Protein detection method based on array type nanopore structure

Abstract

The invention relates to the technical field of protein detection, and discloses a protein detection method based on an array nanopore structure, which comprises the following steps: step 1, array groove processing of a chip film, step 2, connection of the bottom of the chip film with a temperature control element, step 3, fixation of protein at the bottom of the array groove, and step 4, detection of the array nanopore structure. The surface of the chip film is covered with a phospholipid layer; and 5, electrically driving the embedded functional protein. According to the protein detection method based on the array type nano-pore structure, the relative distance between two functional proteins in the nano-pore structure can be adjusted through the thickness of a chip film and the processing depth of a groove, so that the problem of reading length of a peptide chain caused by limitation of a nano space is effectively solved; meanwhile, the array type combined protein sensor can also realize high-throughput detection on peptide chains, and stable, long-reading-length and high-accuracy protein sequencing is realized.

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 array nanopore structure. Background Art

[0002] Modern proteomics relies heavily on tandem mass spectrometry (MS), which is valued for its high precision and ability to identify and quantify proteins in complex mixtures. However, most mass spectrometers are bulky, have high investment costs, are expensive to maintain, and require professional personnel to operate. 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 that can adapt to the native environment. In nanopore analysis, when the analyte passes through a single nanopore, the ionic 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 size differentiation of chemically similar (bio)polymers such as PEG chains, DNA, proteins, and peptides. In addition, nanopores can accurately detect a variety of molecules, including proteins and DNA, and have the advantages of being label-free, rapid, and highly accurate at the single-molecule level.

[0004] At present, biological nanopore detection technology has been proven to achieve highly sensitive detection and resolution of 20 amino acids. Recently, Martin-Baniandres et al. [1] Using an engineered charged selective nanopore, the electroosmosis phenomenon is used to achieve non-enzymatic capture, unfolding and transport of single peptide chains, and to detect post-translational modifications within peptide chains. 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] By using Hel308 helicase to control the peptide chain to pass through the sensing area of ​​the nanopore, the detection of phosphorylation modification at the single molecule level was achieved, and the peptide sequence with one or two close phosphorylation sites could be distinguished with 95% accuracy. 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]By using the ClpX helicase nanopore to pull proteins through CsgG, single protein molecules can be read over long distances and in a single molecule, thus achieving sequencing of complete protein chains. However, this method involves the pre-treatment of the target peptide chain and the problem of ClpX helicase binding efficiency, which limits the application of this technology to a certain extent. Therefore, protein (peptide) sequencing still faces challenges such as short read length, poor stability, and low accuracy. Therefore, achieving stable, long read length, and highly accurate protein sequencing is of great significance to life science research.

[0005] Therefore, a protein sequencing method based on an array-type combined nanopore structure is proposed. First, an array groove is processed on a chip membrane, and a protein with a synthesis or unwinding function for a DNA chain is fixed at the bottom of each groove; then a layer of phospholipid bilayer is laid on the top of the groove, and a protein with a high-resolution reading ability for proteins and DNA molecules is embedded in the phospholipid bilayer on the upper layer of the groove; when the peptide-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-modified part of the DNA molecule to pass through the reading protein embedded in the phospholipid bilayer, generating different blocking current amplitudes, and completing the reading of the peptide chain modified on the DNA molecule. The relative distance between the two functional proteins is a 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 chip membrane thickness and the processing depth of the groove, which effectively solves the problem of short read length of the peptide chain caused by nanospace limitations. At the same time, this array-type combined 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). [2]Nova, IC, Ritmejeris, J., Brinkerhoff, H. et al. Detection of phosphorylation post-translational modifications along single peptides with nanopores. Nat Biotechnol 42, 710–714 (2024). [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

[0007] The purpose of the present invention is to provide a protein detection method based on an array nanopore structure. The relative distance between two functional proteins in the nanopore structure can be adjusted by adjusting the chip membrane thickness and the processing depth of the groove, which effectively solves the problem of short read length of the peptide chain caused by the limitation of nano space; at the same time, the array-type combined protein sensor can also realize high-throughput detection of peptide chains and realize stable, long read length and highly accurate protein sequencing.

[0008] To achieve the above object, the present invention provides a protein detection method based on an array nanopore structure, comprising the following steps: 1) Construction of array nanopore structure: Step 1, processing array grooves on the chip film: processing array grooves with a certain spacing and depth on the chip film; Step 2, connecting the temperature control element to the bottom of the chip film: connecting the temperature control element to the bottom of the chip film; Step 3, fix the protein at the bottom of the array groove: fix the protein with the function of synthesizing and unwinding the DNA chain at the bottom of each array groove; Step 4, covering the chip film surface with a phospholipid layer: applying a phospholipid bilayer on the chip film surface to form a phospholipid bilayer on the surface of the array groove; Step 5, electrically driven embedding of functional proteins: embedding proteins with high-resolution reading capabilities for proteins and DNA molecules into the phospholipid bilayer on the surface of the array grooves to obtain an array nanopore structure; 2) Perform protein detection: Step 6, the DNA modified by the peptide chain is driven by the electric field force through the functional protein embedded in the phospholipid bilayer to generate a blocking current signal; at this time, the temperature control element is started to heat the electrolyte solution until it reaches the normal working temperature of the protein at the bottom of the array groove; when the DNA modified by the 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 to synthesize or unwind the double-stranded DNA, and drives the peptide chain modified on the DNA chain through the protein with high-resolution reading ability to realize the information reading of the peptide chain.

[0009] Furthermore, in step 1, the array grooves are a rectangular array or a circular array, the array spacing is 100nm-100μm, and the depth of the array grooves is 100nm-1mm.

[0010] Furthermore, in step 2, the temperature control element includes a Peltier element.

[0011] Furthermore, in step 3, the proteins having the functions of synthesizing and unwinding the DNA chain include DNA synthase, DNA helicase, DNA topoisomerase and the like.

[0012] Furthermore, the proteins with high-resolution reading capabilities for proteins and DNA molecules in step 5 include MspA, SP1, α-HL, aerolysin, Phi29, CsgG, SPP1, and FraC.

[0013] The detection principle of the present invention is: Driven by the electric field force, the DNA modified with peptide chains passes through the functional protein embedded in the phospholipid bilayer to generate a blocking current signal; at this time, the temperature control element is started to heat 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 peptide chains 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 to synthesize or unwind the double-stranded DNA, driving the peptide chain modified on the DNA chain to pass through the protein with high-resolution reading capability. The relative distance between the two functional proteins is controlled by the chip film thickness and the processing depth of the groove, which can solve the problem of short read length of the peptide chain caused by nano-space limitations.

[0014] The advantages and positive effects of the protein detection method based on the array nanopore structure described in the present invention are: 1. The present invention processes array grooves on a chip film, and fixes a protein with the function of synthesizing or unwinding the DNA chain at the bottom of each groove; then a phospholipid bilayer is laid on the top of the groove, and the protein with high-resolution reading ability of protein and DNA molecules is embedded in the phospholipid bilayer on the upper layer of the groove; when the peptide chain-modified DNA molecule enters the groove through the reading protein embedded in the phospholipid bilayer and combines with 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 of the DNA molecule to pass through the reading protein embedded in the phospholipid bilayer, generating different blocking current amplitudes, and completing the reading of the peptide chain modified on the DNA molecule.

[0015] 2. The relative distance between the two functional proteins in the present invention is a key factor affecting the read length of the peptide chain modified on the DNA molecule. The relative distance between the two functional proteins can be controlled by the chip film thickness and the processing depth of the groove, which effectively solves the problem of short read length of the peptide chain caused by nano-space limitations. At the same time, the array-type combined protein sensor can also achieve high-throughput detection of peptide chains.

[0016] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the array nanopore structure in an embodiment of the present invention; Figure 2 This is a schematic diagram of protein detection in an embodiment of the present invention; Figure 3 This is a flow chart of protein detection in an embodiment of the present invention; Figure 4 The current diagram of the protein detection process in the embodiment of the present invention, wherein A is Figure 3 The current diagram of step 5 in the figure, B is Figure 3 The current diagram of step 6 in the figure, C is Figure 3 Current diagram for step 7 in .

[0018] Reference numerals 1. Thin film chip; 2. Metal layer; 3. Peltier; 4. Protein with the function of synthesizing or unwinding DNA chain; 5. Phospholipid bilayer; 6. Protein with high resolution for protein or DNA. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0020] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0021] Unless otherwise defined, the instruments and reagents used in the present invention are conventionally commercially available.

[0022] A method for preparing an array-type nanopore structure comprises the following steps: Step 1, array groove processing on the chip film: array grooves with a certain pitch and depth are processed on the chip film. The array grooves are rectangular arrays or circular arrays. The array pitch is 100nm-100μm, and the depth of the array grooves is 100nm-1mm.

[0023] Step 2, connecting a temperature control element to the bottom of the chip film: connecting the temperature control element to the bottom of the chip film, wherein the temperature control element is a Peltier; Step 3, fix the protein at the bottom of the array groove: fix the protein with the function of synthesizing and unwinding the DNA chain at the bottom of each array groove; Step 4, covering the chip film surface with a phospholipid layer: applying a phospholipid bilayer on the chip film surface to form a phospholipid bilayer on the surface of the array groove; Step 5, electrically driven embedded functional protein: embed the protein with high-resolution reading capability for protein and DNA molecules into the phospholipid bilayer on the surface of the array groove to obtain an array nanopore structure ( Figure 1 as shown).

[0024] Among them, proteins that have the function of synthesizing and unwinding DNA chains include DNA synthase, DNA helicase, DNA topoisomerase, etc.; proteins that have the ability to read proteins and DNA molecules with high resolution include MspA, SP1, α-HL, aerolysin, Phi29, CsgG, SPP1, FraC, etc.

[0025] The specific process of the detection is as follows: Figure 2 shown): Driven by the electric field force, the DNA modified with peptide chains passes through the functional protein embedded in the phospholipid bilayer to generate a blocking current signal; at this time, the temperature control element is started to heat 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 peptide chains 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 to synthesize or unwind the double-stranded DNA, driving the peptide chain modified on the DNA chain through a protein with high-resolution reading capability to realize information reading of the peptide chain.

[0026] Below is Si 3 N4 The thin film chip made of this material is used to explain in detail the protein phi29-DNAP with the function of synthesizing and unwinding DNA chains and the protein MspA with high-resolution reading ability.

[0027] Example The protein detection method based on the array nanopore structure includes the following steps (the specific process is as follows Figure 3 shown): 1. Preparation of array nanopore structure ( Figure 1 shown): (1) Chip film array groove and solid-state nanopore processing: clean Si 3 N 4 The thin film chip is placed in a vacuum chamber processed by a focused ion beam. A focused gallium ion beam is used to bombard the Si 3 N 4 The film surface is processed to obtain an array of grooves; (2) Connecting the Peltier element to the bottom of the chip: Connect the Peltier element to the bottom of the chip; (3) Fix the protein at the bottom of the array groove: Place the chip with the array groove in the electrolyte solution, and chemically modify the gold layer at the bottom of the groove with 11-Mercaptoundecanoic Acid to provide binding sites for the protein to be fixed at the bottom of the groove. Add the solution containing phi29-DNAP 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 lower gold layer, so that it exists stably at the bottom of the groove; (4) The treated single-stranded DNA molecule with a 12 bp hairpin structure at the front end modified by the peptide chain is added to the electrolyte solution. The hairpin structure recognizes and binds to the phi29-DNAP at the bottom of the groove.

[0028] (5) Covering the surface of the thin film chip with a phospholipid layer: adding phospholipids to the electrolyte solution in the chip environment, and repeatedly absorbing and injecting the electrolyte solution to assemble a phospholipid bilayer on the thin film chip, so that the upper layer of the array groove on the chip is covered with the phospholipid bilayer; (6) Functional protein electrically driven to embed into the chip array groove structure: Electrodes are connected to both ends of the chip array groove, and a protein solution containing MspA is added. Due to the driving effect of the electric field force, MspA is embedded into the phospholipid bilayer on the upper layer of the groove; 2. Protein detection based on arrayed nanopore structures ( Figure 2 shown): (7) Peptide chain read length detection: The single-stranded DNA molecule modified with the peptide chain is driven by the electric field force through the MspA embedded in the phospholipid bilayer, generating a blocking current signal ( Figure 4As shown). At this time, the Peltier element is started to heat the electrolyte solution until it reaches the normal operating temperature of phi29-DNAP at the bottom of the groove. In the process of synthesizing or unwinding double-stranded DNA using single-stranded DNA molecules as templates, phi29-DNAP drives the peptide chain modified on the DNA chain to pass through MspA to read the information of the peptide chain.

[0029] The relative distance between the two functional proteins is a 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 the chip film thickness and the processing depth of the groove, which effectively solves the problem of short read length of the peptide chain caused by nano-space limitations. At the same time, the array groove type combined protein sensor can also achieve high-throughput detection of peptide chains.

[0030] Therefore, the present invention adopts the above-mentioned protein detection method based on the array nanopore structure. The relative distance between the two functional proteins in the nanopore structure can be adjusted by adjusting the chip membrane thickness and the processing depth of the groove, which effectively solves the problem of short read length of the peptide chain caused by the nanospace limitation; at the same time, the array-type combined protein sensor can also realize high-throughput detection of peptide chains, and realize stable, long read length, and highly accurate protein sequencing.

[0031] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A protein detection method based on an array nanopore structure, characterized in that: The following steps are involved: 1) Construction of array nanopore structure: Step 1, processing array grooves on the chip film: processing array grooves with a spacing and depth on the chip film; Step 2, connecting the temperature control element to the bottom of the chip film: connecting the temperature control element to the bottom of the chip film; Step 3, fix the protein at the bottom of the array groove: fix the protein with the function of synthesizing and unwinding the DNA chain at the bottom of each array groove; Step 4, covering the chip film surface with a phospholipid layer: applying a phospholipid bilayer on the chip film surface to form a phospholipid bilayer on the surface of the array groove; Step 5, electrically driven embedding of functional proteins: embedding proteins with high-resolution reading capabilities for proteins and DNA molecules into the phospholipid bilayer on the surface of the array grooves to obtain an array nanopore structure; 2) Perform protein detection: Step 6, the DNA modified by the peptide chain is driven by the electric field force through the functional protein embedded in the phospholipid bilayer to generate a blocking current signal; at this time, the temperature control element is started to heat the electrolyte solution until it reaches the normal working temperature of the protein at the bottom of the array groove; when the DNA modified by the 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 to synthesize or unwind the double-stranded DNA, and drives the peptide chain modified on the DNA chain through the protein with high-resolution reading ability to realize the reading of the peptide chain information.

2. A protein detection method based on an array nanopore structure according to claim 1, characterized in that: In step 1, the array grooves are rectangular arrays or annular arrays, the array spacing is 100nm-100μm, and the depth of the array grooves is 100nm-1mm.

3. A protein detection method based on an array nanopore structure according to claim 1, characterized in that: In step 2, the temperature control element includes a Peltier element.

4. The protein detection method based on array nanopore structure according to claim 1, characterized in that: In step 3, the protein having the function of synthesizing and unwinding the DNA chain includes DNA synthetase, DNA helicase or DNA topoisomerase.

5. The protein detection method based on array nanopore structure according to claim 1, characterized in that: Proteins with high-resolution readout capabilities for both protein and DNA molecules in step 5 include MspA, SP1, α-HL, aerolysin, Phi29, CsgG, SPP1, or FraC.

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