A method for reusing silicon nitride nanopores after they are blocked by proteins

Abstract

The present invention discloses a method for reusing a silicon nitride nanopore after it is blocked by protein, and belongs to the field of nanopore sensing technology. The method uses a square wave voltage to cycle multiple times to push out the protein that blocks the pore and adheres to the pore wall, and performs a patch clamp test on the silicon nitride nanopore after the pore is blocked. The current baseline is restored to the value before the pore is blocked and the baseline is stable, indicating that the nanopore has restored the original normal test state. The problem that the silicon nitride nanopore cannot be used after the protein adheres to the pore wall during the protein detection process is solved. The nanopore after the pore is connected can continue to perform the detection experiment, which not only improves the experimental efficiency, but also reduces the cost of the nanopore experiment. The square wave voltage pore connection method provided by the present invention is simple to operate and has good repeatability. It can also be applied to other solid-state nanopores and has high application value and economic value.

Description

Technical Field

[0001] The invention belongs to the technical field of nanopore sensing, and in particular relates to a method for reusing silicon nitride nanopores blocked by proteins by using a pulse voltage to conduct through-hole reuse. Background Art

[0002] As a new type of pore sensing analysis technology, nanopore detection technology has the advantages of low cost, high throughput, and no labeling. Its working principle is based on resistive pulse sensing. Two reservoirs filled with electrolytes are separated by a thin, impermeable membrane and connected by a nanopore. An external electric field is applied to both ends of the nanopore, and the concentration, charge and structural characteristics of ions or molecules passing through the nanopore are judged by monitoring the changes in the weak current (picoampere level) signal when the electrolyte flows through the nanopore, including the amplitude and residence time of the current blockage, the change frequency and the fingerprint signal.

[0003] Nanopores are single-molecule resolution sensors that are increasingly being used in fields such as genomics, glycomics, lipidomics, and virology. Recently, proteins have become the focus of nanopore research. Dysregulation of certain proteins and enzymes is often considered an indicator of many pathologies, and these disease-associated proteins can be used as biomarkers for diagnosing disease onset and progression. Silicon nitride nanopores are particularly advantageous for protein detection because they are not only mechanically strong but also easy to modify the size, structure, and surface properties of the pores. However, nonspecific adsorption of proteins on solid surfaces is an obstacle in the detection process. Despite many experimental studies of protein adsorption at liquid-solid interfaces, this phenomenon is still not fully understood.

[0004] In the process of protein detection in silicon nitride nanopores, proteins will inevitably adsorb to the pore wall. At this time, we usually choose to apply a voltage slightly larger than the test voltage or a reverse voltage slightly larger than the test voltage to push the protein out of the pore. When these two methods fail to push the protein out, the silicon nitride nanopore will no longer be usable. Faced with this situation that affects test efficiency and increases test costs, we need a method to restore the blocked silicon nitride nanopore to its original normal test state. Summary of the invention

[0005] Purpose of the invention: In order to solve the above technical problems, the purpose of the present invention is to provide a method for reusing silicon nitride nanopores after they are blocked by proteins, so that the silicon nitride nanopores blocked by proteins can be restored to their original normal test state, so that subsequent experiments can be carried out.

[0006] Technical solution: The method for reusing the silicon nitride nanopores after being blocked by proteins according to the present invention comprises the following steps:

[0007] (1) Preparing a silicon nitride nanopore detection platform and performing protein detection;

[0008] (2) after the protein blocks the pore wall, a bias voltage slightly larger than the detection voltage in step (1) is applied to push out part of the protein;

[0009] (2) Using square wave voltage for multiple cycles to push out proteins that block the pores and adhere to the pore walls;

[0010] (3) The silicon nitride nanopore after the hole was opened was subjected to a patch clamp test, and the current baseline returned to the value before the hole was blocked and the baseline was stable, indicating that the nanopore returned to its original normal test state.

[0011] Furthermore, the silicon nitride nanopore chip (NBPT001Z-HR) used in the silicon nitride nanopore was ordered from Norcada, Canada, and the thickness of the silicon nitride film was 12±2 nm. The silicon nitride nanopore chip was cleaned with oxygen plasma on both the front and back sides for 1 minute each, and then the nanopore was formed by dielectric breakdown method (CBD).

[0012] Specifically, the dielectric breakdown method is to apply a voltage of 6.5V for thousands of seconds to reach a threshold current of 300nA, indicating the formation of a nanopore.

[0013] Furthermore, the silicon nitride nanopore detection platform in step (1) is constructed by the following method: a polytetrafluoroethylene detection cell is used, a silicon nitride nanopore chip with nanoscale holes is installed in the detection cell, and an electrolyte solution is added to the liquid cells on both sides until the liquid level exceeds the silicon nitride nanopore chip window to form a silicon nitride nanopore detection platform. When protein is detected, the protein solution is added to the grounded Cis chamber, and a positive transmembrane potential is applied to drive BSA to pass through the hole.

[0014] Furthermore, the slightly larger bias voltage applied in step (2) is a positive / negative transmembrane potential of 1.5-2 times the transmembrane potential when the detection protein blocks the nanopore.

[0015] Furthermore, the square wave voltage in step (2) is positive and negative pulse electricity, for example, 0.5V for several seconds, -0.5V for several seconds, and this cycle is repeated many times until the protein blocked in the pore wall is pushed out.

[0016] Furthermore, in the patch clamp test in step (3), the current when a certain positive / negative transmembrane potential is applied after the nanopore is opened returns to the current value before the nanopore is blocked, and the baseline is stable.

[0017] Beneficial effects: Compared with the prior art, the technical solution of the present invention has the following significant advantages: the present invention uses square wave voltage to perform through-holes, which can effectively avoid the problem that the silicon nitride nanopore cannot be used after being blocked by proteins, and the pore size of the nanopore after through-holes can be controlled according to the level of the square wave voltage. When a smaller voltage is used to perform through-holes, the pore size of the nanopore hardly changes, and when the voltage is larger, the pore size of the nanopore may become larger. The method of the present invention can effectively increase the use time of the silicon nitride nanopore after the pore is blocked by proteins, which not only improves the test efficiency but also reduces the experimental cost. The square wave voltage through-hole method provided by the present invention is simple to operate and has good repeatability. It can also be applied to other solid-state nanopores and has high application value and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a diagram of the device for dielectric breakdown drilling and the process of nanopore formation;

[0019] Figure 2 is a through-hole current trajectory diagram after plugging during the BSA detection process in Example 1 of the present invention, wherein Figure 2 a is the current trajectory diagram of the hole blocking state when the reverse voltage is applied. Figure 2 b is a schematic diagram of the square wave voltage applied when the hole is through and the change of current over time under this square wave voltage. Figure 2 c is the current trajectory diagram of the patch clamp test at different voltages after the through hole;

[0020] Figure 3 This is a through-hole current trajectory diagram after hole blocking during the detection of IgG (immunoglobulin G) in Example 2 of the present invention, wherein Figure 3 a is the current trajectory diagram of the hole blocking state when the reverse voltage is applied; Figure 3 b is a schematic diagram of the square wave voltage applied when the hole is through and the change of current over time under this square wave voltage. Figure 3 c is the current trajectory diagram of the patch clamp test at different voltages after the through hole;

[0021] Figure 4 This is a through-hole current trajectory diagram after plugging the hole in the process of detecting Papain (papain) in Example 3 of the invention, wherein Figure 4 a is the current trajectory diagram of the Papain plugging state when reverse voltage is applied. Figure 4 b is a schematic diagram of the square wave voltage applied when the hole is through and the change of current over time under this square wave voltage. Figure 4 c is the current trajectory diagram of the patch clamp test at different voltages after the hole is opened. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but it is necessary to point out that the following embodiments are only used to describe the content of the invention and do not constitute a limitation on the scope of protection of the present invention. In addition, the well-known structures and their descriptions that are well known to researchers in the field are partially omitted.

[0023] Example 1

[0024] Fabrication of silicon nitride nanopores for BSA detection

[0025] (1) Preparation of silicon nitride nanopore detection platform

[0026] ① Take a silicon nitride (SiNx) nanochip with a thickness of 12±2nm and clean it with oxygen plasma on both the front and back sides for one minute each.

[0027] ② Install the processed chip into the detection pool, add pH=8, 1M KCl, 10mM Tris-HCl solution into the liquid pool of the detection device until the liquid level exceeds the silicon nitride window, and insert Ag / AgCl electrodes into the two liquid pools respectively. Apply 6.5V voltage and reach the threshold current of 300nA after thousands of seconds, indicating the formation of nanopores. Figure 1 shown.

[0028] (2) BSA detection using silicon nitride nanopores

[0029] ①Put the completed experimental device in the shielding cage. At this time, the two Ag / AgCl electrodes inserted into the liquid pool are connected to the positive and negative electrodes of the patch clamp amplifier (Axon 200B) respectively. The positive electrode of the patch clamp amplifier is inserted into the Trans end of the liquid pool, and the negative electrode is inserted into the Cis end.

[0030] ② Add BSA solution to the solution of pH=8, 1M KCl, 10mM Tris-HCl to a final BSA concentration of 135nM. It is known that the isoelectric point of BSA is about 4.5, and BSA is negatively charged as a whole in the prepared test solution. Add this mixed solution to the Cis end of the detection device, and the solution at the Trans end is pH=8, 1M KCl, 10mM Tris-HCl. Apply a positive bias voltage to observe the BSA perforation signal.

[0031] ③If Figure 2As shown in a, when BSA is blocked in the pore, a negative bias voltage (-200mV) is applied. When 100mV is applied again, the current does not return to the normal value and the baseline noise becomes very large. This indicates that the negative bias voltage can no longer push BSA out of the nanopore. At this time, the BSA solution in the liquid pool is sucked out and rinsed, and then replaced with pH=8, 1M KCl, 10mM Tris-HCl solution and placed in the Keithley 6487 meter to apply as shown in the figure. Figure 2 The square wave voltage shown in c, that is, 0.5V for 3s and then -0.5V for 3s, is cycled in sequence. It can be seen that the current gradually increases with the change of applied voltage.

[0032] ④ The device after through-hole was placed in a shielding cage and current was tested using a patch clamp. The current trajectory diagrams under different voltages showed that the current returned to the level before blockage, and the baseline was stable, indicating that the silicon nitride nanopore had recovered from the blocked state to the original normal test state, proving that this square wave pressure through-hole method is feasible for detecting BSA in silicon nitride.

[0033] Example 2

[0034] Preparation of Silicon Nitride Nanopores for IgG (Immunoglobulin G) Detection

[0035] (1) Preparation of silicon nitride nanopore detection platform

[0036] ① Take a silicon nitride (SiNx) nanochip with a thickness of 12±2nm and clean it with oxygen plasma on both the front and back sides for one minute each.

[0037] ② Install the processed chip into the detection pool, add pH=8, 1M KCl, 10mM Tris-HCl solution into the liquid pool of the detection device until the liquid level exceeds the silicon nitride window, and insert Ag / AgCl electrodes into the two liquid pools respectively. Apply 6.5V voltage and reach the threshold current of 300nA after thousands of seconds, indicating the formation of nanopores. Figure 1 shown.

[0038] (2) Detection of IgG (Immunoglobulin G) using silicon nitride nanopores

[0039] ① Add the IgG solution to a solution of pH=8, 1M KCl, 10mM Tris-HCl until the final IgG concentration is 440nm. It is known that the isoelectric point of IgG is about 8.5. In the prepared test solution, IgG is positively charged as a whole. Add this mixed solution to the Cis end of the detection device. The solution at the Trans end is pH=8, 1M KCl, 10mM Tris-HCl. Apply a negative bias voltage to observe the IgG trans-hole signal.

[0040] ② If Figure 3As shown in a, if the current decreases during the experiment and cannot return to the normal baseline current value, it means that IgG is blocked in the pore. At this time, a positive bias voltage (200mV) is applied, and it can be seen that the baseline noise becomes larger and the nanopore is still in a blocked state.

[0041] ③ This indicates that the positive bias voltage can no longer push IgG out of the nanopore. At this time, we aspirate the IgG solution in the liquid pool and rinse it clean, then replace it with pH=8, 1M KCl, 10mM Tris-HCl solution and place it in the Keithley6487 meter to apply the following voltage: Figure 3 The square wave voltage shown in b, i.e. 0.5V for 3s and then -0.5V for 3s, is cycled sequentially. It can be seen that the current gradually increases with the change of applied voltage, indicating that the silicon nitride nanopore has recovered from the blocked state to the original normal test state.

[0042] ④ The device after through-hole was placed in a shielding cage for current testing. The current trajectory diagrams under different voltages showed that the current level before blockage returned, and the baseline was stable, indicating that the silicon nitride nanopore had recovered from the blocked state and the nanopore had returned to its original normal test state, proving that this square wave pressure through-hole method is feasible for silicon nitride detection of IgG.

[0043] Example 3

[0044] Preparation of Silicon Nitride Nanopores for Papain Detection

[0045] (1) Preparation of silicon nitride nanopore detection platform

[0046] ① Take a silicon nitride (SiNx) nanochip with a thickness of 12±2nm and clean it with oxygen plasma on both the front and back sides for one minute each.

[0047] ② Install the processed chip into the detection pool, add pH=8, 1M KCl, 10mM Tris-HCl solution into the liquid pool of the detection device until the liquid level exceeds the silicon nitride window, and insert Ag / AgCl electrodes into the two liquid pools respectively. Apply 6.5V voltage and reach the threshold current of 300nA after thousands of seconds, indicating the formation of nanopores. Figure 1 shown.

[0048] (2) Papain detection using silicon nitride nanopores

[0049] ① Add the Papain solution to the solution of pH = 8, 1M KCl, 10mM Tris-HCl until the final concentration of Papain is 640nm. It is known that the isoelectric point of Papain is about 8.75, and it carries a positive charge as a whole in the prepared test solution, so this mixed solution is added to the Cis end of the detection device, and the solution at the Trans end is pH = 8, 1M KCl, 10mM Tris-HCl. A negative bias voltage is applied to observe the Papain through-hole signal.

[0050] ② If Figure 4 As shown in a, a small amount of perforation signal appears when a -50mV bias voltage is applied (the local enlarged image is located at Figure 4 a), the upper right corner, then Papain adheres to the nanopore wall causing the current to decrease slightly and then there is no perforation signal. Continuously applying bias voltages of 100mV and 200mV and then switching to -100mV, it can be seen that the baseline noise increases but the nanopore is still in a blocked state.

[0051] ③ This indicates that the positive bias voltage can no longer push Papain out of the nanopore. At this time, we suck out the Papain solution in the liquid pool and rinse it clean, then replace it with 1M KCl solution and place it in the Keithley 6487 meter to apply the following voltage: Figure 4 The square wave voltage shown in c, i.e. 0.2V for 3s and then -0.2V for 3s, is cycled sequentially. It can be seen that the current gradually increases with the change of applied voltage, indicating that the silicon nitride nanopore has recovered from the blocked state to the original normal test state.

[0052] ④ The device after the through-hole was placed in a shielding cage for current testing. The current trajectory diagram under different voltages showed that the current level before the blockage returned, and the baseline was stable, indicating that the silicon nitride nanopore had recovered from the blocked state to the original normal test state. This proves that this square wave voltage through-hole method is feasible for silicon nitride to detect Papain.

[0053] In summary, the present invention provides a method for opening a silicon nitride nanopore after it is blocked during protein detection. The present invention aims to solve the disadvantage that the silicon nitride nanopore cannot be used after being blocked during protein detection. The nanopore after opening can continue to perform detection experiments, which not only improves the experimental efficiency but also reduces the cost of nanopore experiments. The square wave voltage opening method provided by the present invention is simple to operate and has good repeatability, and has a good effect when opening a silicon nitride nanopore after protein blockage is detected.

[0054] Finally, it should be noted that the above preferred examples and embodiments are only used to illustrate the technical solution of the present invention and are not intended to limit it. This description does not need to and cannot list all possible implementation plans. For researchers in related fields, the technical solution can be modified, replaced or improved in different forms based on the above description, but it should be noted that all modifications and improvements made within the purpose and scope of the technical solution should be included in the scope of the claims of the present invention.

Claims

1. A method for reusing silicon nitride nanopores after they have been blocked by proteins, characterized in that: The following steps are involved: (1) Prepare a silicon nitride nanopore detection platform and perform protein detection; (2) After the protein blocks the pore wall, a bias voltage slightly larger than the detection voltage in step (1) is applied to push out part of the protein; (3) Use square wave voltage to cycle multiple times to push out proteins that block the pores and adhere to the pore walls; (4) The silicon nitride nanopore after the hole was opened was tested for current using a patch clamp. The current baseline returned to the value before the hole was blocked and the baseline was stable, indicating that the nanopore returned to its original normal test state. The slightly larger bias voltage applied in step (2) is a positive / negative transmembrane potential of 1.5-2 times the transmembrane potential when the protein blocks the nanopore. The square wave voltage in step (3) is a positive and negative pulse voltage. The specific operation of step (3) is 0.5 V for several seconds, -0.5 V for several seconds, and this cycle is repeated many times until the protein blocked in the pore wall is pushed out.

2. The method for reusing the silicon nitride nanopores after being blocked by proteins according to claim 1, characterized in that: The thickness of the silicon nitride nanopore chip is 12±2 nm.

3. The method for reusing the silicon nitride nanopores after being blocked by proteins according to claim 1, characterized in that: In step (1), the silicon nitride nanopore detection platform is constructed by the following method: a polytetrafluoroethylene detection cell is used, a silicon nitride nanopore chip with nanoscale holes is installed in the detection cell, and an electrolyte solution is added to the liquid cells on both sides until the liquid level exceeds the silicon nitride nanopore chip window, thereby forming a silicon nitride nanopore detection platform.

4. The method for reusing the silicon nitride nanopores after being blocked by proteins according to claim 3, characterized in that: The silicon nitride nanopore in the silicon nitride nanopore detection platform in step (1) is prepared by a dielectric breakdown method.

5. The method for reusing the silicon nitride nanopores after being blocked by proteins according to claim 4, characterized in that: The dielectric breakdown method is to apply a voltage of 6.5V for several thousand seconds to reach a threshold current of 300nA.

6. The method for reusing the silicon nitride nanopores after being blocked by proteins according to claim 1, characterized in that: In the patch clamp test in step (4), when a certain positive / negative transmembrane potential is applied after the nanopore is opened, the current returns to the current value before the nanopore is blocked, and the baseline is stable.

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