Preparation method and application of a nanopore sensor based on specific antibodies

Abstract

The application discloses a preparation method of a nanopore sensor based on specific antibodies and application thereof, and relates to the field of heavy metal monitoring.The method comprises the following steps: based on an electron beam exposure micro-nano processing technology, Si3N4 nanopores are prepared on a Si3N4 thin film substrate to obtain a Si3N4 nanopore array; and surface functionalization is performed on the Si3N4 nanopore array to fix specific antibodies on the nanopore surface; wherein the specific antibodies can be combined with target heavy metals to produce electric signal characteristic changes under a bias voltage.Compared with the prior art, the nanopore sensor prepared by the application has low cost and shows high selectivity and high sensitivity.

Description

Technical Field

[0001] This invention relates to the field of heavy metal monitoring technology, and more specifically, to a method for preparing a nanopore sensor based on specific antibodies and its application. Background Technology

[0002] Traditional heavy metal detection methods, such as atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS), have high sensitivity, but they suffer from problems such as complex instruments, cumbersome operation, long time and high cost, making it impossible to achieve rapid on-site detection.

[0003] In recent years, nanotechnology-based sensors have demonstrated great potential in environmental monitoring, especially solid-state nanopore sensors, which have gradually become an important tool for detecting heavy metal pollutants due to their advantages such as high sensitivity, label-free detection, and real-time analysis. Si3N4 nanopores possess good stability and applicability, making them one of the materials widely used in biological and chemical sensing. Si3N4 nanopore arrays fabricated using high-intensity electron beam lithography (HIM) can provide accurate channels for molecules, effectively monitoring the penetration behavior of heavy metal ions. However, current Si3N4 nanopore sensors are susceptible to the influence of non-target metal ions when detecting heavy metal pollutants, exhibiting poor selectivity and sensitivity towards target metal ions. Summary of the Invention

[0004] To overcome the shortcomings of poor selectivity and sensitivity in the prior art, this invention provides a method for preparing a nanopore sensor based on a specific antibody and its application.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] Firstly, a method for preparing a nanopore sensor based on a specific antibody includes:

[0007] Based on electron beam micro-nano fabrication technology, Si3N4 nanopores were fabricated on Si3N4 thin film substrates to obtain Si3N4 nanopore arrays.

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

[0009] In a second aspect, a nanopore sensor based on specific antibodies includes a Si3N4 nanopore array, wherein specific antibodies are immobilized on the nanopore surface of the Si3N4 nanopore array.

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

[0011] Thirdly, a method for detecting heavy metal pollutants, comprising:

[0012] The nanopore sensor as described in the second aspect is fixed in a fluid pool with a cross-section adapted to the size of the nanopore sensor, such that the nanopores on the nanopore sensor become the only channels in the fluid pool, and the fluid pool is divided into two independent fluid pools by the nanopore sensor.

[0013] Electrolyte solution is injected into the two independent fluid pools, and the entire fluid pool and the nanopore sensor are placed in a Faraday shielded box;

[0014] An Ag / AgCl electrode was inserted into the electrolyte solution, a voltage was applied across the nanopore sensor, and a reference current was recorded based on a patch-clamp amplifier.

[0015] Replace the electrolyte solution at the CIS end in the fluid cell with the sample solution to be tested, apply a bias voltage and record the time-current graph;

[0016] Based on the time-current graph and the reference current, it is determined whether molecular translocation has occurred, and the qualitative / quantitative detection results of the target heavy metal in the sample to be tested are determined based on the molecular translocation results.

[0017] Fourthly, a detection device for heavy metal pollutants, comprising:

[0018] A Faraday shielded box, wherein a fluid pool and a nanopore sensor as described in claim 8 are placed in the Faraday shielded box, the nanopore sensor is fixed between the fluid pools, and the nanopores on the nanopore sensor become the only channels in the fluid pools, the fluid pools are divided into two independent fluid pools by the nanopore sensor; wherein the fluid pools are also used to contain electrolyte solution and / or sample solution to be tested;

[0019] A power source is provided to apply electrical signals to both sides of the nanopore sensor.

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

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

[0022] This application discloses a method for preparing a nanopore sensor based on specific antibodies and its application. Si3N4 nanopores are prepared using electron beam micro-nano fabrication technology, which offers low cost and high operability in both laboratory and large-scale production. Specific antibody modification optimizes the selectivity of the nanopores and improves the sensitivity of the nanopore sensor for heavy metal detection. Furthermore, the prepared nanopore sensor exhibits high mechanical strength, chemical stability, and good electrical performance, enabling long-term stable operation in complex environments with good repeatability. It has broad application prospects in the detection of heavy metal pollutants in mining areas, other environmental heavy metal pollutants, food safety testing, and biomedical fields. Attached Figure Description

[0023] Figure 1 This is a schematic flowchart of the fabrication method of the nanopore sensor in Embodiment 1 of this application;

[0024] Figure 2 This is an imaging schematic diagram of the nanopores fabricated based on HIM in Example 1 of this application;

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

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

[0027] Figure 5 This is a schematic flowchart of the method for detecting heavy metal pollutants in Embodiment 2 of this application;

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

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

[0030] Figure 8 This is a schematic diagram of the heavy metal pollutant detection device in Embodiment 2 of this application. Detailed Implementation

[0031] The terms "first," "second," etc., used in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such terms are interchangeable where appropriate; this is merely a way of distinguishing objects with the same attributes in the description of embodiments of this application. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion, so that a process, method, system, product, or apparatus that comprises a series of units is not necessarily limited to those units, but may include other units not explicitly listed or inherent to those processes, methods, products, or apparatuses. The term "determine" broadly covers a wide variety of actions, including acquiring, calculating, processing, deriving, investigating, searching (e.g., searching in a table, database, or other data structure), probing, and similar actions; it may also include receiving (e.g., receiving information), accessing (e.g., accessing data in memory), and similar actions; it may also include generating, creating, establishing, and similar actions; and parsing, selecting, choosing, and similar actions, etc. Definitions of other terms will be given in the following description.

[0032] It should be noted that when one 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 intermediary element. Furthermore, in the following embodiments, "connection" should be understood as "electrical connection," "communication connection," etc., if there is transmission of electrical signals or data between the connected objects.

[0033] The accompanying drawings are for illustrative purposes only and should not be construed as limiting the scope of this patent.

[0034] To better illustrate this embodiment, some parts in the accompanying drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions;

[0035] It will be understood by those skilled in the art that certain well-known structures and their descriptions may be omitted in the accompanying drawings.

[0036] The technical solution of the present invention will be further described below with reference to 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 attached document.) Figure 1 ,include:

[0039] Based on electron beam micro-nano fabrication technology, Si3N4 nanopores were fabricated on Si3N4 thin film substrates to obtain Si3N4 nanopore arrays.

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

[0041] It should be noted that using Si3N4 material as the nanoporous substrate provides high mechanical strength, chemical stability, and good electrical properties, enabling it to operate stably for extended periods in complex environments. Furthermore, the antibody-modified nanoporous sensor exhibits excellent repeatability, ensuring consistency across multiple detections.

[0042] It should also be noted that the fabrication process employs simple and mature micro-nano fabrication techniques, which offer low cost and high operability in both laboratory and large-scale production. Furthermore, surface functionalization technology simplifies the sensor assembly process and improves operational efficiency.

[0043] It is important to emphasize that antibody modification can enhance the sensor's ability to identify heavy metals such as lead, cadmium, arsenic, copper, and zinc by specifically binding to target heavy metals. In this modified nanopore sensor, heavy metal ions induce current changes as they pass through the nanopores, thus providing direct electrical signal feedback for detection. By optimizing the selectivity of the nanopores through specific antibody modification, combined with current signal analysis methods, 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 detect heavy metal pollutants in a short time and can achieve real-time monitoring throughout the detection process. Compared with the disadvantages of traditional methods that require a long time for sample pretreatment or complex analysis procedures, the detection of heavy metals (such as lead, cadmium, copper, and zinc) based on the nanopore sensor has a significant speed advantage 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 considered 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 such as lead, cadmium, copper, zinc, and arsenic.

[0046] More specifically, as a non-limiting example, 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, and Mouse polyclonal to Pb12 antibody.

[0047] In some preferred embodiments, the fabrication of Si3N4 nanopores on a Si3N4 thin film substrate includes:

[0048] A helium ion microscope was used to etch a Si3N4 thin film substrate to obtain nanopores with sizes within a predetermined diameter range. The etching process employed an accelerating voltage of 20kV to 30kV and a helium working pressure of 1.5 × 10⁻⁶ kJ / m³. -10 Torr up to 2.0×10 -10 Torr, and / or a base vacuum pressure of 3.5 × 10⁻⁶ -10 Torr up to 4.5×10 -10 The conditions of Torr.

[0049] It should be noted that using helium ion microscopy (HIM) to precisely process the Si3N4 substrate enables precise control of the nanopore size, thereby adjusting the sensor's sensitivity and adapting to different concentration ranges of heavy metal pollutants, ensuring stability, high sensitivity, and reliability during the detection process.

[0050] In some specific implementations, helium ion microscopy (HIM) is used to etch the Si3N4 thin film substrate to obtain nanopores with diameters ranging from 250 nm to 350 nm.

[0051] In some optional embodiments, the Si3N4 thin film substrate is pretreated before the Si3N4 nanopores are fabricated on the Si3N4 thin film substrate, 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 to clean it. Then, deionized water is used to remove the residual solution on the Si3N4 thin film substrate, and it is placed in an oven to dry for a second preset time.

[0053] Alternatively, after immersing the Si3N4 thin film substrate in acetone for a third preset time, the Si3N4 thin film substrate is then immersed in anhydrous ethanol for a fourth preset time and in deionized water for a fifth preset time, and then placed in an oven to dry 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 washed in a water bath at 90℃±5℃ (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 thin film substrate is removed with deionized water, and the substrate is dried in an oven at 100℃±10℃ for 20 min to 30 min (i.e., the second preset time).

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

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

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

[0058] In some preferred embodiments, the preparation process of the Si3N4 thin film substrate includes, as described in [reference] 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, a low-stress silicon nitride thin film with a second thickness is deposited on the upper and lower surfaces of the substrate by LPCVD (low-pressure chemical vapor deposition).

[0060] Photoresist is coated on one side of the silicon nitride film, referred to as the back side of the chip, and a square photoresist window is formed using photolithography.

[0061] Using CHF3, SiF6, and He as etching gases, reactive ion etching (RIE) is employed to remove silicon nitride protected by photoresist, exposing the silicon substrate, and then removing the photoresist.

[0062] A wet etching process was adopted, using a 30% potassium hydroxide solution, and heated in a second water bath environment for a fifth preset time to etch the silicon substrate along the (111) crystal plane until it was etched to another layer of silicon nitride, resulting in a square silicon nitride film of preset size.

[0063] At least one Si3N4 thin film substrate is obtained by laser scribing on the square silicon nitride thin film.

[0064] In some specific implementations, SiH2Cl2 and NH3 are used as reactant gases in low-pressure chemical vapor deposition.

[0065] In some specific implementation processes, an n-type medium-resistivity silicon wafer with a thickness of 200μm±10μm (i.e., the first thickness), double-sided polishing, and a crystal phase plane of (100) is selected as the substrate; low-pressure chemical vapor deposition (LPCVD) is used to deposit low-stress silicon nitride thin films with a thickness of 100nm±10nm (i.e., the second thickness) on the upper and lower surfaces of the silicon wafer, respectively, using SiH2Cl2 and NH3 as reaction gases; photoresist is coated on one side of the silicon nitride thin film, which is designated as the back side of the chip, and a square photoresist window is formed using photolithography, with the window size ranging from 10μm×10μm to 50μm×50μm; Reactive ion etching (RIE) was used with CHF3, SiF6, and He as etching gases to remove silicon nitride protected by photoresist, exposing the silicon substrate and removing the photoresist. A wet etching process was used with a 30% potassium hydroxide solution, heated in a water bath at 80℃±5℃ for 5 to 10 hours to etch the silicon substrate along the (111) crystal plane until the other layer of silicon nitride was etched, resulting in a square silicon nitride film with dimensions of 5μm×5μm±0.5μm. Finally, laser scribing was used to obtain individual Si3N4 film substrates in units of 2.4mm×2.4mm±0.1mm.

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

[0067] After cleaning the Si3N4 nanopore array, the Si3N4 nanopore array is subjected to oxygen plasma treatment within a first preset power range 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 were activated using CDI (1-chloro-3-chloromethyl-1,3,5-triazine) solution, and the specific antibody was bound to the surface of the Si3N4 nanopore array via an EDC / NHS crosslinking reaction to form a highly specific sensing interface.

[0069] In some alternative embodiments, the step of activating the carboxyl groups on the surface of the Si3N4 nanoporous array with CDI solution and covalently binding the specific antibody to the surface of the Si3N4 nanoporous array via an EDC / NHS crosslinking reaction includes:

[0070] Prepare a 0.1M to 1M CDI solution, place the Si3N4 nanopore array into 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 seventh preset time of the EDC / NHS crosslinking reaction; wherein the solution ratio is 1mM EDC and 1mM NHS.

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

[0073] After the reaction was completed, the sample was washed with PBS solution to remove any unbound specific antibodies, thus obtaining the functionalized nanoporous sensor.

[0074] In some specific implementation processes, the Si3N4 nanoporous array is first cleaned in deionized water and then soaked in acetone and ethanol sequentially for 5 to 15 minutes to remove surface organic matter and contaminants. Next, the cleaned Si3N4 nanoporous array is placed in an oxygen plasma treatment device and subjected to plasma treatment at 20 W to 40 W for 10 to 20 minutes to introduce oxygen-containing functional groups such as carboxyl groups (-COOH) and hydroxyl groups (-OH). Then, the surface carboxyl groups are activated using 1-chloro-3-chloromethyl-1,3,5-triazine (CDI), with a 0.1 M to 1 M CDI solution prepared and reacted at room temperature for 1 to 2 hours. Furthermore, the activated carboxyl groups are converted into active esters via an EDC / NHS crosslinking reaction, with a solution ratio of 1 mM EDC and 1 mM NHS, and a reaction time of 30 minutes to 1 hour. Finally, specific antibodies (such as those targeting the target molecule) were dissolved in an appropriate buffer solution at a concentration of 0.1 mg / mL to 1 mg / mL and added to the surface-activated Si3N4 nanoporous array. The reaction was allowed to proceed for 4 to 12 hours to complete the covalent binding of the antibodies. After the reaction, the surface was washed with PBS solution to remove unbound antibodies, yielding the functionalized Si3N4 nanoporous 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 immobilized on the nanopore surface of the Si3N4 nanopore array.

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

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

[0079] The aforementioned nanopore sensor is fixed in a fluid pool with a cross-section adapted to the size of the nanopore sensor, so that the nanopores on the nanopore sensor become the only channels in the fluid pool, and the fluid pool is divided into two independent fluid pools by the nanopore sensor.

[0080] Electrolyte solution is injected into the two independent fluid pools, and the entire fluid pool and the nanopore sensor are placed in a Faraday shielded box;

[0081] An Ag / AgCl electrode was inserted into the electrolyte solution, a voltage was applied across the nanopore sensor, and a reference current was recorded based on a patch-clamp amplifier.

[0082] Replace the electrolyte solution at the CIS end in the fluid cell with the sample solution to be tested, apply a bias voltage and record the time-current graph;

[0083] Based on the time-current graph and the reference current, it is determined whether molecular translocation has occurred, and the qualitative / quantitative detection results of the target heavy metal in the sample to be tested are determined based on the molecular translocation results.

[0084] It should be noted that label-free detection using solid-state nanopore sensors reduces reliance on labels or reagents compared to traditional immunoassays or chromatography, avoiding potential errors and high costs associated with the labeling process. Furthermore, nanopore arrays enable multi-channel, high-throughput detection, further improving experimental efficiency.

[0085] In some specific implementations, the Si3N4 nanopore chip and a silicone gasket are clamped between an acrylic glass liquid pool (i.e., the fluid pool) and secured with bolts to ensure that the nanopores are the only channels. The acrylic glass liquid pool is divided into two independent fluid pools (commonly referred to as the CIS end and the trans end). Next, both sides of the liquid pool are wetted with anhydrous ethanol and rinsed with deionized water using a 100 μL pipette, followed by the injection of potassium chloride solution. The entire device is placed in a Faraday shielded box, and the Ag / AgCl electrode is inserted into the electrolyte solution. The shielded box is then covered to isolate external interference. A patch-clamp amplifier and a data acquisition system are used to record the reference current. The sampling frequency is set to 250 kHz, and a low-pass Bessel filter is used at 10 kHz to ensure signal stability and a high signal-to-noise ratio.

[0086] In some specific implementations, heavy metal ions (such as Pb) are driven by applying a voltage ranging from 100mV to 150mV across the Si3N4 solid nanopores. 2+ Cd 2+ Cu 2+ Zn 2+ (etc.) through nanopores. During this process, the presence of heavy metal ions or their complexes can have a blocking effect on the nanopore current, causing a change in the current amplitude (i.e., the blocking current amplitude ΔI), see [reference]. Figure 6 .

[0087] Those skilled in the art should understand that, based on the time-current plot (which reflects the trend of current amplitude changing over time, and can be a curve or scatter plot) and the reference current, an ion current plot (reflecting the trend of ion current amplitude changing over time after removing the influence of the reference current) can be determined. Based on the ion current plot, by real-time monitoring and recording the current change (ΔI) and passage time (Δt) during passage through the nanopore, it can be determined whether molecular translocation has occurred.

[0088] As a non-limiting example, Clampfit software was used to plot time-current graphs and statistically analyze parameters such as signal current amplitude and via time. Next, data filtering and distribution plotting were performed using Excel and Origin. The filtering criteria were: current amplitude greater than three times the via current noise (based on RMS calculations) and signal-to-noise ratio greater than 3. Meeting these conditions effectively distinguishes the reference current from the via signal, ensuring signal accuracy and thus identifying whether molecular translocation has occurred.

[0089] It should be noted that, based on the fitted ion current graph, the concentration of each heavy metal ion can be further estimated. The concentration of each heavy metal pollutant in the sample can be determined by comparing the detection results with the standard concentration range. For example, a heavy metal solution with a concentration range of 0.5 mol / L to 1.5 mol / L is prepared as a standard solution, added to a fluid cell, and brought into full contact with the electrodes on both sides of the fluid cell. A standard curve with a known concentration is obtained by fitting the ion current graph of the standard solution, and compared with the ion current graph of the actual sample. The concentration of each heavy metal pollutant in the sample can then be determined. This method has high sensitivity and can distinguish between different heavy metal ions, making it suitable for efficient and rapid analysis in environmental monitoring and pollutant detection.

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

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

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

[0093] This embodiment also provides a device for detecting heavy metal pollutants, see reference. Figure 8 ,include:

[0094] Faraday shielding box ( Figure 8 (d) The Faraday shielding box contains a fluid pool and a nanopore sensor as described in claim 8, the nanopore sensor being fixed in the fluid pool. Figure 8 Between (e), and the nanopores on the nanopore sensor become the only channels within the fluid pool, the fluid pool is divided into two independent fluid pools by the nanopore sensor; wherein, the fluid pool is also used to contain electrolyte solution and / or 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) is used in a patch-clamp amplifier. Figure 8c) Acquire electrical signals from the nanopore sensor to determine the reference current and time-current map; also used to determine whether molecular translocation has occurred based on the time-current map and the reference current, and to determine the qualitative / quantitative detection results of the target heavy metal in the sample solution to be tested based on the molecular translocation results.

[0097] It is understood that the apparatus of this embodiment corresponds to the method of the foregoing embodiment, and the options in the foregoing embodiment are also applicable to this embodiment, so they will not be described again here.

[0098] The same or similar labels correspond to the same or similar parts;

[0099] The terms used to describe positional relationships in the accompanying drawings are for illustrative purposes only and should not be construed as limiting this application.

[0100] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

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

[0102] Obviously, the above embodiments of this application are merely examples for clearly illustrating this application, and are not intended to limit the implementation of this application, nor are they intended to limit this application. For those skilled in the art, other variations or modifications can be made based on the above description. The separate structural / functional modules or units can be integrated together to form an independent part, or each module can exist independently, or two or more modules can be integrated to form an independent part. The structure and function of the separate components can be implemented as a combined structure or component. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the scope of protection of the claims of this application.

Claims

1. A method for preparing a nanopore sensor based on specific antibodies, characterized in that, The application relates to a method for preparing a nanometer-pore sensor for heavy metal ions. The method comprises the following steps: Preparation of Si3N4 nanometer-pores on a Si3N4 film substrate based on electron beam exposure micro-nano processing technology to obtain a Si3N4 nanometer-pore array; Surface functionalization of the Si3N4 nanometer-pore array to fix specific antibodies on the surface of the nanometer-pores; wherein the specific antibodies can react with target heavy metals to produce characteristic changes in electrical signals under a bias voltage after reaction and combination; The surface functionalization of the Si3N4 nanometer-pore array comprises the following steps: After the Si3N4 nanometer-pore array is cleaned, the Si3N4 nanometer-pore array is subjected to oxygen plasma treatment in a first preset power range to introduce oxygen-containing functional groups; wherein the oxygen-containing functional groups include carboxyl groups; Activation of the carboxyl groups on the surface of the Si3N4 nanometer-pore array by using a CDI solution, and covalent combination of the specific antibodies to the surface of the Si3N4 nanometer-pore array by EDC / NHS cross-linking reaction to form a sensing interface with high specificity; The activation of the carboxyl groups on the surface of the Si3N4 nanometer-pore array by using a CDI solution, and the covalent combination of the specific antibodies to the surface of the Si3N4 nanometer-pore array by EDC / NHS cross-linking reaction comprises the following steps: A 0.1 M to 1 M CDI solution is configured, the Si3N4 nanometer-pore array is placed in the CDI solution, and the carboxyl groups on the surface of the Si3N4 nanometer-pore array are activated by reacting at room temperature for a sixth preset time length; The activated carboxyl groups are converted into active esters by EDC / NHS cross-linking reaction for a seventh preset time length; wherein the solution ratio is 1 mM EDC to 1 mM NHS; The specific antibodies are dissolved in a buffer solution at a specified concentration range, the buffer solution containing the specific antibodies is added to the surface-activated Si3N4 nanometer-pore array, and the specific antibodies are covalently combined to the surface of the Si3N4 nanometer-pore array by reacting for an eighth preset time length; 2. The method for preparing a nanoporous sensor based on a specific antibody according to claim 1, characterized in that, After the reaction is completed, a PBS solution is used for cleaning to remove the uncombined specific antibodies, and the functionalized nanometer-pore sensor is obtained. The Si3N4 thin film substrate is etched by using a helium ion microscope to obtain a nanopore with a size within a preset diameter interval; wherein, during the etching process, the conditions are as follows: an acceleration voltage of 20 kV to 30 kV, a helium gas working pressure of 1.5 × 10 - ¹ 0 Torr to 2.0 × 10 - ¹ 0 Torr, and / or a basic vacuum pressure of 3.5 × 10 - ¹ 0 Torr to 4.5 × 10 - ¹ 0 Torr.

3. The method of claim 2, wherein the antibody is a specific antibody. The preparation of Si3N4 nanometer-pores on a Si3N4 film substrate comprises the following steps: ​ Before the preparation of Si3N4 nanometer-pores on a Si3N4 film substrate, the Si3N4 film substrate is pretreated, which comprises the following steps: In a first water bath environment, the Si3N4 film substrate is cleaned in a highly corrosive oxidizing agent solution for a first preset time length, deionized water is used to remove residual solution on the Si3N4 film substrate, and the Si3N4 film substrate is dried in an oven for a second preset time length; Or, the Si3N4 film substrate is soaked in acetone for a third preset time length, then the Si3N4 film substrate is sequentially soaked in anhydrous ethanol for a fourth preset time length and deionized water for a fifth preset time length, and then the Si3N4 film substrate is dried in an oven for a second preset time length.

4. The method of claim 2, wherein the antibody is a specific antibody. When etching the Si3N4 thin film substrate by using a helium ion microscope, a processing pattern is a circle corresponding to the preset diameter interval, an ion beam current is 10 pA to 20 pA, an ion dosage is 60 nC / μm² to 100 nC / μm², a residence time is 800 μs to 1200 μs, and / or a pixel size is 8 nm² to 12 nm².

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

6. A specific antibody-based nanopore sensor, prepared by the method of any one of claims 1-5, wherein the specific antibody-based nanopore sensor comprises a specific antibody and a nanopore. The Si3N4 nanopore array has specific antibodies fixed on a nanopore surface of the Si3N4 nanopore array; The specific antibodies are used to generate electric signal feature changes under a bias voltage after being combined with target heavy metals.

7. A method of detecting heavy metal contaminants, characterized by, The method comprises the following steps: The nanopore sensor is fixed in a fluid pool with a cross section that is suitable for the size of the nanopore sensor, so that the nanopores on the nanopore sensor become the only channel in the fluid pool, and the fluid pool is divided into two independent fluid pools by the nanopore sensor; Electrolyte solution is injected into the two independent fluid pools, and the entire fluid pool and the nanopore sensor are placed in a Faraday shield box; An Ag / AgCl electrode is inserted into the electrolyte solution, a voltage is applied on both sides of the nanopore sensor, and a reference current is recorded based on a patch clamp amplifier; The electrolyte solution in the cis end of the fluid pool is replaced with a sample solution to be tested, a bias voltage is applied, and a time-current graph is recorded based on the electric signal of the nanopore sensor; Based on the time-current graph and the reference current, it is determined whether molecular translocation occurs, and a qualitative / quantitative detection result of the target heavy metal in the sample solution to be tested is determined according to the molecular translocation result.

8. A device for detecting heavy metal contaminants, characterized in that, The method comprises the following steps: The Faraday shield box contains a fluid pool and the nanopore sensor of claim 6, the nanopore sensor is fixed between the fluid pools, and the nanopores on the nanopore sensor become 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 electrolyte solution and / or sample solution to be tested. a power supply for applying an electrical signal to both sides of the nanopore sensor; a data processing module for collecting the nanopore sensor electrical signal via a patch clamp amplifier, determining a reference current and a time-current graph; and for determining whether a molecular translocation has occurred according to the time-current graph and the reference current, and determining a qualitative / quantitative detection result of the target heavy metal in the sample solution to be detected according to the molecular translocation result.

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