A single-molecule protein and post-translational modification sensor based on cysteine-functionalized MoS2 nanopore and application thereof in nanopore detection technology

Abstract

The application relates to a cysteine functionalized MoS2 nanopore and application thereof in single-molecule protein and post-translational modification detection. The device realizes the regulation of the interaction between molecules in the pore, the gating state and the transport rate by constructing a peptide chain valve structure fixed by cysteine in the MoS2 nanopore and introducing positively charged and aromatic amino acids. By adjusting the composition of the peptide chain and the voltage condition, the van der Waals force, the electrostatic force and the hydrogen bond force between the protein molecule and the pore can be enhanced, the single-molecule protein can be slowly transported through the pore and the high-resolution ion current detection can be realized. The application can be used for distinguishing different amino acids, protein sequence characteristics and post-translational modification sites such as phosphorylation, and has high sensitivity, high selectivity and good scalable application prospect in protein sequencing, post-translational modification positioning and biomolecular genomics analysis.

Description

Technical Field

[0001] This invention relates to the field of nanopore sensing and single-molecule detection technology, specifically to a single-molecule protein and post-translational modification sensor based on cysteine-functionalized MoS2 nanopores and its application in nanopore detection technology. Background Technology

[0002] Proteins and their post-translational modifications play crucial roles in living organisms; their primary structure and specific post-translational modification sites determine cellular biological functions and signal transduction characteristics. High-resolution detection of protein sequences and post-translational modifications is of great significance for disease diagnosis, drug development, and basic biological research. Traditional protein detection and sequencing methods mainly include Edman degradation and mass spectrometry. Edman degradation relies on chemical labeling and stepwise degradation, which is complex and has low throughput; while mass spectrometry is sensitive, it usually requires high-purity samples, making it difficult to achieve single-molecule resolution, and it also presents challenges in locating complex post-translational modifications. Furthermore, these methods lack the ability to achieve rapid, real-time, and high-throughput analysis, limiting their application at the single-molecule level.

[0003] In recent years, nanopore technology has attracted widespread attention as a novel single-molecule detection method due to its advantages of low cost, high throughput, and no need for chemical labeling. Single-molecule resolved information can be directly obtained by measuring the blocking of ion currents as molecules pass through nanopores. However, existing nanopore detection still faces several technical challenges: the migration speed of proteins and peptides in traditional nanopores is too fast, making single-molecule events difficult to resolve, resulting in short signal duration and limited resolution; unfunctionalized nanopores have limited ability to recognize proteins and post-translational modifications, making it difficult to accurately distinguish amino acids or post-translational modification sites; relying solely on ion current signals is insufficient to resolve amino acid types at different sites on peptides, especially isolating amino acids and different post-translational modifications; existing nanopores cannot simultaneously regulate the protein molecule passage speed, pore wall interaction strength, and molecular channel selectivity, limiting the analytical capabilities for complex biomolecular systems; furthermore, traditional methods have limited utilization of ion current changes, making it difficult to fully extract sequence information and post-translational modification features from single-molecule events.

[0004] Two-dimensional nanopores (such as MoS2) provide an ideal platform for high-resolution single-molecule detection due to their atomic-level thickness, tunable pore size, excellent mechanical strength, and electronic properties. MoS2 nanopores can provide short channel lengths, extending the passage time of single molecules and improving spatial resolution. Simultaneously, surface chemical modification can enhance the non-covalent interactions between proteins and the pore walls, enabling precise recognition of amino acids and post-translational modification sites. Cysteine ​​(Cys) covalently functionalized MoS2 nanopores further fix the functionalized molecular structure through disulfide bonds, making the molecule-pore wall interaction controllable. Combined with adjustments to the applied electric field and solution conditions, "slow passage" of protein molecules can be achieved, enhancing signal resolution accuracy.

[0005] However, existing nanoporous protein detection technologies are still generally limited by problems such as excessively fast molecular permeation speed, insufficient recognition sites within the pores, and subtle differences in protein amino acid and post-translational modification structures, making it difficult to achieve high-resolution resolution of protein sequence information and modification sites at the single-molecule level. In particular, traditional cysteine ​​modification is mostly used only as a means of surface fixation or chemical linkage, lacking the ability to finely regulate the local charge distribution, molecular interaction strength, and permeation dynamics within the pores. Summary of the Invention

[0006] To address the aforementioned issues, this invention discloses a single-molecule protein and post-translational modified sensor based on cysteine-functionalized MoS2 nanopores. Unlike conventional cysteine ​​modification, this invention proposes a cysteine-mediated amino acid-functionalized MoS2 nanopore sensor. Stable cysteine ​​covalent linkage sites are constructed at the edges of the MoS2 nanopores, and functional amino acids with different charges, polarities, hydrophobicities, and aromatic structures are further introduced to form a programmable Cys-X intrapore recognition interface. This structure can synergistically regulate van der Waals interactions, electrostatic interactions, and hydrogen bonding between the protein molecule and the pore wall, and generate a controllable hydrogen bond gating effect under an applied electric field, thereby achieving slow molecular passage through the pore, prolonged residence time, and enhanced ion current signal.

[0007] Based on the aforementioned tunable molecular recognition mechanism, this invention can effectively improve the signal discrimination of different amino acids, isomeric residues, and post-translational modifications such as phosphorylation, providing a highly sensitive, highly selective, and scalable functionalized solid-state nanopore detection platform for single-molecule protein sequencing, post-translational modification localization, and proteomics analysis.

[0008] By adding peptides to the solution, the amino acid sites on the peptides and amino acids at specific sites can be identified through transient hydrogen bonding gating and non-covalent interactions between residues on the peptide chain and pore-edge modifying molecules, resulting in current blocking of specific amplitudes and different degrees of pore passage time.

[0009] The biosensor includes MoS2, cysteine, amino acids, peptides, trans-side, cis-side, working electrode one, working electrode two, ground electrode, probe, and wires;

[0010] The same electrolyte solution is distributed in both the cis and trans sides. Here, monovalent cations are selected as the main ion species, including but not limited to K+. + Li + and Na + The concentration range is 0.1M to 3.0M;

[0011] The cis and trans sides are connected by nanopores in the center of MoS2. The MoS2 is a single-layer or multi-layer structure with central nanopores, serving as a core supporting substrate; the cysteine ​​is anchored and confined to the edge of the MoS2 nanopores due to the S-Mo covalent bond between it and MoS2, thus constructing a chemically selective sensing interface.

[0012] Furthermore, the cysteine ​​can be linked to other amino acid molecules to modulate the physicochemical properties of the sensing interface. Working electrodes one and two, respectively located on the trans and cis sides, together with a ground electrode connected to an external circuit, form a stable sensing loop; a highly sensitive probe is connected to a back-end signal processing system via wires.

[0013] This invention discloses a method for fabricating a cysteine-functionalized MoS2 nanopore sensor, specifically including the following steps:

[0014] (1) Preparation and transfer of MoS2 nanofilms: Monolayer or few-layer MoS2 was prepared on the growth substrate by chemical vapor deposition; MoS2 nanofilms were transferred to a silicon nitride or silicon dioxide support framework with micron-sized pores (diameter of 100nm-500nm) using polymethyl methacrylate as a support layer; then polymethyl methacrylate was removed with acetone and vacuum annealing was performed to obtain a clean suspended MoS2 film.

[0015] (2) Precision fabrication of nanopores: The substrate loaded with MoS2 is placed in a transmission electron microscope or a focused ion beam device, and a high-energy beam is used to bombard the central region of MoS2 to fabricate nanopores with a diameter of 1nm-5nm; during the fabrication process, real-time imaging is used to ensure the accuracy and symmetry of the pore size.

[0016] (3) Surface active site treatment: The processed nanoporous substrate is cleaned with oxygen plasma for 30s-120s to remove carbon contamination adsorbed on the surface and activate the Mo atoms at the edge of the MoS2 pores to provide active sites for subsequent covalent bonding.

[0017] (4) Cysteine ​​covalent functionalization modification: The substrate is immersed in a degassed aqueous solution of L-cysteine, preferably at a concentration of 5 mM-50 mM; the reaction is carried out at 25℃-40℃ for 12-24 h under nitrogen protection. At this time, the thiol group (-SH) of the cysteine ​​side chain is directionally anchored to the Mo atom at the edge of MoS2 through sulfur-molybdenum (S-Mo) covalent coupling.

[0018] (5) Purification and drying: After the reaction is completed, rinse the substrate surface with deionized water and anhydrous ethanol alternately 3-5 times each to completely remove the non-covalently bound molecules that are physically adsorbed; then slowly dry it with high-purity nitrogen and place it in a vacuum drying oven for later use.

[0019] (6) Assembly of the sensing system: The modified MoS2 sheet is sealed in a polytetrafluoroethylene cavity and separated into cis side and trans side; an electrolyte solution containing monovalent cations (such as KCl) is injected into both sides; working electrode one and working electrode two are installed through the microelectrode interface, and the ground electrode is connected to the external low noise amplifier circuit.

[0020] (7) Sample addition and signal acquisition: Dissolve the polypeptide to be tested in the electrolyte solution on the cis side; apply a transpore bias voltage through the electrode to drive the polypeptide and its carried amino acid residues to translocate; and use the probe to acquire the ion current signal in real time via the wire.

[0021] In the application of the biosensor based on cysteine-functionalized MoS2 nanopores in nanopore detection technology, the analyte to be detected is one or more of polymers, proteins, peptides, drugs, or diagnostic agents; the analyte is detected by passing through the MoS2 nanopores through electrophoresis or electroosmosis under the action of the electric field generated by the working electrode.

[0022] In the application of the biosensor based on cysteine-functionalized MoS2 nanopores in nanopore detection technology, when the analyte is a peptide, the specific steps are as follows:

[0023] (1) The polypeptide is brought into contact with the MoS2 nanopores and moved relative to the MoS2 nanopores under the drive of the transpore bias voltage;

[0024] (2) When the polypeptide moves relative to the MoS2 nanopores, transient hydrogen bond gating and non-covalent interactions occur between the amino acid residues on the polypeptide chain and the cysteine ​​or other amino acids connected to it at the pore edge. When the polypeptide passes through the cysteine-modified pores, the interaction causes a specific amplitude of blocking of the ion current. The amplitude and duration of this characteristic weak fluctuation signal are measured, wherein the measurement indicates one or more physicochemical characteristics of the polypeptide and thus characterizes the amino acid sites of the polypeptide and the amino acid class of a specific site.

[0025] (3) When the peptide completely leaves the MoS2 nanopore, the ion current will return to the open-pore baseline level. When the next peptide moves through the MoS2 nanopore, the sensor will perform the next signal capture and measurement.

[0026] Among the features obtained during the detection process, characterization includes indicators of whether a polypeptide passes through, different amino acid sites of the polypeptide, and the amino acid class at specific sites (such as the distinction between natural amino acids and modified amino acids). The current blocking rate and residence time exhibit significant characteristic differentiation due to the differences in hydrogen bond lifetime and binding energy between amino acid residues and cysteine ​​residues.

[0027] The beneficial effects of this invention are:

[0028] (1) This invention constructs a chemically selective intrapore recognition interface by introducing cysteine ​​covalent modification at the edge of MoS2 nanopores, which can significantly enhance the non-covalent interaction between the target peptide or protein molecule and the nanopore, thereby improving the signal sensitivity and molecular discrimination ability in the nanopore sensing process.

[0029] (2) The present invention can effectively reduce the permeation speed of polypeptide or protein molecules and prolong their residence time in nanopores, which is beneficial to obtaining a more stable and distinguishable ion current blocking signal.

[0030] (3) Unlike ordinary cysteine ​​modification, this invention not only uses cysteine ​​as a fixed group, but also uses it as a connecting bridge to further introduce terminal amino acids with different physicochemical properties to form Cys3-X-MoS2 functionalized nanopores. By changing the charge, polarity, hydrophobicity and aromatic structure of the terminal amino acids, the local electrostatic potential, ion distribution and non-covalent interaction strength of the nanopores can be adjusted, thereby realizing programmable control of molecular transport dynamics and sensing signals.

[0031] (4) This invention utilizes the formation and breaking of hydrogen bonds within the pores to achieve gating regulation of molecular transport. Hydrogen bonds can restrict peptide movement and regulate the pore speed under a moderate electric field, while they can be weakened or destroyed under a stronger electric field. Therefore, voltage-responsive molecular transport regulation can be achieved, which helps to improve the recognition resolution of peptides with different sequences.

[0032] (5) This invention belongs to label-free single-molecule detection method. It does not require fluorescent labeling, enzyme digestion and fragmentation or complex pretreatment of the protein or peptide to be tested. It can directly obtain information on molecular transport, interaction and structural differences through nanopore ion current signal. It is suitable for protein sequence recognition, post-translational modification detection and proteomics analysis.

[0033] (8) This invention has good scalability. By adjusting the nanopore size, the number of cysteine ​​modifications, the type of terminal amino acids, the applied voltage, and the solution conditions, the single-molecule recognition resolution, pore dynamics, and detection throughput can be further optimized, providing a new technical solution for constructing a highly sensitive, highly selective, and tunable solid-state nanopore protein detection platform. The conclusion of the paper also points out that this Cys covalently functionalized MoS2 nanopore platform can slow down peptide pore permeation, enhance peptide-nanopore interactions, and achieve tunable sensing performance through different amino acid functionalizations. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the cysteine-functionalized MoS2 nanopore sensor of the present invention (where a is an isometric view and b is a top view).

[0035] Figure 2 The image shows the recognition results of different peptides and their modified variants by Cys3-MoS2 nanopores. Figure 3 Figure showing the effect of Cys3-X-MoS2 nanopores modified with different terminal amino acids on peptide transport time;

[0036] Figure 4 A schematic diagram of the regulation of peptide transport by hydrogen bonding (where (a) shows the hydrogen bonding between the peptide and the terminal modified amino acid; (b) shows the difference in transport time of different peptides under hydrogen bonding regulation).

[0037] List of reference numerals in the attached diagram:

[0038] 1-MoS2, 2-cysteine, 3-amino acid / peptide chain, 4-polypeptide, 5-cis-side, 6-trans-side, 7-working electrode one, 8-working electrode two, 9-ground electrode, 10-probe, 11-lead wire. Detailed Implementation

[0039] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. It should be noted that the terms "front," "rear," "left," "right," "up," and "down" used in the following description refer to directions in the accompanying drawings, and the terms "inner" and "outer" refer to directions toward or away from the geometric center of a specific component, respectively.

[0040] See Figure 1 This embodiment proposes a single-molecule protein and post-translational modified sensor based on cysteine-functionalized MoS2 nanopores, including MoS21, cysteine ​​2, amino acid 3, polypeptide 4, trans side 5, cis side 6, working electrode 1 7, working electrode 2 8, ground electrode 9, probe 10 and wire 11.

[0041] The core sensing unit is a monolayer MoS21, with nanopores at its center serving as the core support substrate. The pore size of the nanopores can be adjusted according to the size, conformational characteristics, and required ion current resolution of the analyte protein, peptide, or post-translational modified molecule. In a preferred embodiment, the diameter of the nanopores is 1.6 nm to enhance the interaction between the pore walls and the molecules while ensuring the smooth passage of the analyte molecules. However, this invention is not limited to this pore size; any nanopore size that enables the restricted passage of a single protein or peptide molecule and generates a resolvable ion current signal should be included within the scope of protection of this invention.

[0042] At the edge of the nanopores, cysteine ​​2 is precisely modified via a sulfur-molybdenum (S-Mo) covalent bond to construct a chemically selective sensing interface. To avoid excessive steric hindrance caused by over-functionalization, this embodiment preferably uses a Cys3-MoS2 modification system. To achieve precise control over the local electrostatic potential and non-covalent interactions of the nanopores, the cysteine ​​2 residue is further covalently linked (Cys3-X) to amino acid 3 residues with different physicochemical properties, including but not limited to: arginine (Arg), tyrosine (Tyr), leucine (Leu), serine (Ser), valine (Val), alanine (Ala), isoleucine (Ile), threonine (Thr), and glutamine (Gln).

[0043] Physically, the device is divided into an upper cis-side 6 and a lower trans-side 5 cavity by a MoS21 substrate, which are connected by nanopores in the center of the MoS21 substrate. Both cavities are filled with a 2.0M KCl electrolyte solution. Working electrode 7 and working electrode 8 are placed on the trans-side 5 and cis-side 6, respectively, and connected to a low-noise ion current monitoring system consisting of a probe 10 and wires 11, along with a ground electrode 9.

[0044] This embodiment describes the fabrication and verification of a single-molecule protein and a post-translational modified sensor based on cysteine-functionalized MoS2 nanopores.

[0045] In addition to providing the aforementioned sensor, this embodiment specifically includes the following steps in its fabrication process:

[0046] (1) Preparation and transfer of MoS2 nanofilm: Monolayer MoS21 was prepared by chemical vapor deposition (CVD) and transferred to the suspended region on the supporting membrane;

[0047] (2) Precision fabrication of nanopores: High-energy beams are used to fabricate nanopores with a diameter of 1.6 nm in the center of monolayer MoS21, so that they are precisely matched in physical size to the steric hindrance of single-chain polypeptide molecules.

[0048] (3) Cysteine ​​covalent functionalization modification: The processed chip is immersed in an L-cysteine ​​solution for reaction. Cysteine ​​2 is introduced into the pore edge through S-Mo covalent bonds, which enhances the local negative charge density and causes a downward shift in electrostatic potential. The reaction conditions are strictly controlled in this step to form the optimal Cys3 modification degree. Furthermore, amino acids such as Gln or Tyr can be introduced to construct Cys3-Gln3 or Cys3-Tyr3 composite modification layers;

[0049] (4) Assembly and verification of the sensing system: The functionalized chip was sealed and installed in a liquid cavity unit, and a 2.0M KCl solution was injected. The IV characteristic curve was measured under a bias voltage ranging from -2.0V to 2.0V (in 0.4V steps). The experiment confirmed that the modified channel exhibited obvious ohmic behavior, and due to the introduction of the modification layer, its resistance to ion transport was significantly higher than that of the bare via, thus verifying the success of covalent modification.

[0050] An application of a single-molecule protein and post-translational modification sensor based on cysteine-functionalized MoS2 nanopores in single-molecule nanopore detection technology is described. In this embodiment, the analyte is polypeptide 4, specifically the sequence N-DDDTTTEEEIIIEEE-C (DTEIE) and its mutants. The specific detection steps are as follows:

[0051] Step 1: Dissolve the polypeptide 4 (such as DTEIE, DTELE, DLELE, or DTE(pS)E with post-translational modification, etc.) in the electrolyte solution on the cis side 6 and contact it with the MoS2 nanopores; Since the polypeptide carries a negative charge of -9e as a whole, under the drive of the transpore bias voltage (a constant external bias voltage of 1.5V or higher up to 2.0V applied along the Z-axis), the electric field force will effectively drive the polypeptide 4 to trans side 5 to translocate;

[0052] Step 2: As the polypeptide 4 moves relative to the MoS2 nanopores, transient hydrogen bond gating and non-covalent interactions occur between the amino acid residues on the polypeptide chain and cysteine ​​2 or other amino acids 3 (such as Gln in the Cys3-Gln3 modified layer) at the pore edge. For example, the longer side chain of the Gln residue and its amide group (-CONH2) can simultaneously act as hydrogen bond donors and acceptors, forming a strong hydrogen bond gating effect with the polypeptide. When the polypeptide passes through the cysteine-modified pores, the interaction causes a specific amplitude of ion current blocking; the amplitude and duration of this characteristic weak fluctuation signal are captured in real time by probe 10, and the measurement data directly indicates the physicochemical characteristics of different residues in polypeptide 4 (such as the translocation residence time and current characteristics of the extracted polypeptide in the pores), thereby characterizing and directly distinguishing different residue categories and modification sites;

[0053] Step 3: Once peptide 4 has completely exited and left the MoS2 nanopores, the ion current returns to the open-pore baseline level. When the next peptide 4 enters the MoS2 nanopores, the sensor will perform the next single-molecule signal capture and measurement.

[0054] This embodiment demonstrates high-resolution identification of isomeric amino acids:

[0055] Reference Figure 2 As shown, cysteine ​​functionalization can prolong the peptide pore passage time and improve signal discrimination.

[0056] Cys3-MoS2 nanopores exhibit extremely high resolution for peptides with highly similar sequences (such as DTELE and DTEIE, where Leu and Ile are isomers). Experiments show that the deceleration events during the translocation process mainly occur in uncharged residue regions (especially the substitution sites of Ile and Leu). Due to the stronger non-covalent interaction between the side chain of Leu and the Cys-modified layer, the residence time of peptide DLELE in the pore is almost twice that of DTELE (39.8 ns longer at 1.5 V and 28.5 ns longer at 2 V). In contrast, unmodified bare MoS2 nanopores cannot distinguish between these two isomer residues at the same voltage.

[0057] Reference Figure 3As shown, the physicochemical properties of terminal amino acids can regulate the pore-passing behavior of peptides. Connecting terminal amino acids with different properties (Cys3-X) can completely change the electrostatic potential distribution and ion dynamics of the pore opening. For example, connecting positively charged Arg significantly increases the local potential of the pore opening, enhances electroosmotic flow, and induces ion selectivity for Cl-; while connecting Tyr or Thr, which have strong hydrophobicity and van der Waals forces, can even "drag" the modified residues into the depth of the pore during translocation, inducing extremely strong interaction forces, thus providing a sufficient signal amplification window for detecting weak post-translational modifications such as phosphorylation (pSer).

[0058] (2) Voltage sensitivity of hydrogen bond gating effect:

[0059] Reference Figure 4 As shown, in Cys3-Gln3 modified nanopores, the formation and breaking of hydrogen bonds dominate the translocation dynamics of peptides. Experiments demonstrate that the hydrogen bond lifetime is shorter than that of van der Waals forces and is extremely sensitive to external electric fields. At a moderate bias voltage of 1.5V, the transient but effective hydrogen bond network greatly restricts peptide movement, resulting in a significant "deceleration zone." However, when the voltage increases to 2.0V, a strong external electric field rapidly drives the peptide to perforate the pores, directly disrupting the hydrogen bond network and significantly weakening the hydrogen bond-gated deceleration effect.

[0060] The effects of this invention will be further illustrated below with specific examples:

[0061] Example 1: High-resolution label-free recognition of isomeric amino acids and post-translational modifications based on Cys3-MoS2 nanopores

[0062] Objective: To verify the high-resolution physical recognition capability of this invention for isomeric amino acids with extremely similar spatial structures (such as leucine Leu and isoleucine Ile) and post-translational modifications of proteins that induce slight changes in physicochemical characteristics (such as phosphorylation) without relying on any chemical or fluorescent labeling.

[0063] Specific implementation and results: First, by controlling the reaction conditions, three cysteine ​​molecules were covalently linked to the edge of a 1.6 nm MoS2 nanopore via sulfur-molybdenum bonds to prepare a Cys3-MoS2 sensing channel. Subsequently, peptides with single-residue isomerism (sequences DTEIE and DTELE) and peptides with phosphorylation modification differences (sequences DTESE and DTE(pS)E) were synthesized and dissolved in a cis-side cavity filled with 2.0 MkCl electrolyte solution. Finally, a constant bias voltage of 1.5 V was applied through the connected electrode system to drive the peptides to translocate and perforate. The microscopic ion current trajectory of a single molecule passing through the channel was collected in real time using a probe and a low-noise amplifier.

[0064] By extracting and comparing data features, it was found that: (1) For isomers, due to the stronger van der Waals forces and steric hindrance interactions between the leucine side chain and the cysteine ​​modification layer, the average translocation residence time of the mutant peptide containing leucine (DTELE) in the pore was significantly longer than that of DTEIE by about 39.8 ns; (2) For post-translational modifications, when the phosphorylated residue (pS) passes through the cysteine ​​modification layer, due to the additional charge repulsion and expanded steric hindrance effect brought about by the introduction of the phosphate group, it produces specific physical interference with the modification layer, thereby forming a characteristic blocking peak with unique amplitude and residence time on the ion current trajectory. This embodiment successfully verified that the sensor has excellent label-free targeted recognition ability for both single-residue isomers that are extremely difficult to distinguish and weak post-translational modifications due to its keen physical response.

[0065] Example 2: On-demand control of physicochemical properties based on end-group functionalization (Cys3-X)

[0066] Objective: To verify that the detection platform of the present invention has high design flexibility and can reconstruct the electrostatic and physical environment of the channel by replacing the covalent end groups, thereby achieving customization of local physicochemical properties.

[0067] Specific Implementation and Results: To address different targeted detection needs, this embodiment fabricated Cys3-Arg3 nanopore sensors coupled with positively charged arginine and Cys3-Tyr3 nanopore sensors coupled with strongly hydrophobic aromatic tyrosine, respectively. When peptides containing the corresponding target residues were used for transpore detection, it was found that the introduction of arginine significantly increased the local electrostatic potential at the pore opening and effectively enhanced local electroosmotic flow, resulting in excellent selectivity for chloride ions in the solution. When tyrosine was introduced, its strong hydrophobic effect and van der Waals forces caused the peptide to drag the modified residues deep into the pore during translocation, causing the interaction force to reach its peak and significantly increasing the blocking amplitude of the characteristic current. This embodiment conclusively verifies that the present invention can achieve customized control of the hydrophilicity / hydrophobicity and electric field distribution of the pores without reconstructing the underlying physical hardware structure; only the terminal amino acid residues need to be replaced.

[0068] Example 3: Precise regulation of peptide translocation dynamics using hydrogen bond gating effect

[0069] Objective: To verify the feasibility of precisely controlling the translocation rate of peptides through a microscopic hydrogen bond network and its sensitivity to driving voltage.

[0070] Specific implementation and results: First, glutamine was covalently coupled to the cysteine ​​residues at the pore edge to prepare Cys3-Gln3-MoS2 functionalized nanopores. A highly sensitive sensing interface was constructed by utilizing the amide group of the glutamine side chain as both a hydrogen bond donor and acceptor. Next, the analyte peptide with the sequence DQEQE was added to the cis-side cavity. During the detection phase, translocation signals were recorded at bias voltages of 1.5V and 2.0V. Experiments showed that at 1.5V, a strong transient hydrogen bond network formed between the peptide residues and the glutamine-modified layer, producing a significant gating deceleration effect and outputting a very wide current blocking pulse. However, when the voltage was increased to 2.0V, the strong external electric field instantly disrupted the hydrogen bond network, causing a sharp increase in the peptide's translocation speed. This result fully verifies that the sensor can flexibly control the hydrogen bond gating effect by adjusting the external driving voltage to meet the detection resolution requirements of different analytical scenarios.

[0071] The technical means disclosed in this invention are not limited to those disclosed in the above embodiments, but also include technical solutions composed of any combination of the above technical features.

Claims

1. A single-molecule protein and post-translational modified sensor based on cysteine-functionalized MoS2 nanopores, characterized in that, It includes MoS2 (1), cysteine ​​(2), amino acid or peptide chain (3), polypeptide (4), trans side (5), cis side (6), working electrode one (7), working electrode two (8), ground electrode (9), probe (10) and wire (11); wherein the MoS2 (1) is a single-layer or multi-layer structure with a central nanopore channel, serving as a core support substrate; Cysteine ​​(2) is anchored to the edge of the MoS2 (1) nanopores via S-Mo covalent bonds to construct a chemically selective sensing interface. Cysteine ​​(2) can also connect to other amino acids (3). Working electrode one (7) and working electrode two (8) are respectively set in the cavity of the trans side (5) and the cis side (6), and together with the ground electrode (9) connected to the external circuit, they form a stable sensing circuit. The probe (10) is placed near the nanopores of the MoS2 (1) and connected to the back-end signal processing system via wire (11) to capture the characteristic weak current fluctuation signal generated when the peptide (4) to be tested perforates in real time. When the peptide (4) to be tested is initially located in the cis side (6) solution and passes through the MoS2 nanopores from top to bottom into the trans side (5) under the drive of the electric field, the sensing circuit and the probe (10) work together to realize high-resolution, tagged identification and analysis of single protein sequences and their post-translational modifications.

2. The single-molecule protein and post-translational modified sensor based on cysteine-functionalized MoS2 nanopores according to claim 1, characterized in that, The cis-side (6) and trans-side (5) are filled with the same electrolyte solution containing 0.1 M to 3.0 M of monovalent cations selected from K. + Li + and Na + At least one of them.

3. The single-molecule protein and post-translational modified sensor based on cysteine-functionalized MoS2 nanopores according to claim 1, characterized in that, The cysteine ​​(2) and the MoS2 (1) nanopores are connected by S-Mo covalent bonds to form a chemically stable functionalized layer.

4. The single-molecule protein and post-translational modified sensor based on cysteine-functionalized MoS2 nanopores according to claim 1, characterized in that, The MoS2 (1) modifies cysteine ​​(2) by covalently modifying it, thereby regulating the non-covalent interaction between the polypeptide (4) molecules and the nanopores. This increases the friction between the molecules and the pore walls or forms transient binding, thereby slowing down the passage speed of single molecules and enhancing the blocking current signal.

5. A single-molecule protein and post-translational modified sensor based on cysteine-functionalized MoS2 nanopores according to claim 4, characterized in that: When cysteine ​​(2) interacts with amino acid residues with benzene rings, it increases the residence time of the residues in the pores by enhancing hydrophobic interactions and van der Waals forces; when it interacts with charged amino acids, it regulates the translocation rate by changing the local electroosmotic flow state at the pore opening; when it interacts with amino acids with donor and acceptor groups, it precisely controls the translocation kinetics by forming hydrogen bonds.

6. A method for preparing a single-molecule protein and a post-translational modified sensor based on cysteine-functionalized MoS2 nanopores, characterized in that, Its preparation steps include the following: Step 1: Sensor substrate preparation: MoS2(1) thin layer is obtained by CVD or mechanical exfoliation, transferred to a support frame with micron-sized pores and surface activated; Step 2: Nanopore processing: pores with a diameter of 1-5 nm are processed in the center of MoS2(1) using a focused electron beam or ion beam, and the edge Mo atoms are activated by oxygen plasma cleaning. Step 3: Surface covalent functionalization: The substrate is immersed in a 5-50 mM cysteine ​​(2) solution and reacted for 12-24 hours under nitrogen protection to achieve directional anchoring through S-Mo bonds, followed by purification and drying; Step 4: Sensor system assembly: The modified MoS2 sheet is installed between the cis side (6) and the trans side (5) cavity, electrolyte is injected and the working electrode (7, 8) and the ground electrode (9) are connected. Step 5: Pore size modification verification and detection: The modification success and the thickness of the modified layer are verified by measuring the change in ionic conductivity before and after modification. Then, the current signal generated by the pores of the peptide (4) is collected by probe (10).

7. An application of a single-molecule protein and post-translational modification sensor based on cysteine-functionalized MoS2 nanopores in nanopore detection technology, characterized in that, Includes the following steps: Step 1: Place the analyte in the electrolyte solution on the cis side (6) of the sensor; Step 2: Apply a transpore bias voltage through working electrode one (7) and working electrode two (8) to drive the analyte to pass through the nanopores in the center of MoS2 (1); Step 3: During the passage of the analyte, transient hydrogen bonds and non-covalent interactions are generated between the functional groups on its molecules and cysteine ​​(2) or other amino acids (3) anchored at the edge of the pore, causing the ion current to generate a characteristic blocking signal; Step 4: The amplitude, duration and frequency of the blocking signal are collected in real time using the probe (10), and analyzed by the back-end signal processing system to indicate the type, structure, sequence or post-translational modification state of the analyte.

8. The application of a single-molecule protein and post-translational modification sensor based on cysteine-functionalized MoS2 nanopores according to claim 7 in nanopore detection technology, characterized in that: The analyte to be tested is a polypeptide (4) or a protein. When the polypeptide or protein passes through the nanopore, the interaction between its amino acid residues and cysteine ​​(2) causes the current blocking rate and residence time to show characteristic differences depending on the hydrophobicity, charge or hydrogen bond acceptor ability of the amino acid residues, thereby realizing the identification of amino acid sites and specific amino acid categories.

9. The application of a single-molecule protein and post-translational modification sensor based on cysteine-functionalized MoS2 nanopores according to claim 8 in nanopore detection technology, wherein the identification includes distinguishing between natural amino acids and post-translational modified amino acids.

10. The application of a single-molecule protein and post-translational modified sensor based on cysteine-functionalized MoS2 nanopores according to claim 7 in nanopore detection technology, characterized in that: The analyte to be tested is selected from one or more polymers, drugs, or diagnostic agents.

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