffer solution at a specified concentration range, adding the buffer solution containing the specific antibody to the surface-activated Si3N4 nanopore array, and performing a reaction for an eighth preset time to covalently bind the specific antibody to the surface of the Si3N4 nanopore array; After the reaction is completed, PBS solution is used for washing to remove the unbound specific antibody to obtain the functionalized nanopore sensor.

8. A nanopore sensor based on specific antibodies, characterized in that: It comprises a Si3N4 nanopore array, wherein specific antibodies are fixed on the nanopore surface of the Si3N4 nanopore array; The specific antibody is used to generate characteristic changes of electrical signals under a bias voltage after binding with the target heavy metal.

9. A method for detecting heavy metal pollutants, characterized in that: include: Fixing the nanopore sensor as claimed in claim 8 in a fluid pool whose cross section is adapted to the size of the nanopore sensor, so that the nanopore on the nanopore sensor becomes the only channel in the fluid pool, and the fluid pool is divided into two independent fluid pools by the nanopore sensor; Injecting electrolyte solution into the two independent fluid pools, and placing the entire fluid pool and the nanopore sensor in a Faraday shielding box; Inserting Ag / AgCl electrodes into the electrolyte solution, applying voltage across the nanopore sensor and recording a reference current based on a patch clamp amplifier; Replacing the electrolyte solution at the cis end of the fluid pool with the sample solution to be tested, applying a bias voltage and recording a time-current diagram based on the electrical signal of the nanopore sensor; According to the time-current diagram and the reference current, it is determined whether molecular translocation occurs, and according to the molecular translocation result, a qualitative / quantitative detection result of the target heavy metal in the sample solution to be detected is determined.

10. A heavy metal pollutant detection device, characterized in that: include: A Faraday shielding box, in which a fluid pool and the nanopore sensor as claimed in claim 8 are placed, the nanopore sensor is fixed between the fluid pools, and the nanopore on the nanopore sensor becomes the only channel in the fluid pool, and the fluid pool is divided into two independent fluid pools by the nanopore sensor; wherein the fluid pool is also used to contain an electrolyte solution and / or a sample solution to be detected; A power source for applying an electrical signal to both sides of the nanopore sensor; The data processing module is used to collect the electrical signal of the nanopore sensor through a patch clamp amplifier to determine the reference current and the time-current diagram; it is also used to determine whether molecular translocation occurs based on the time-current diagram and the reference current, and determine the qualitative / quantitative detection results of the target heavy metal in the sample solution to be detected based on the molecular translocation results.

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