A protein detection method based on a combined nanopore structure

Abstract

The present application relates to the technical field of protein detection, and discloses a protein detection method based on a combined nanopore structure, comprising the following steps: processing a sandwich film chip surface to form a nanopore, preparing a "hole-cavity-hole" structure, and embedding a functional protein into the sandwich film chip of the "hole-cavity-hole" structure under the action of an electric field for protein detection. The protein detection method based on the combined nanopore structure can effectively overcome the problem of protein detection read length caused by the spatial limitation of the nanopore. Compared with the method of embedding a combined protein with relevant functions into a phospholipid bilayer, embedding a functional protein into a solid nanopore combines the advantages of a biological nanopore and a solid nanopore, reduces the strict requirements of the sensor on the detection environment, and improves the resolution and stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein detection, and particularly relates to a protein detection method based on a combined nanopore structure. BACKGROUND

[0002] Modern proteomics relies heavily on tandem mass spectrometry (MS) technology, which is valued for its high precision and ability to identify and quantify proteins in complex mixtures. However, most mass spectrometry analyzers are bulky, costly to invest, expensive to maintain, and require professional operation. With the growing demand for high-throughput proteomics research and personalized medicine, there is an urgent need to develop scalable and low-cost protein analysis technology.

[0003] Compared with mass spectrometry equipment, nanopore-based detection technology provides a low-cost and high-throughput platform and can adapt to the native environment. In nanopore analysis, when the analyte passes through a single nanopore, it will block the ionic current passing through the nanopore under the action of an applied potential. Importantly, the size of the current block is mainly proportional to the excluded volume of the analyte, which allows size discrimination of (bio)polymers with similar chemical properties such as PEG chains, DNA, proteins, and peptide segments. In addition, nanopores can accurately detect various molecules including proteins and DNA, and have the advantages of label-free, fast, and high precision at the single molecule level.

[0004] Currently, biological nanopore detection technology has been proven to be able to achieve high-sensitivity detection and discrimination of 20 amino acids. Recently, Martin-Baniandres et al. [1] Using an engineered charged selective nanopore, the phenomenon of electroosmosis is used to achieve non-enzymatic capture, unfolding and transport of single polypeptide chains, which can be more than 1200 amino acid residues long, to achieve detection of post-translational modifications inside the polypeptide chain, providing a possibility for nanopore long-read protein sequencing. Nova et al. [2] Using Hel308 helicase to control the sensing region of the peptide chain through the nanopore, detection of post-translational modifications at the single molecule level is achieved, and peptides with one or two closely spaced phosphorylation sites can be distinguished with 95% accuracy, but due to the spatial limitation of the nanopore, the length of the detected chain can only be 15-20 amino acids. Motone et al. [3]Using the ClpX helicase nanopore pulling protein through CsgG, long-distance, single-molecule reading of individual protein molecules can be achieved, and sequencing of intact protein chains can be achieved. However, this method involves the problem of pretreatment of the target peptide chain and the binding efficiency of the ClpX helicase, which to some extent limits the application of this technology. Therefore, in protein (peptide segment) sequencing, there are still challenges such as short read length, poor stability, and low accuracy. Therefore, it is of great significance to realize stable, long-read, and high-accuracy protein sequencing for life science research.

[0005] Therefore, the present application proposes a combined nanopore structure protein detection device and sequencing method to solve the above technical problems.

[0006] [1]Martin-Baniandres, P., Lan, WH., Board, S. et al. Enzyme-lessnanopore detection of post-translational modifications within longpolypeptides. Nat. Nanotechnol. 18, 1335–1340 (2023).

[0007] [2]Nova, I.C., Ritmejeris, J., Brinkerhoff, H. et al. Detection ofphosphorylation post-translational modifications along single peptides withnanopores. Nat Biotechnol 42, 710–714 (2024).

[0008] [3]Motone, K., Kontogiorgos-Heintz, D., Wee, J. et al. Multi-pass,single-molecule nanopore reading of long protein strands. Nature 633, 662–669(2024). SUMMARY

[0009] The application aims to provide a protein detection method based on a combined nanopore structure, which can effectively overcome the protein detection read length problem caused by the spatial limitation of the nanopore. Compared with the method of embedding the combined protein with relevant functions into the phospholipid bilayer, embedding the functional protein into the solid nanopore combines the advantages of the biological nanopore and the solid nanopore, which not only reduces the strict requirements of the sensor on the detection environment, but also improves the resolution and stability.

[0010] To achieve the above-mentioned purpose, the application provides a protein detection method based on a combined nanopore structure, comprising the following steps:

[0011] 1) Constructing a protein detection device of a combined nanopore structure:

[0012] Step 1: Processing the surface of the sandwich film chip, and milling to form a nanopore;

[0013] Step 2: Preparing a "hole-cavity-hole" structure: placing the sandwich film chip with the nanopore prepared in step 1 in a BOE solution in an inclined manner, so that the buffer oxide etchant etches the intermediate layer, and a sandwich film chip with a "hole-cavity-hole" structure is obtained;

[0014] Step 3: Functionally embedding the "hole-cavity-hole" structure sandwich film chip under the action of an electric field: after the sandwich film chip obtained in step 2 is cleaned by the piranha solution, it is loaded into a liquid pool chamber, the liquid pool chamber is filled with electrolyte containing two functional proteins on both sides, and a bias voltage is applied. Under the action of the electric field force, the two functional proteins are embedded into the solid nanopores on both sides of the sandwich film chip in sequence, and a protein detection device based on a combined nanopore structure is obtained;

[0015] 2) Protein detection:

[0016] The functional protein with the function of controlling the translocation speed of biomolecules drives the DNA molecule modified by the peptide chain to pass through the protein with high resolution reading capability, and reads and detects the peptide sequence in the DNA molecule modified by the peptide chain. When the peptide sequence enters the protein with high resolution reading capability, the reading of the peptide sequence shows a change in the current signal.

[0017] Further, in step 2, the etching time is controlled to adjust the volume of the cavity in the intermediate layer. The etching time is 0.5s-30min, and the volume of the cavity in the intermediate layer is 100nm 3 -1×10 5 nm 3 .

[0018] Further, the two functional proteins in step 3 are respectively a functional protein with a function of controlling the translocation speed of biomolecules and a protein with a high-resolution reading capability. Preferably, the functional protein with the function of controlling the translocation speed of biomolecules includes phi29 DNA P, hel308, ClpX helicase, etc. The protein with the high-resolution reading capability includes MspA, alpha-HL, SP1, aerolysin, Phi29, CsgG, SPP1, FraC, etc.

[0019] The detection principle of the present application is as follows:

[0020] The functional protein with the function of controlling the translocation speed of biomolecules drives the peptide chain modified DNA molecule to pass through the protein with the high-resolution reading capability to read and detect the peptide chain sequence in the peptide chain modified DNA molecule. There is a cavity between the two functional proteins, and the thickness of the cavity structure can be adjusted by the manufacturing process, so that different length proteins can be detected, and the problem of protein detection read length caused by the space limitation of the nanopore can be effectively overcome. When the peptide chain sequence enters the protein with the high-resolution reading capability, the reading of the peptide chain sequence shows a change in the current signal.

[0021] The advantages and positive effects of the protein detection method based on the combined nanopore structure according to the present application are as follows:

[0022] In the present application, there is a cavity between the two functional proteins, and the thickness of the cavity structure can be adjusted by the manufacturing process. The prepared combined nanopore can effectively overcome the problem of protein detection read length caused by the space limitation of the nanopore. At the same time, compared with the method of embedding the combined protein with related functions into the phospholipid bilayer, the functional protein is embedded in the solid nanopore in the present application, which combines the advantages of the biological nanopore and the solid nanopore. Not only does it reduce the strict requirements of the sensor on the detection environment, but also improves the resolution and stability.

[0023] The technical solutions of the present application will be further described in detail below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a schematic diagram of the sandwich film chip structure in the embodiment of the present application.

[0025] Figure 2 It is a schematic diagram of the protein molecule detection in the embodiment of the present application.

[0026] Figure 3Figure 1 is a current change graph of the protein sequencing stage in the embodiment of the present application, wherein (1) is the current graph of the sandwich film chip, (2) is the current graph of the sandwich film chip embedded with proteins with high-resolution reading capacity, (3) and (4) are the current graphs of the sandwich film chip embedded with proteins with high-resolution reading capacity, and the proteins with the control of the speed of biomolecular translocation are being embedded, (5) and (6) are the current graphs of the proteins during the sequencing.

[0027] Reference signs

[0028] 1, sandwich film chip; 2, phi29 DNA P; 3, MspA. DETAILED DESCRIPTION

[0029] The technical solutions of the present application are further described below through the accompanying drawings and examples.

[0030] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those skilled in the art to which the present application belongs.

[0031] Unless otherwise defined, the instruments and reagents used in the present application are all commercially available.

[0032] The combined nanopore protein sequencing method comprises the following steps:

[0033] Step 1, processing the surface of the sandwich film chip to mill the nanometer through hole; the processing method is not limited, and the existing processing method is selected according to the needs until the nanometer through hole is drilled.

[0034] Step 2, preparing a "hole-cavity-hole" structure: placing the sandwich film chip with the nanometer through hole prepared in step 1 in a BOE solution at an angle to etch the intermediate layer with a buffer oxide etchant, thereby obtaining a sandwich film chip with a "hole-cavity-hole" structure; the optimal etching time is 3-5 min, and the volume of the intermediate layer cavity is 100 nm 3 -1×10 5 nm 3 .

[0035] Step 3, functional protein electric field driven insertion of sandwich film chip with "hole-cavity-hole" structure: the sandwich film chip obtained in step 2 is loaded into a liquid pool chamber after cleaning with an aiptasia solution, the liquid pool chamber is filled with electrolyte containing two functional proteins on both sides, and a bias voltage is applied, under the action of electric field force, the two functional proteins are inserted into the solid state nanopores on both sides of the sandwich film chip in sequence; the two functional proteins are a functional protein with the function of controlling the translocation speed of biomolecules and a protein with high resolution reading ability. The functional protein with the function of controlling the translocation speed of biomolecules includes DNA synthesis enzyme, DNA helicase, DNA topoisomerase, etc., such as phi29DNAP, hel308, ClpX helicase, etc.; the protein with high resolution reading ability includes MspA, alpha-HL, SP1, aerolysin, Phi29, CsgG, SPP1, FraC, etc.

[0036] A protein with high resolution reading ability for biomolecules is inserted into the first solid state nanopore, and another protein with the function of controlling the translocation speed of the peptide chain modified DNA molecule is driven by electric field force and inserted into the second solid state nanopore (the protein is combined with the peptide chain modified DNA molecule before being inserted into the solid state nanopore). A combined nanopore protein detection device is formed, and then combined nanopore protein sequencing is performed.

[0037] Step 4, sequence read length detection: the protein with the function of biomolecular motor drives the peptide chain modified DNA molecule to pass through the protein with high resolution reading ability to detect the sequence of the target peptide chain.

[0038] The following takes the chip material of the sandwich film as Si3N4 / SiO2 / Si3N4, selects phi29DNAP as the functional protein for controlling the translocation speed of DNA molecules, and selects MspA as the reading protein for biomolecules as an example to explain the present application in detail.

[0039] Embodiment

[0040] The combined nanopore protein sequencing method comprises the following steps:

[0041] (1) Si3N4 / SiO2 / Si3N4 sandwich film chip pretreatment: a piece of Si3N4 / SiO2 / Si3N4 sandwich film chip is selected, and the Si3N4 / SiO2 / Si3N4 sandwich film needs to be pretreated before processing. The specific pretreatment steps include: heating the Si3N4 / SiO2 / Si3N4 sandwich film in an aiptasia solution at 90°C for 30 minutes to remove organic contaminants and other impurities on the surface of the Si3N4 / SiO2 / Si3N4 sandwich film.

[0042] (2) Use relevant processing methods to mill the Si3N4 / SiO2 / Si3N4 sandwich film chip to prepare nano-through holes: The pre-treated Si3N4 / SiO2 / Si3N4 sandwich film chip is processed using a focused ion beam to obtain a through-hole structure.

[0043] (3) Etching silicon oxide cavities with buffered oxide etchant (BOE) to prepare a "hole-cavity-hole" structure: The Si3N4 / SiO2 / Si3N4 sandwich film chip with nano-through holes prepared in step (2) is placed obliquely in the BOE solution and etched for 3-5 minutes to obtain SiO2 cavities of a certain diameter, thereby obtaining a Si3N4 / SiO2 / Si3N4 sandwich film chip with a "hole-cavity-hole" structure.

[0044] (4) Functional protein electric field drives solid-state nanopores embedded in the “pore-cavity-pore” structure: The Si3N4 / SiO2 / Si3N4 sandwich film chip prepared in step (3) is cleaned with piranha solution and loaded into the liquid pool chamber. Electrolyte is filled on both sides of the liquid pool chamber, and a bias voltage is applied, such as Figure 1 As shown, Figure 1 The chip material of the sandwich membrane is Si3N4 / SiO2 / Si3N4. The lower pore of the "pore-cavity-pore" structure contains the protein MspA, which has high-resolution readout capabilities, and the upper pore contains the protein phi29DNAP, which functions to control the translocation rate of biomolecules. A prepared MspA solution is added to one end of the liquid pool. Driven by electrophoretic force, MspA embeds into one of the two solid-state nanopores. Then, a peptide-modified DNA molecule and a complementary DNA template to the peptide-modified DNA are mixed and annealed, making the tail end of the peptide-modified DNA template partially double-stranded. Next, the protein phi29DNAP, which functions as a biomolecular motor, is added and bound to the double-stranded portion of the template tail. It is then added to the other end of the liquid pool. Driven by electrophoretic force, the peptide-modified DNA molecule and the motor protein complex enter and embed into the other solid-state nanopore.

[0045] (5) Protein molecule read length detection ( Figure 2 As shown in the figure: By synthesizing the single-stranded portion of the DNA template chain, the peptide chain modification portion of the DNA is controlled by the protein MspA with high-resolution reading ability to start reading and detecting the sequence of the target protein peptide chain. When the peptide chain sequence enters the process of reading the protein MspA, the reading of the peptide sequence is manifested as a change in the current signal ( Figure 3The peptide segment modified on the DNA strand enters the cavity and moves towards the phi29 DNA P, and the cavity distance between the two proteins determines the reading length of the peptide segment. When the cavity distance is sufficient, the method can complete long-distance, single-molecule reading of a single protein molecule, and realize sequencing of the entire protein chain.

[0046] Figure 3 The current change diagram for each stage of protein sequencing, when the peptide segment modified on the DNA strand enters the cavity and moves towards the driving protein, due to the cavity between the two functional proteins, and the distance of the cavity can be determined according to the actual situation, the problem of reading length caused by the limitation of nanospace of the protein molecule can be effectively overcome, long-distance, single-molecule reading of a single protein molecule can be completed, and protein sequencing can be realized.

[0047] Therefore, the protein detection method based on the combined nanopore structure can effectively overcome the problem of protein detection reading length caused by the limitation of the nanopore space. At the same time, compared with the method of embedding the combined protein with related functions into the phospholipid bilayer, embedding the functional protein into the solid nanopore combines the advantages of the biological nanopore and the solid nanopore, not only reduces the strict requirements of the sensor on the detection environment, but also improves the resolution and stability.

[0048] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application and not to limit it, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand: it can still modify or replace the technical solutions of the present application, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A protein detection method based on a combined nanopore structure, characterized in that: The following steps are involved: 1) Construction of a protein detection device with a modular nanopore structure: Step 1: Processing the surface of the sandwich film chip by milling to form nano-through holes; Step 2, preparing a "pore-cavity-pore" structure: the sandwich film chip with nanopores prepared in step 1 is placed obliquely in a BOE solution, and the buffered oxide etchant etches the intermediate layer to obtain a sandwich film chip with a "pore-cavity-pore" structure; Step 3: Electric field-driven embedding of functional proteins into a sandwich film chip with a "pore-cavity-pore" structure: The sandwich film chip obtained in Step 2 is cleaned with piranha solution and then loaded into a liquid cell chamber. Electrolytes containing the two functional proteins are filled on both sides of the liquid cell chamber. A bias voltage is applied. Under the action of the electric field, the two functional proteins are embedded in the solid-state nanopores on both sides of the sandwich film chip, respectively, to obtain a protein detection device with a modular nanopore structure. Among them, the two functional proteins are a protein that controls the translocation speed of biomolecules and a protein with high-resolution reading capabilities; 2) Perform protein detection: The functional protein that controls the translocation speed of biological molecules drives the peptide-modified DNA molecules to pass through the protein with high-resolution reading ability to read and detect the peptide chain sequence in the peptide-modified DNA molecules. When the peptide chain sequence enters the protein with high-resolution reading ability, the reading of the peptide chain sequence is manifested as a change in the current signal.

2. The protein detection method based on a combined nanopore structure according to claim 1, characterized in that: During etching in step 2, the etching time is controlled to adjust the volume of the middle layer cavity. The etching time is 0.5s-30min, and the volume of the middle layer cavity is 20nm. 3 -1×10 5 nm 3 .

3. The protein detection method based on a combined nanopore structure according to claim 1, characterized in that: Functional proteins that control the translocation rate of biomolecules include phi29DNAP, hel308, or ClpX helicases, and proteins with high-resolution reading capabilities include MspA, α-HL, SP1, aerolysin, Phi29, CsgG, SPP1, or FraC.

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