A protein detection method based on an array-type nanopore structure

Abstract

The present invention relates to the technical field of protein detection, and discloses a protein detection method based on an arrayed nanopore structure, comprising the following steps: Step 1, processing array grooves on a chip thin film; Step 2, connecting a temperature control element to the bottom of the chip thin film; Step 3, fixing a protein at the bottom of the array grooves; Step 4, covering a phospholipid layer on the surface of the chip thin film; Step 5, electrically driving the embedding of functional proteins. By adopting the above-mentioned protein detection method based on an arrayed nanopore structure, the relative distance between two functional proteins in the nanopore structure can be adjusted by controlling the thickness of the chip film and the processing depth of the grooves, effectively solving the problem of short read lengths caused by the nano-space limitation of the peptide chain. At the same time, the arrayed combined protein sensor can also achieve high-throughput detection of peptide chains, realizing stable, long-read-length, and highly accurate protein sequencing.

Description

Technical Field

[0001] The present invention relates to the technical field of protein detection, and in particular to a protein detection method based on an arrayed nanopore structure. Background Art

[0002] Modern proteomics largely relies on tandem mass spectrometry (MS) technology, which is valued for its high precision and the ability to identify and quantify proteins in complex mixtures. However, most mass spectrometers are bulky, have high investment costs, expensive maintenance fees, 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 technologies.

[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 an analyte passes through a single nanopore, an ion current passing through the nanopore is blocked under the action of an applied potential. Importantly, the magnitude of the current blockage is mainly proportional to the volume excluded by the analyte, which allows for size discrimination of chemically similar (bio)polymers such as PEG chains, DNA, proteins, and peptides. 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, it has been proven that bio-nanopore detection technology can achieve highly sensitive detection and resolution of 20 amino acids. Recently, Martin-Baniandres et al. [1] used an engineered charge-selective nanopore and utilized electroosmosis to achieve non-enzymatic capture, unfolding, and translocation of a single peptide chain, and realized the detection of post-translational modifications inside the peptide chain. The length of these peptide chains can exceed 1200 amino acid residues, providing a possibility for nanopores to achieve long-read protein sequencing. Nova et al. [2] utilized the Hel308 helicase to control the passage of peptide chains through the sensing region of the nanopore, realized the detection of phosphorylation modifications at the single-molecule level, and could distinguish peptide sequences with one or two close phosphorylation sites with an accuracy of 95%. However, due to the spatial limitation of the nanopore, the detection length of this technology can only be 15-20 amino acids. Motone et al. [3]Using the ClpX helicase nanopore to pull proteins through CsgG enables long-distance, single-molecule reading of individual protein molecules and realizes the sequencing of complete protein chains. However, this method involves the pre-treatment of the target peptide chain and the binding efficiency of the ClpX helicase, which to a certain extent limits the application of this technology. Therefore, challenges such as short read lengths, poor stability, and low accuracy still exist in protein (peptide segment) sequencing. Therefore, achieving stable, long-read, and highly accurate protein sequencing is of great significance for life science research.

[0005] Therefore, a protein sequencing method based on an arrayed composite nanopore structure is proposed. First, array grooves are fabricated on a chip membrane, and a protein with DNA strand synthesis or unwinding function is fixed at the bottom of each groove; then, a phospholipid bilayer is laid on top of the grooves, and a protein with high-resolution reading ability for protein and DNA molecules is embedded in the phospholipid bilayer on the upper layer of the grooves; when the DNA molecule modified with a peptide chain enters the groove through the reading protein embedded in the phospholipid bilayer and binds to the functional protein at the bottom of the groove, the functional protein fixed at the bottom of the groove starts to synthesize or unwind the DNA molecule, driving the peptide chain-modified part on the DNA molecule through the reading protein embedded in the phospholipid bilayer, generating different blocked current amplitudes, and completing the reading of the peptide chain modified on the DNA molecule. The relative distance between the two functional proteins is the key factor affecting the read length of the peptide chain modified on the DNA molecule. In this method, the relative distance between the two functional proteins can be controlled by the thickness of the chip membrane and the processing depth of the grooves, effectively solving the problem of short read lengths caused by the nano-space limitation of the peptide chain. At the same time, this arrayed composite protein sensor can also achieve high-throughput detection of peptide chains.

[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 of the Invention

[0009] The object of the present invention is to provide a protein detection method based on an arrayed nanopore structure, in which the relative distance between two functional proteins in the nanopore structure can be adjusted by controlling the thickness of the chip membrane and the processing depth of the grooves, effectively solving the problem of short read lengths caused by nano-space limitations of peptide chains; at the same time, the arrayed combined protein sensor can also achieve high-throughput detection of peptide chains, realizing stable, long-read, and highly accurate protein sequencing.

[0010] To achieve the above object, the present invention provides a protein detection method based on an arrayed nanopore structure, comprising the following steps:

[0011] 1) Construct an arrayed nanopore structure:

[0012] Step 1, perform array groove processing on the chip film: Process array grooves with a certain pitch and depth on the chip film;

[0013] Step 2, connect a temperature control element to the bottom of the chip film: Connect the temperature control element to the bottom of the chip film;

[0014] Step 3, fix the protein at the bottom of the array grooves: Fix the protein with the functions of synthesizing and unwinding DNA strands at the bottom of each array groove;

[0015] Step 4, cover the surface of the chip film with a phospholipid layer: Apply a phospholipid bilayer on the surface of the chip film to form a phospholipid bilayer on the surface of the array grooves;

[0016] Step 5, electrically drive the embedding of functional proteins: Embed proteins with high-resolution reading ability for proteins and DNA molecules into the phospholipid bilayer on the surface of the array groove, obtaining an array-type nanopore structure;

[0017] 2) Perform protein detection:

[0018] Step 6, the peptide-chain modified DNA is driven by an electric field force to pass through the functional protein embedded in the phospholipid bilayer, generating a blocked current signal; at this time, start the temperature control element to heat the electrolyte solution until the normal operating temperature of the protein at the bottom of the array groove is reached; when the peptide-chain modified DNA moves to bind to the protein at the bottom of the array groove, during the process of the protein at the bottom of the array groove synthesizing or unwinding double-stranded DNA using the single-stranded DNA molecule as a template, drive the peptide chain modified on the DNA strand to pass through the protein with high-resolution reading ability, realizing the information reading of the peptide chain.

[0019] Further, in Step 1, the array grooves are rectangular arrays or circular arrays, the spacing of the arrays is 100 nm - 100 μm, and the depth of the array grooves is 100 nm - 1 mm.

[0020] Further, in Step 2, the temperature control element includes a Peltier element.

[0021] Further, in Step 3, the proteins with the functions of synthesizing and unwinding DNA strands include DNA polymerase, DNA helicase, DNA topoisomerase, etc.

[0022] Further, in Step 5, the proteins with high-resolution reading ability for proteins and DNA molecules include MspA, SP1, α-HL, aerolysin, Phi29, CsgG, SPP1, FraC.

[0023] The detection principle of the present invention is:

[0024] The peptide-chain modified DNA is driven by an electric field force to pass through the functional protein embedded in the phospholipid bilayer, generating a blocked current signal; at this time, start the temperature control element to heat the electrolyte solution until the normal operating temperature of the protein at the bottom of the array groove is reached; when the peptide-chain modified DNA moves to bind to the protein at the bottom of the array groove, during the process of the protein at the bottom of the array groove synthesizing or unwinding double-stranded DNA using the single-stranded DNA molecule as a template, drive the peptide chain modified on the DNA strand to pass through the protein with high-resolution reading ability. The relative distance between the two functional proteins is controlled by controlling the chip film thickness and the processing depth of the groove, which can solve the problem of short reading length caused by the nano-space limitation of the peptide chain.

[0025] The advantages and positive effects of the protein detection method based on the array-type nanopore structure described in the present invention are:

[0026] 1. In the present invention, array grooves are processed on a chip thin film, and a protein with the function of synthesizing or unwinding DNA strands is fixed at the bottom of each groove. Then, a phospholipid bilayer is laid on top of the grooves, and a protein with high-resolution reading ability for proteins and DNA molecules is embedded in the phospholipid bilayer on the upper layer of the grooves. When the peptide chain-modified DNA molecule enters the groove through the reading protein embedded in the phospholipid bilayer and binds to the functional protein at the bottom of the groove, the functional protein fixed at the bottom of the groove begins to synthesize or unwind the DNA molecule, driving the peptide chain-modified part on the DNA molecule through the reading protein embedded in the phospholipid bilayer, generating different blocked current amplitudes, and completing the reading of the peptide chain modified on the DNA molecule.

[0027] 2. The relative distance between the two functional proteins in the present invention is the key factor affecting the reading length of the peptide chain modified on the DNA molecule. The relative distance between the two functional proteins can be controlled by controlling the film thickness of the chip and the processing depth of the grooves, effectively solving the problem of short reading length caused by the nano-space limitation of the peptide chain. At the same time, the arrayed combined protein sensor can also achieve high-throughput detection of the peptide chain.

[0028] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings

[0029] Figure 1 Schematic diagram of the arrayed nanopore structure in the embodiment of the present invention;

[0030] Figure 2 Schematic diagram of protein detection in the embodiment of the present invention;

[0031] Figure 3 Flow chart of protein detection in the embodiment of the present invention;

[0032] Figure 4 Current diagram during protein detection in the embodiment of the present invention, where A is Figure 3 the current diagram of step 5 in Figure 3 the current diagram of step 6 in Figure 3 the current diagram of step 7 in

[0033] Reference Signs

[0034] 1. Thin film chip; 2. Metal layer; 3. Peltier; 4. Protein with the function of synthesizing or unwinding DNA strands; 5. Phospholipid bilayer; 6. Protein with high resolution for proteins or DNA. Detailed Description of the Specific Embodiment

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0036] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the field to which the present invention pertains.

[0037] Unless otherwise defined, the instruments and reagents used in the present invention are all commercially available as conventional products.

[0038] A method for preparing an arrayed nanopore structure includes the following steps:

[0039] Step 1, processing array grooves on the chip film: Array grooves with a certain spacing and depth are processed on the chip film. The array grooves are a rectangular array or a circular array. The spacing of the array is 100 nm - 100 μm, and the depth of the array grooves is 100 nm - 1 mm.

[0040] Step 2, connecting a temperature control element to the bottom of the chip film: Connect the temperature control element to the bottom of the chip film, where the temperature control element is a Peltier.

[0041] Step 3, fixing the protein at the bottom of the array grooves: Fix the protein having the functions of synthesizing and unwinding DNA strands at the bottom of each array groove.

[0042] Step 4, covering the surface of the chip film with a phospholipid layer: Apply a phospholipid bilayer on the surface of the chip film to form a phospholipid bilayer on the surface of the array grooves.

[0043] Step 5, electrically driving the embedding of functional proteins: Embed the protein having a high-resolution reading ability for protein and DNA molecules into the phospholipid bilayer on the surface of the array grooves to obtain the arrayed nanopore structure ( Figure 1 as shown).

[0044] Among them, the proteins having the functions of synthesizing and unwinding DNA strands include DNA polymerase, DNA helicase, DNA topoisomerase, etc. The proteins having a high-resolution reading ability for protein and DNA molecules include MspA, SP1, α-HL, aerolysin, Phi29, CsgG, SPP1, FraC, etc.

[0045] The specific process of detection is ( Figure 2 as shown):

[0046] The peptide chain-modified DNA generates a blocked current signal under the drive of an electric field force through a functional protein embedded in the phospholipid bilayer. At this time, the temperature control element is activated to heat up the electrolyte solution until the normal operating temperature of the protein at the bottom of the array groove is reached. When the peptide chain-modified DNA moves to bind to the protein at the bottom of the array groove, during the process of the protein at the bottom of the array groove synthesizing or unwinding double-stranded DNA using single-stranded DNA molecules as templates, the peptide chain modified on the DNA strand is driven through a protein with high-resolution reading ability to achieve the information reading of the peptide chain.

[0047] Taking a thin film chip made of Si3N4 material, the protein phi29-DNAP with the functions of synthesizing and unwinding DNA strands, and the protein MspA with high-resolution reading ability as examples, the following is a detailed description.

[0048] Example

[0049] A protein detection method based on an array-type nanopore structure includes the following steps (the specific process is as Figure 3 shown):

[0050] 1. Prepare an array-type nanopore structure ( Figure 1 shown):

[0051] (1) Process the array grooves and solid-state nanopores on the chip thin film: Place a clean Si3N4 thin film chip in the vacuum chamber of a focused ion beam processor. Use a focused gallium ion beam to bombard the surface of the Si3N4 thin film to process the array grooves;

[0052] (2) Connect a Peltier element to the bottom of the chip: Connect the Peltier element to the bottom of the chip;

[0053] (3) Fix the protein at the bottom of the array groove: Place the chip with processed array grooves in the electrolyte solution. Chemically modify the gold layer at the bottom of the groove through 11-Mercaptoundecanoic Acid to provide a binding site for the fixation of the protein at the bottom of the groove. Add the phi29-DNAP solution to the electrolyte solution, and through the NHS / EDC reaction, make phi29-DNAP move to the bottom of the groove and bind to the corresponding position of the bottom gold layer, stably existing at the bottom of the groove;

[0054] (4) Add the processed single-stranded DNA molecule with a 12bp hairpin structure at the front end modified with a peptide chain to the electrolyte solution, and the hairpin structure recognizes and binds to phi29-DNAP at the bottom of the groove.

[0055] (5) Cover the surface of the thin-film chip with a phospholipid layer: Add phospholipids to the electrolyte solution in the environment where the chip is located, and repeatedly suck-in and inject the electrolyte solution to assemble a phospholipid bilayer on the thin-film chip, so that the upper layer of the array grooves on the chip is covered by the phospholipid bilayer;

[0056] (6) Electrically drive functional proteins into the groove structure of the chip array: Connect electrodes to both ends of the chip array grooves, and add a protein solution containing MspA. Due to the driving force of the electric field, MspA is embedded into the phospholipid bilayer on the upper layer of the grooves;

[0057] 2. Protein detection based on the arrayed nanopore structure ( Figure 2 as shown):

[0058] (7) Detection of the read length of the peptide chain: The single-stranded DNA molecule modified with the peptide chain passes through MspA embedded in the phospholipid bilayer under the driving force of the electric field, generating a blocking current signal ( Figure 4 as shown). At this time, start the Peltier element to heat up the electrolyte solution until the normal operating temperature of phi29-DNAP at the bottom of the groove is reached. When phi29-DNAP synthesizes or unwinds double-stranded DNA using the single-stranded DNA molecule as a template, it drives the peptide chain modified on the DNA strand through MspA to realize the information reading of the peptide chain.

[0059] The relative distance between the two functional proteins is the key factor affecting the read length of the peptide chain modified on the DNA molecule. In this application, the relative distance between the two functional proteins can be controlled by controlling the film thickness of the chip and the processing depth of the grooves, effectively solving the problem of short read length caused by the nano-space limitation of the peptide chain. At the same time, the arrayed groove-type combined protein sensor can also achieve high-throughput detection of the peptide chain.

[0060] Therefore, the present invention adopts the above-mentioned protein detection method based on the arrayed nanopore structure. The relative distance between the two functional proteins in the nanopore structure can be adjusted by controlling the film thickness of the chip and the processing depth of the grooves, effectively solving the problem of short read length caused by the nano-space limitation of the peptide chain; at the same time, the arrayed combined protein sensor can also achieve high-throughput detection of the peptide chain, realizing stable, long-read-length, and highly accurate protein sequencing.

[0061] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, 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 invention.

Claims

1. A protein detection method based on an array nano - pore structure, characterized in that, It includes the following steps: 1) Construct an array-type nanopore structure: Step 1, perform array groove processing on the chip thin film: Process array grooves with spacing and depth on the chip thin film; Step 2, connect a temperature control element to the bottom of the chip thin film: Connect the temperature control element to the bottom of the chip thin film; Step 3, fix the protein at the bottom of the array groove: Fix the protein with the function of synthesizing and unwinding DNA strands at the bottom of each array groove; Step 4, cover the surface of the chip thin film with a phospholipid layer: Apply a phospholipid bilayer on the surface of the chip thin film to form a phospholipid bilayer on the surface of the array groove; Step 5, electro-drive the embedding of functional proteins: Embed the protein with high-resolution reading ability for protein and DNA molecules into the phospholipid bilayer on the surface of the array groove to obtain an array-type nanopore structure; 2) Perform protein detection: Step 6, the DNA modified with a peptide chain passes through the functional protein embedded in the phospholipid bilayer under the drive of an electric field force, generating a blocking current signal; at this time, start the temperature control element to heat up the electrolyte solution until it reaches the normal working temperature of the protein at the bottom of the array groove; when the DNA modified with a peptide chain moves to bind to the protein at the bottom of the array groove, the protein at the bottom of the array groove uses the single-stranded DNA molecule as a template, and in the process of synthesizing or unwinding double-stranded DNA, drives the peptide chain modified on the DNA strand through the protein with high-resolution reading ability to realize the information reading of the peptide chain.

2. The protein detection method based on an array nano - pore structure according to claim 1, wherein: In Step 1, the array grooves are rectangular arrays or circular arrays, the spacing of the arrays is 100 nm - 100 μm, and the depth of the array grooves is 100 nm - 1 mm.

3. The protein detection method based on an array nano-pore structure according to claim 1, wherein: In Step 2, the temperature control element includes a Peltier element.

4. A protein detection method based on an arrayed nanopore structure according to claim 1, characterized in that: In Step 3, the protein with the function of synthesizing and unwinding DNA strands includes DNA polymerase, DNA helicase or DNA topoisomerase.

5. A protein detection method based on an array-type nanopore structure according to claim 1, characterized in that: In Step 5, the protein with high-resolution reading ability for protein and DNA molecules includes MspA, SP1, α-HL, aerolysin, Phi29, CsgG, SPP1 or FraC.

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