Controllable biological nanopore sensing detection system and application thereof

Abstract

The invention relates to a controllable biological nanopore sensing detection system and application thereof, and belongs to the technical field of nanopore detection. A traditional biological nanopore detection technology is tedious in detection process, limited in flux and difficult to realize multi-sample continuous detection due to poor mechanical stability of a phospholipid membrane, easy falling of nanopores and low solution replacement efficiency, and application of the traditional biological nanopore detection technology in scenes such as rapid diagnosis is seriously restricted. The invention provides a controllable biological nanopore sensing detection system. The controllable biological nanopore sensing detection system comprises a three-dimensional nanometer control platform, a biological nanopore probe and an array detection pool. The system provided by the invention has the advantages of high stability, high throughput detection, simplified operation and wide applicability, is compatible with DNA, protein and small molecule drug detection, has a detection limit reaching a pM level, provides an efficient tool for precision medical treatment, and can be used in life science, disease diagnosis, biomolecule detection, DNA sequencing and other aspects.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanopore detection, and relates to a controllable biological nanopore sensing detection system and applications thereof. Background Art

[0002] As a new generation of single-molecule analysis, nanopore detection technology has shown great potential in gene sequencing, biomolecular recognition, and disease diagnosis due to its advantages such as label-free, high throughput, and high sensitivity. In existing technologies, nanopores are mainly divided into two categories: biological nanopores (such as α-hemolysin and MspA protein pores) and solid-state nanopores (such as silicon nitride and two-dimensional material film pores). In recent years, solid-state nanopores have attracted much attention due to their high environmental stability and adjustable pore size. However, they still face significant challenges in terms of biocompatibility, detection throughput, and operational flexibility.

[0003] Biological nanopores are a key branch of the nanopore field. Their formation mechanism involves the spontaneous embedding of porins into lipid membranes through hydrophobic interactions, forming stable pores. In addition to sequencing applications, biological nanopores are also widely used in sensor construction, successfully detecting biomarkers, metal ions, and small molecules. Currently, a key challenge facing this technology lies in its limited environmental adaptability. Once a biological nanopore is embedded in a phospholipid bilayer, mechanical vibrations in the detection device can easily lead to membrane damage and nanopore detachment. Furthermore, excessive amounts of porins must be added to improve protein embedding efficiency. The mechanical shock caused by repeated pipetting during solution exchange exacerbates membrane vibrations and causes nanopore detachment, requiring specialized techniques for experimental operation and significantly reducing construction efficiency. In biomarker detection, frequent solution exchange is required to test different samples or concentrations in a single channel, making it difficult to maintain the stability of the nanopore until the experiment is completed. With this in mind, the present invention proposes a controllable biological nanopore sensor for base number detection. Through its innovative design, this sensor enables rapid solution exchange at one end of the biological nanopore, significantly improving sample exchange convenience and experimental efficiency. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide a controllable biological nanopore sensing detection system, and the second objective is to provide an application of a controllable biological nanopore sensing detection system.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a controllable biological nanopore sensing detection system, which includes a three-dimensional nano-manipulation platform, an array sample detection pool, and a biological nanopore probe;

[0007] The three-dimensional nano-manipulation platform includes a three-axis micron drive module and a three-axis nano-drive module, wherein the step resolution of the micron drive module is 1-15 μm, and the travel range of the nano-drive module in the vertical Z-axis direction is 200-400 μm with a step resolution of 50-1000 nm;

[0008] The biological nanopore probe comprises a glass capillary, the tip of which is hydrophobized to construct a phospholipid bilayer and embed a porin to form a single-channel biological nanopore probe;

[0009] The array detection pool is a multi-well plate structure with a single hole diameter of ≥5mm, a depth of 100μm-1cm, and a solution depth in the hole of ≥50μm;

[0010] The controllable biological nanopore sensing detection system also includes a low-noise, high-speed current detection amplifier and a host controller. The three-dimensional nano-manipulation platform is provided with a detection cell installation area for installing an array detection cell. The electrode holder of the current detection device is fixed on the three-dimensional micron manipulation platform. The detection electrode is fixed in the electrode holder, and the reference electrode is placed in the small hole of the detection cell.

[0011] Preferably, the biological nanopore probe preparation method is as follows:

[0012] S1: Use a glass capillary puller to prepare a glass probe with a tip diameter of 50nm-10μm, heat seal it, and polish it to prepare a micron pore with a diameter of 10-100μm;

[0013] S2: Activate the hydroxyl groups on the surface of the glass probe hole by treating it with piranha wash or nitric acid, and then hydrophobize the glass probe hole with a fluorosilane reagent; inject electrolyte into the interior of the glass probe, fix the hydrophobic glass hole on the holder, and connect it to the detection electrode;

[0014] S3: constructing a phospholipid bilayer in the glass probe hole by dipping or blowing, applying a triangular wave voltage to detect the film formation, adding a porin to the detection pool outside the nanopore, and driving the porin under a relatively low negative voltage (<100mV) to self-assemble into the phospholipid bilayer on the glass probe hole and embedding the protein pore channel to construct a single-channel biological nanopore probe, thereby preparing the biological nanopore probe;

[0015] Preferably, the fluorosilane reagent has a fluoroalkylsilane compound with a carbon chain structure, which is any one of 3-cyanopropyldimethylfluorosilane, (tridecafluoro-1,1,2,2-tetrahydrooctyl)dimethylchlorosilane, and (heptadecafluoro-1,1,2,2-tetrahydrodecyl)dimethylchlorosilane;

[0016] Preferably, the phospholipid is a polymerizable polyenoyl lipid, which is any one of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine.

[0017] Preferably, the biological nanopore is a channel protein, which is any one of α-hemolysin, Mycobacterium smegmatis porin (MspA), bacteriophage phi29 connector protein, Aerolysin, curli pilus σS-dependent growth subunit G pore (CsgG), Fragaceatoxin C (FraC), cytotoxin K (CytK), outer membrane protein F (OmpF), and engineered transmembrane proteins;

[0018] Preferably, the array detection pool is a commercial microplate or a custom-made array detection pool. The bottom of the array detection pool is horizontal, and the plane size is suitable for forming a three-dimensional manipulation platform. The depth (h) of the detection pool pores is 100 μm-1 cm, the pore diameter (d) is greater than 5 mm, and the solution depth in the pores is greater than 50 μm. The material of the array detection pool is an insulating material such as plastic or glass.

[0019] Furthermore, the detection method of the controllable biological nanopore sensing detection system includes the following detection steps:

[0020] S1: Add the sample to be tested into the array detection pool, which is placed on a three-dimensional nanomanipulation platform;

[0021] S2: Manipulate the three-axis micrometer drive module to control the biological nanopore probe to enter the detection cell pore of the detection cell;

[0022] S3: Manipulate the three-axis nano-drive module to slowly insert the biological nanopore probe into the solution in the detection cell pore, adjust the voltage to capture the analyte into the biological nanopore, and obtain the current blockade signal of the analyte;

[0023] S4: Obtain the concentration of the test sample through signal processing and analysis;

[0024] Furthermore, the application of the controllable biological nanopore sensing detection system in detecting biological molecules;

[0025] Preferably, the biomolecule is a nucleic acid, a protein, or a polypeptide.

[0026] The beneficial effects of the present invention are:

[0027] This invention utilizes manipulation technology to achieve the movement of the biological nanopore. The capillary phospholipid support platform provides the necessary conditions for the movement of the biological nanopore, increasing its controllability and reducing the risk of phospholipid membrane rupture. This enables controllable detection of the nanopore and improves the detection efficiency of the biological nanopore.

[0028] 1. High stability: The probe movement stability error is ≤5nm / s, combined with the capillary tip solution protection, the phospholipid membrane rupture rate is reduced;

[0029] 2. High-throughput detection: The array detection pool works in conjunction with the multi-axis drive, and a single probe can continuously detect multiple samples, improving efficiency;

[0030] 3. Simplified operation: The probe is completely separated from the original solution to avoid residual interference, reduce background noise, and eliminate the need for complex pipetting operations;

[0031] 4. Wide applicability: Compatible with DNA, protein and small molecule drug detection, with a detection limit of pM level, providing an efficient tool for precision medicine. The present invention can be used in life sciences, disease diagnosis, biomolecule detection, DNA sequencing and other aspects.

[0032] Other advantages, objects, and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art upon examination of the following description or may be learned from practice of the present invention. The objects and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:

[0034] Figure 1 Schematic diagram of the controllable biological nanopore sensing detection system of the present invention;

[0035] Figure 2 Schematic diagram of the probe-type three-dimensional nanomanipulation and signal detection system;

[0036] Figure 3 Schematic diagram for the preparation of biological nanopore probes;

[0037] Figure 4 Schematic diagram of array detection pool;

[0038] Figure 5 This is the current signal diagram after the biopore probe enters the electrolyte;

[0039] Figure 6 The current signal and linear fitting diagram of the manipulable biopore probe for a series of DNA sample concentrations.

[0040] Figure numerals: 101, Faraday shield; 102, three-axis micron drive platform; 103, three-axis nano drive platform; 104, low-noise, high-speed current detection amplifier; 105, host controller; 106, detection cell; 107, electrode holder of the current detection device; 108, detection electrode, 109, reference electrode; 110, detection cell pore; 201, glass micro / nanotube; 202, hydrophobic treatment; 203, construction of phospholipid bilayer; 204, embedded channel protein; 301, array detection cell; 302, detection cell pore. DETAILED DESCRIPTION

[0041] The following describes the embodiments of the present invention by means of specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0042] Among them, the accompanying drawings are only for illustrative purposes and represent only schematic diagrams rather than actual pictures, and should not be understood as limiting the present invention. In order to better illustrate the embodiments of the present invention, some parts of the accompanying drawings may be omitted, enlarged or reduced, and do not represent the dimensions of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions may be omitted in the accompanying drawings.

[0043] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right", "front", "back", etc. indicating directions or positional relationships, they are based on the directions or positional relationships shown in the drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operate in a specific direction. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0044] Example 1

[0045] Step 1: Build a 3D nanomanipulation and signal detection platform

[0046] A three-dimensional nano-manipulation and signal detection platform is constructed, which is equipped with a Faraday shield (101), a micron drive platform with a travel range of 15 cm in the XY direction, a travel range of 5 cm in the Z axis direction, and a step resolution of 5 μm; a three-axis nano-drive platform (102), a travel range of 10 μm in the Z axis direction, and a step resolution of 50 nm (103), a low-noise high-speed current detection amplifier (104) for pA-level current sensitivity detection, a host controller (105), etc. The nano-piezoelectric driving platform is provided with a detection cell installation area for fixing the detection cell (107). The array detection cell is a commercial 24-well plate with a size of 125×85×23 mm (length×width×height), a single hole diameter of 16 mm, and a depth of 17 mm. The electrode holder (108) of the current detection device is fixed on the three-dimensional micron manipulation platform. The detection electrode (109) is an Ag / AgCl electrode fixed in the electrode holder, and the reference electrode (110) is an Ag / AgCl electrode placed in the detection cell hole (110).

[0047] Step 2: Preparation of biological nanopore probes

[0048] First, a borosilicate glass probe (201) with a tip diameter of approximately 100 nm was prepared by drawing a glass capillary using a drawing instrument. The probe was then sealed by heating and polished to prepare a 50 μm micropore. The prepared glass pore was treated with piranha solution to activate the hydroxyl groups on the surface of the glass pore. The activated hydroxyl group glass pore was then hydrophobized using 3-cyanopropyldimethylfluorosilane reagent to form a glass pore (202) with a hydrophobic structure. 2M LiCl electrolyte was injected into the glass probe, and the hydrophobic glass pore was fixed on a holder and connected to a detection electrode. A 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine phospholipid bilayer (203) was constructed on the surface of the glass nanopore using an immersion method. A triangular wave voltage was applied to detect the film formation. α-hemolysin pore protein is added to the detection pool outside the nanopore, and the pore protein is driven to embed into the protein pore channel through the phospholipid bilayer self-assembled on the glass pore under a negative voltage of more than 50mV to construct a biological nanopore probe (204).

[0049] Step 3: Sample testing

[0050] S1, a series of samples to be tested are added to the array detection, and the three-axis nano-drive platform (103) is used to slowly allow the biological nanopore probe (204) to enter the detection pool (302) solution, and the current is turned on ( Figure 5 ), the approach is stopped and the voltage is adjusted to capture the analyte into the nanopore;

[0051] S2, using the three-axis nano-driving platform (103), the biological nanopore probe (204) is gradually moved to the liquid level of the detection pool solution.

[0052] In step S3, the micrometer-driven stage is used to move the bionanopore probe into the next detection cell and repeat step 3 to obtain current blockade information of the molecule to be detected.

[0053] S4, through subsequent signal processing technology and analysis technology, the concentration of the detected sample is obtained ( Figure 6 ).

[0054] like Figure 6 As shown in the linear fitting diagram of the measured current signal and concentration of the DNA sample, the DNA sample concentration is linearly related to the current signal, R 2 =0.993.

[0055] Example 2

[0056] Step 1: Build a 3D nanomanipulation and signal detection platform

[0057] A three-dimensional nano-manipulation and signal detection platform is constructed, which is equipped with a Faraday shield (101), a micron drive platform with a travel range of 15 cm in the XY direction, a travel range of 5 cm in the Z axis direction, and a step resolution of 10 μm; a three-axis nano-drive platform (102), a travel range of 20 μm in the Z axis direction, and a step resolution of 50 nm (103), a low-noise high-speed current detection amplifier (104) for pA-level current sensitivity detection, a host controller (105), etc. The nano-piezoelectric driving platform is provided with a detection cell installation area for fixing the detection cell (107). The array detection cell is a commercial 12-microwell plate with a size of 125×85×23 mm (length×width×height), a single hole diameter of 22.5 mm, and a depth of 17.4 mm. The electrode holder (108) of the current detection device is fixed on the three-dimensional micron manipulation platform. The detection electrode (109) is an Ag / AgCl electrode fixed in the electrode holder, and the reference electrode (110) is an Ag / AgCl electrode placed in the detection cell hole (110).

[0058] Step 2: Preparation of biological nanopore probes

[0059] First, a borosilicate glass probe (201) with a tip diameter of approximately 200 nm is prepared by drawing a glass capillary using a drawing instrument. The probe is then sealed by heating and then polished to prepare a 70 μm micropore. The prepared glass pore is treated with piranha wash solution to activate the hydroxyl groups on the surface of the glass pore. The activated hydroxyl glass pore is then hydrophobized using a fluorosilane reagent, wherein the fluorosilane reagent is any one of (tridecafluoro-1,1,2,2-tetrahydrooctyl)dimethylchlorosilane and (heptadecafluoro-1,1,2,2-tetrahydrodecyl)dimethylchlorosilane, to form a glass pore (202) with a hydrophobic structure. 1M LiCl electrolyte is injected into the interior of the glass probe, and the hydrophobic glass pore is fixed on a holder and connected to a detection electrode. A phospholipid bilayer (203) is constructed on the surface of the glass nanopore by an immersion method, wherein the phospholipid is any one of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. A triangular wave voltage is applied to detect the film formation. A pore protein is added to the detection pool outside the nanopore, which is any one of the following: Mycobacterium smegmatis pore protein (MspA), bacteriophage phi29 connector protein, Aerolysin, curli fimbriae σS-dependent growth subunit G pore (CsgG), Fragaceatoxin C (FraC), cytotoxin K (CytK), outer membrane protein F (OmpF), and engineered transmembrane protein. Under a negative voltage of more than 40 mV, the pore protein is driven to embed into the protein pore channel on the phospholipid bilayer self-assembled on the glass pore to construct a biological nanopore probe (204).

[0060] Step 3: Sample testing

[0061] S1, a series of samples to be tested (proteins or peptides) are added to the array detection, and a three-axis nano-drive platform (103) is used to slowly allow the biological nanopore probe (204) to enter the detection pool (302) solution. After the current is turned on, the approach is stopped and the voltage is adjusted to capture the sample to enter the nanopore;

[0062] S2, using the three-axis nano-driving platform (103), the biological nanopore probe (204) is gradually moved to the liquid level of the detection pool solution.

[0063] In step S3, the micrometer-driven stage is used to move the bionanopore probe into the next detection cell and repeat step 3 to obtain current blockade information of the molecule to be detected.

[0064] S4, obtaining the detection sample concentration through subsequent signal processing technology and analysis technology.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should all be included in the scope of the claims of the present invention.

Claims

1. A controllable biological nanopore sensing detection system, characterized by: The controllable biological nanopore sensing detection system includes a three-dimensional nanomanipulation platform, an array sample detection pool, and a biological nanopore probe; The three-dimensional nano-manipulation platform includes a three-axis micron drive module and a three-axis nano-drive module, wherein the step resolution of the micron drive module is 1-15 μm, and the travel range of the nano-drive module in the vertical Z-axis direction is 200-400 μm with a step resolution of 50-1000 nm; The biological nanopore probe comprises a glass capillary, the tip of which is hydrophobized to construct a phospholipid bilayer and embed a porin to form a single-channel biological nanopore probe; The array detection pool is a multi-well plate structure, with a single hole diameter of ≥5 mm, a depth of 100 μm-1 cm, and a solution depth in the hole of ≥50 μm.

2. The controllable biological nanopore sensing detection system according to claim 1, characterized in that: It also includes a low-noise, high-speed current detection amplifier and a host controller. The three-dimensional nano-manipulation platform is provided with a detection cell installation area for installing an array detection cell; the electrode holder of the current detection device is fixed on the three-dimensional micron manipulation platform; the detection electrode is fixed in the electrode holder, and the reference electrode is placed in the small hole of the detection cell.

3. The controllable biological nanopore sensing detection system according to claim 2, characterized in that: The biological nanopore probe preparation method is as follows: S1: Use a glass capillary puller to prepare a glass probe with a tip diameter of 50nm-10μm, heat seal it, and polish it to prepare a micron pore with a diameter of 10-100μm; S2: Activate the hydroxyl groups on the surface of the glass probe hole by treating it with piranha wash or nitric acid, and then hydrophobize the glass probe hole with a fluorosilane reagent; inject electrolyte into the interior of the glass probe, fix the hydrophobic glass hole on the holder, and connect it to the detection electrode; S3: A phospholipid bilayer is constructed in the glass probe hole by the immersion method or the blowing method, and a triangular wave voltage is applied to detect the membrane formation. After the phospholipid bilayer membrane is formed, a pore protein is added to the detection pool outside the nanopore. Under a relatively low negative voltage (<100mV), the pore protein is driven to self-assemble on the phospholipid bilayer on the glass probe hole to embed the protein pore channel, thereby constructing a single-channel protein biological nanopore and preparing the biological nanopore probe.

4. The controllable biological nanopore sensing detection system according to claim 3, characterized in that: The fluorosilane reagent is a fluoroalkylsilane compound having a carbon chain structure, which is any one of 3-cyanopropyldimethylfluorosilane, (tridecafluoro-1,1,2,2-tetrahydrooctyl)dimethylchlorosilane, and (heptadecafluoro-1,1,2,2-tetrahydrodecyl)dimethylchlorosilane; The phospholipid is a polymerizable polyenoyl lipid, which is any one of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine.

5. The controllable biological nanopore sensing detection system according to claim 4, characterized in that: The biological nanopore is a channel protein, which is any one of α-hemolysin, Mycobacterium smegmatis porin, bacteriophage phi29 connector protein, Aeromonas lysin, curli pilus σS-dependent growth subunit G pore, Fragaceatoxin C, cytotoxin K, outer membrane protein F, and engineered transmembrane protein.

6. The detection method of the controllable biological nanopore sensing detection system according to any one of claims 1 to 5, characterized in that: The detection steps are as follows: S1: Add the sample to be tested into the array detection pool, which is placed on a three-dimensional nanomanipulation platform; S2: Manipulate the three-axis micrometer drive module to control the biological nanopore probe to enter the detection cell pore of the detection cell; S3: Manipulate the three-axis nano-drive module to slowly insert the biological nanopore probe into the solution in the detection cell pore, adjust the voltage to capture the analyte into the biological nanopore, and obtain the current blockade signal of the analyte; S4: Obtain the concentration of the test sample through signal processing and analysis.

7. Use of the controllable biological nanopore sensing detection system according to any one of claims 1 to 5 in detecting biological molecules.

8. The use according to claim 7, characterized in that: The biomolecules are nucleic acids, proteins, and polypeptides.

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