Single molecule in-situ recognition and detection method based on intermolecular hydrogen bonds

Abstract

The invention relates to a monomolecular in-situ recognition and detection method based on intermolecular hydrogen bonds, and belongs to the technical field of monomolecular detection.The method comprises the steps that a gold nanoelectrode is prepared through an electrochemical corrosion method, then a bridging molecule solution with sulfydryl is combined with the surface of the gold nanoelectrode, meanwhile, gold nanoparticles are mixed with the bridging molecule solution, and the gold nanoparticles are obtained; the method comprises the following steps: modifying a gold nano-electrode, placing the modified gold nano-electrode in a modified gold nano-particle solution, adding a target molecule solution into the gold nano-particle solution, forming molecular knots by virtue of an electrostatic adsorption effect between gold nano-particles and the gold nano-electrode, dynamically adjusting the molecular knots by virtue of electrochemical regulation, and finally monitoring in real time by virtue of an SERS (Surface Enhanced Raman Scattering) signal. And obtaining identification information of the target molecule. Therefore, the method has the advantages of high identification precision, high sensitivity and real-time dynamic monitoring.

Description

Technical Field

[0001] The present invention belongs to the field of single molecule detection technology, and in particular relates to a single molecule in situ recognition and detection method based on intermolecular hydrogen bonds. Background Art

[0002] In recent years, the critical role of single-molecule detection technology in fields such as bioanalysis, disease diagnosis, and molecular biology has become increasingly prominent. However, current detection technologies suffer from numerous limitations, primarily manifested in three key areas: insufficient specific recognition capabilities, limited dynamic response dimensions, and insufficient detection throughput to meet complex requirements. Traditional fluorescence detection techniques lack specific recognition capabilities due to interference from background fluorescence. While surface plasmon resonance (SPR) technology can monitor molecular interactions in real time, its sensitivity for microscopic intermolecular forces, such as hydrogen bonds, is low, and it cannot provide fingerprint information about molecular structure. In addition, common single-molecule detection technologies include microfluidics and mass spectrometry. Microfluidics has a low detection throughput, making it difficult to meet the high-throughput requirements for detecting trace biomolecules in complex biological systems. Mass spectrometry, however, requires high sample purity, requires complex pre-processing, and struggles to achieve in situ, real-time detection of single molecules. In molecular biology, the precise identification and detection of hydrogen-bonding networks between biomolecules is crucial for in-depth understanding of their functions and interaction mechanisms. However, traditional methods often struggle to achieve ultrasensitive, multi-dimensional detection of trace biomolecules in complex biological systems.

[0003] Over the past decade, surface-enhanced Raman scattering (SERS) has demonstrated significant advantages in studying the chemical reaction dynamics of metal-molecule-metal structures at the single-molecule level. It has significantly advanced our understanding of metal nanoparticle catalysis, photocatalysis, and molecular electronics, and has become a mature vibrational spectroscopic technique with rapidly growing applications in chemistry, materials science, and especially the life sciences. When gold particles collide with and adsorb onto a gold electrode, a nanogap forms, where molecules connect to form a "GNE-molecule-GNP" junction. This nanogap generates a strong electric field, enhancing the Raman spectroscopy signal by more than a million times. The standard SERS enhancement factor ranges widely (103-1010), enabling clear observation of molecular interactions and structural changes at the single-molecule level. However, in order to achieve a regular arrangement of the target molecules on the metal electrode surface for single-molecule SERS detection, the target molecules must possess anchoring groups, such as thiol groups. Many biomolecules lack anchoring groups, making single-molecule signal observation difficult.

[0004] The present invention aims to break through these technical bottlenecks and propose a hydrogen-bond-mediated plasmon molecular pair detection system to achieve efficient capture and signal amplification of biological single-molecule hydrogen bond networks. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a single molecule in situ recognition and detection method based on intermolecular hydrogen bonds.

[0006] To solve the above technical problems, the present invention adopts a technical solution: a single molecule in situ recognition and detection method based on intermolecular hydrogen bonds, comprising the following steps: S1. Preparation and surface treatment of gold nanoelectrode substrate: Gold nanoelectrodes are prepared by electrochemical etching: Gold wire is used as the raw material and treated with electrochemical etching to obtain nanostructured gold nanoelectrodes. Electrochemical etching can form a uniform nanostructure, thereby increasing the surface area and molecular binding capacity of the gold nanoelectrode.

[0007] S2. Modify the bridging molecules on the gold nanoelectrode substrate: A bridging molecule solution with a thiol group (-SH) is combined with the surface of the gold nanoelectrode. The bridging molecule provides both hydrogen bond donors and acceptors, and can form stable molecular pairs with target molecules (such as DNA bases) to form a stable modification layer.

[0008] S3. Modification of the surface of gold nanoparticles with bridging molecules: By mixing the bridging molecule solution of step S2 with gold nanoparticles, the modified gold nanoparticles can bind to target molecules (such as DNA bases) through hydrogen bonds, thereby providing structural support for the subsequent assembly of molecular junctions.

[0009] S4. Construction of molecular junctions: The gold nanoelectrode modified in step S2 is placed in the gold nanoparticle solution modified in step S3, and then the target molecule solution is added to the gold nanoparticle solution to form a stable molecular junction by utilizing the electrostatic adsorption effect between the gold nanoparticles and the gold nanoelectrode.

[0010] S5. Electrochemical Control and Dynamic Regulation of Molecular Binding: By adjusting the electrode potential, the nanogap between the gold nanoparticles and the gold nanoelectrode is regulated, thereby affecting the state of the molecular junction. During this process, the formation and breaking of hydrogen bonds directly affect the intensity and peak position of the SERS signal. By monitoring these changes in real time, dynamic monitoring of intermolecular interactions can be achieved. This electrochemical control method provides a precise operating method for single-molecule detection, enabling the control of the state of the molecular junction in real-time experiments, thereby optimizing signal acquisition.

[0011] S6. Real-time monitoring of SERS signals and molecular recognition: As the electrode potential is adjusted, the gap between the gold nanoelectrode and the gold nanoparticles changes, resulting in changes in the SERS signal intensity. Therefore, through SERS signal analysis, the dynamic changes in intermolecular hydrogen bonding interactions can be accurately captured, and the identification information of the target molecule can be obtained in real time. This technology not only improves the sensitivity of molecular recognition, but also makes dynamic molecular detection possible.

[0012] As can be seen, by adjusting the electrode potential, the nanogap between the gold nanoelectrode and the gold nanoparticle can be precisely controlled, enabling highly sensitive detection of single biological molecules. The dynamic changes in hydrogen bonds enable even higher sensitivity in the identification and detection of target molecules. By monitoring the state of molecular bonding in real time, dynamic monitoring can be performed at the millisecond level, providing powerful technical support for molecular recognition and real-time analysis.

[0013] The SERS technique analyzes the hydrogen bonding interactions between target molecules and bridging molecules, recording changes in the SERS signal at different electrode potentials. Changes in signal intensity and peak position reflect the "opening" and "closing" states of the molecular binding, providing a basis for dynamic identification and detection of target molecules.

[0014] Furthermore, in step S1, the purity of the gold wire is 99.9% and the diameter is 0.2 mm.

[0015] Furthermore, in step S1, the surface of the gold nanoelectrode is cleaned to remove impurities and then coated with high-density polyethylene (HDPE). This design ensures the stability and reproducibility of the gold nanoelectrode during experiments. The resulting gold nanoelectrode is not only stable but also has efficient molecular binding capacity, making it suitable for subsequent modification with bridging molecules.

[0016] Furthermore, in step S2, the bridging molecule is 4-mercaptobenzoic acid (4-MBA) or cysteine ​​(Gys). 4-MBA and cysteine ​​have abundant hydrogen bond donors and acceptors, enabling efficient hydrogen bonding interactions between the gold nanoelectrode and the target molecule, thereby enhancing molecular recognition. By modifying the gold nanoelectrode surface with these bridging molecules, the binding capacity of the target molecule can be effectively regulated, enhancing the efficiency and accuracy of molecular recognition. The modification process of the bridging molecule utilizes methods such as ultrasound-assisted dissolution to ensure stable attachment of the molecule to the electrode surface.

[0017] Furthermore, in step S3, the bridging molecules are modified onto the surface of the gold nanoparticles by centrifugation, standing and extraction to ensure that the bridging molecules are evenly distributed on the surface of the gold nanoparticles and that each gold nanoparticle surface can provide sufficient hydrogen bond donors and acceptors to enhance the ability of molecular recognition.

[0018] The beneficial effects of the present invention are: (1) Achieving high-precision recognition and electrochemical regulation at the molecular level: By introducing 4-MBA or cysteine ​​molecules onto the surface of gold nanoelectrodes and gold nanoparticles, respectively, a stable hydrogen bond recognition network is constructed, enabling precise binding of target molecules with specific structural features. Combined with nanogap regulation under electrochemical control, the interaction between molecules can be mechanically regulated, enabling the present invention to possess sub-nanometer spatial resolution, making it possible to capture tiny conformational changes at the single-molecule level.

[0019] (2) Significantly improve signal intensity and detection response: The aromatic ring structure in the bridging molecule helps to enhance the degree of conjugation of the molecular layer. The highly sensitive movement of charge produces a strong induced dipole moment, which greatly enhances the Raman signal. Combined with the plasmon "hotspot" structure, the signal amplification factor can reach 10 9 level, significantly surpassing the response capability of traditional SERS substrates.

[0020] (3) Qualitative and quantitative multidimensional analysis capabilities: This method not only obtains the spectral characteristics of the target molecule, but also analyzes its orientation, binding state, and number of hydrogen bonds in the probe structure through subtle changes in characteristic peaks. The integration of multidimensional signals makes this method highly adaptable in the quantitative identification, structural analysis, and interaction mechanism research of complex molecular systems.

[0021] (4) Good versatility and expansion potential: The present invention is highly modular, and the choice of bridging molecules can be customized according to the functional groups of different target molecules. By adjusting the molecular recognition elements and their arrangement, it can be applied to different types of biomolecules, supporting single-molecule recognition and monitoring of a variety of targets, from nucleic acids and proteins to small molecule metabolites. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be described in detail below with reference to the accompanying drawings and in combination with examples, and the advantages and implementation modes of the present invention will become more apparent. The contents shown in the accompanying drawings are only used to illustrate the present invention and do not constitute any limitation to the present invention. In the accompanying drawings: Figure 1 Schematic diagram of the modified molecule of the present invention being modified to gold nanoparticles (GNPs) via a bridge molecule.

[0023] Figure 2 Schematic diagram of the connection of the device of the present invention combined with electrochemically controlled surface enhanced Raman spectroscopy technology.

[0024] Figure 3 This is a diagram of the hydrogen bond structure in the GNP-4-mercaptobenzoic acid-adenine-4-mercaptobenzoic acid-GNE molecular pair in Example 1 of the present invention.

[0025] Figure 4This is a diagram of the hydrogen bond structure in the GNP-4-mercaptobenzoic acid-thymine-4-mercaptobenzoic acid-GNE molecular pair in Example 2 of the present invention.

[0026] Figure 5 This is a diagram of the hydrogen bond structure in the GNP-cysteine-guanine-cysteine-GNE molecular pair in Example 3 of the present invention. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and the best embodiments.

[0028] A single molecule in situ recognition and detection method based on intermolecular hydrogen bonds comprises the following steps: S1. Preparation and surface treatment of gold nanoelectrode substrate: Gold nanoelectrodes were prepared by electrochemical corrosion: gold wire (0.2 mm in diameter) with a purity of 99.9% was used as the raw material, and the gold wire was treated by electrochemical corrosion to obtain gold nanoelectrodes with nanostructures.

[0029] Electrochemical etching can form a uniform nanostructure, thereby increasing the surface area and molecular binding capacity of the gold nanoelectrode. The gold nanoelectrode surface is rigorously cleaned to remove impurities and then coated with high-density polyethylene (HDPE) to ensure stability and reproducibility during experiments. The resulting gold nanoelectrode is not only stable but also possesses efficient molecular binding capacity, making it suitable for subsequent modification with bridging molecules.

[0030] S2. Modification of the bridging molecules on the gold nanoelectrode substrate (modification of the bridging molecules and construction of molecular junctions): 4-Mercaptobenzoic acid (4-MBA) or cysteine ​​(Gys) are used as bridging molecules. These bridging molecules bind to the surface of the gold nanoelectrode through their thiol groups (-SH), while providing hydrogen bond donors and acceptors, and can form stable molecular pairs with target molecules (such as DNA bases) to form a stable modification layer.

[0031] 4-MBA and cysteine ​​are abundant hydrogen bond donors and acceptors, providing efficient hydrogen bonding interactions between the gold nanoelectrode and the target molecule, thereby enhancing molecular recognition. By modifying the gold nanoelectrode surface with these bridging molecules, the binding capacity of the target molecule can be effectively modulated, enhancing the efficiency and precision of molecular recognition. The modification of the bridging molecules utilizes methods such as ultrasound-assisted dissolution to ensure stable attachment of the molecules to the electrode surface.

[0032] S3. Modification of the surface of gold nanoparticles with bridging molecules: like Figure 1As shown, the surface of gold nanoparticles also needs to be modified with bridging molecules. By mixing a 4-MBA solution or a cysteine ​​molecule solution with gold nanoparticles, the bridging molecules are modified onto the surface of the gold nanoparticles through steps such as centrifugation and extraction, ensuring that the bridging molecules are evenly distributed on the surface of the gold nanoparticles. These modified gold nanoparticles can bind to target molecules (such as DNA bases) through hydrogen bonds, providing structural support for the subsequent assembly of molecular junctions.

[0033] In step S3, centrifugation, standing, extraction and other methods are used to ensure that the bridging molecules are evenly attached to the surface of the gold nanoparticles and that each gold nanoparticle surface can provide sufficient hydrogen bond donors and acceptors to enhance the ability of molecular recognition.

[0034] S4. Construction of molecular junctions: The gold nanoelectrode modified in step S2 is placed in the gold nanoparticle solution modified in step S3, and then the target molecule solution is added to the gold nanoparticle solution to form a stable molecular junction by utilizing the electrostatic adsorption effect between the gold nanoparticles and the gold nanoelectrode.

[0035] The formation of hydrogen bonds stabilizes the molecular junction, preparing it for SERS detection.

[0036] S5. Electrochemical Control and Dynamic Regulation of Molecular Binding: By electrochemically regulating the gap between the gold nanoelectrode and the gold nanoparticles, the "opening and closing" state of the molecular junction can be precisely controlled. By adjusting the electrode potential, the nanogap between the gold nanoparticles and the gold nanoelectrode can be effectively adjusted, thereby affecting the state of the molecular junction. In this process, the formation and breaking of hydrogen bonds will directly affect the intensity and peak position of the SERS signal. By monitoring these changes in real time, dynamic monitoring of intermolecular interactions can be achieved. This electrochemical regulation method provides a precise operating means for single-molecule detection, which can control the state of the molecular junction in real-time experiments, thereby optimizing signal acquisition.

[0037] S6. Real-time monitoring of SERS signals and molecular recognition: SERS technology enables real-time monitoring of changes in the characteristic peak positions of target molecules in molecular junctions. As the electrode potential is adjusted, the gap between the gold nanoelectrode and the gold nanoparticles changes, resulting in variations in the SERS signal intensity. This variation is closely related to the "opening" and "closing" state of the molecular junction. Therefore, analysis of the SERS signal accurately captures the dynamic changes in intermolecular hydrogen bonding interactions, providing real-time identification information for the target molecule. This technology not only improves the sensitivity of molecular recognition but also enables dynamic molecular detection.

[0038] As can be seen, by adjusting the electrode potential, the nanogap between the gold nanoelectrode and the gold nanoparticle can be precisely controlled, enabling highly sensitive detection of single biological molecules. The dynamic changes in hydrogen bonds enable even higher sensitivity in the identification and detection of target molecules. By monitoring the state of molecular bonding in real time, dynamic monitoring can be performed at the millisecond level, providing powerful technical support for molecular recognition and real-time analysis.

[0039] The SERS technique analyzes the hydrogen bonding interactions between target molecules and bridging molecules, recording changes in the SERS signal at different electrode potentials. Changes in signal intensity and peak position reflect the "opening" and "closing" states of the molecular binding, providing a basis for dynamic identification and detection of target molecules.

[0040] Among them, such as Figure 2 As shown, the device for the surface-enhanced Raman spectroscopy technology combined with electrochemical regulation used in steps S5 and S6 includes a solution pool, in which a modified gold nanoelectrode and a reference electrode (silver / silver chloride electrode) are placed. The gold nanoelectrode is connected to an ammeter, and the modified gold nanoparticle solution is placed in the solution pool.

[0041] Finally, data processing and result analysis are carried out: The SERS signals were analyzed using software such as MATLAB, and the effect of electrochemical potential changes on molecular binding was combined to further optimize the experimental conditions and improve detection sensitivity and accuracy.

[0042] Example 1: like Figure 3 As shown, a single molecule in situ recognition and detection method based on intermolecular hydrogen bonds comprises the following steps: S1. Preparation and surface treatment of gold nanoprobe electrode substrate: Gold nanoprobe electrodes were prepared by electrochemical corrosion: 99.9% pure gold wire (0.2 mm in diameter) was used as the raw material. The gold wire was placed in a mixed solution of 37% HCl and anhydrous ethanol in a ratio of 1:1, and the gold wire was corroded by electrochemical corrosion to form a gold nanoprobe with a tip curvature radius of approximately 300 nm as an electrode.

[0043] The surface of the gold nanoprobe was cleaned to remove impurities and wrapped with high-density polyethylene (HDPE) to ensure the stability and repeatability of the gold nanoprobe electrode in the experiment.

[0044] S2. Modify the bridging molecules on the gold nanoprobe electrode substrate: The gold nanoprobe electrode was immersed in a 10mM 4-MBA solution (this concentration ensures a sufficient layer of 4-MBA molecules on the gold nanoprobe electrode surface without excessive adsorption, thus forming a well-defined molecular film). 40kHz ultrasound was used to assist in dissolution, and the solution was allowed to stand for 12 hours. The 4-MBA molecules were regularly modified on the gold nanoprobe electrode surface via thiol groups, forming a well-defined molecular film.

[0045] S3. Modification of the surface of gold nanoparticles with bridging molecules: 2µL of a 10mM 4-MBA solution was added dropwise to 1mL of a 150pM gold nanoparticle solution. The solution was allowed to stand for 12 hours to allow the molecules to fully adhere to the gold nanoparticle surface. To purify the modified solution, the solution was centrifuged, the supernatant removed, and fresh deionized water added. This process was repeated three times to fully remove any residual molecules.

[0046] S4. Construction of plasmon-molecule pairs based on intermolecular hydrogen bonds: The 4-MBA-modified gold nanoprobe electrode was fixed in the Raman test solution pool, and 200µL of 4-MBA-modified gold nanoparticle solution was injected into the solution pool. Then, a small amount (10µL) of adenine molecule solution (concentration of 10mM) was injected into the solution pool and allowed to stand for about 30 minutes to form a stable plasmon exciton molecular pair structure through hydrogen bond interaction.

[0047] S5. Electrochemical Control and Dynamic Regulation of Molecular Binding: By adjusting the electrode potential, the nanogap between the gold nanoparticles and the gold nanoelectrodes is regulated, thereby affecting the state of the molecular junction.

[0048] S6. Real-time monitoring and identification of SERS signals: A 633nm laser was used as the excitation light source, with a laser power of 40mW and an exposure time of 0.1s. In situ identification and detection of adenine single molecules were achieved through the characteristic peaks of Raman spectroscopy.

[0049] Example 2: like Figure 4 As shown, a single molecule in situ recognition and detection method based on intermolecular hydrogen bonds comprises the following steps: S1. Preparation and surface treatment of gold nanoprobe electrode substrate: Gold nanoelectrodes were prepared by electrochemical corrosion: 99.9% pure gold wire (0.2 mm in diameter) was used as the raw material. The gold wire was placed in a 1:1 mixed solution of 37% HCl and anhydrous ethanol. The gold wire was corroded by electrochemical corrosion to form a gold nanoprobe with a tip curvature radius of about 300 nm as an electrode.

[0050] The surface of the gold nanoprobe was cleaned to remove impurities and wrapped with high-density polyethylene (HDPE) to ensure the stability and repeatability of the gold nanoelectrode in the experiment.

[0051] S2. Modify the bridging molecules on the gold nanoprobe electrode substrate: The gold nanoprobe electrode was immersed in a 10mM 4-MBA solution, dissolved using 40kHz ultrasound, and allowed to stand for 12 hours. The 4-MBA molecules were regularly modified on the surface of the gold nanoprobe electrode through thiol groups, forming a well-defined molecular film.

[0052] S3. Modification of the surface of gold nanoparticles with bridging molecules: 2µL of a 10mM 4-MBA solution was added dropwise to 1mL of a 150pM gold nanoparticle solution. The solution was allowed to stand for 12 hours to allow the molecules to fully adhere to the gold nanoparticle surface. To purify the modified solution, the supernatant was removed and fresh deionized water was added. This process was repeated three times to fully remove any residual molecules.

[0053] S4. Construction of plasmon-molecule pairs based on intermolecular hydrogen bonds: The 4-MBA-modified gold nanoprobe electrode was fixed in the Raman test solution pool, and 200µL of 4-MBA-modified gold nanoparticle solution was injected into the solution pool. Then, a small amount (10µL) of thymine molecule solution (concentration of 10mM) was injected into the solution pool and allowed to stand for about 30 minutes to form a stable plasmon exciton molecular pair structure through hydrogen bond interaction.

[0054] S5. Electrochemical Control and Dynamic Regulation of Molecular Binding: By adjusting the electrode potential, the nanogap between the gold nanoparticles and the gold nanoelectrodes is regulated, thereby affecting the state of the molecular junction.

[0055] S6. Real-time monitoring and identification of SERS signals: A 633nm laser was used as the excitation light source, with a laser power of 40mW and an exposure time of 0.1s. In situ identification and detection of single thymine molecules was achieved through the characteristic peaks of the Raman spectrum.

[0056] Example 3: like Figure 5As shown, a single molecule in situ recognition and detection method based on intermolecular hydrogen bonds comprises the following steps: S1. Preparation and surface treatment of gold nanoprobe electrode substrate: Gold nanoelectrodes were prepared by electrochemical corrosion: 99.9% pure gold wire (0.2 mm in diameter) was used as the raw material. The gold wire was placed in a 1:1 mixed solution of 37% HCl and anhydrous ethanol. The gold wire was corroded by electrochemical corrosion to form a gold nanoprobe with a tip curvature radius of about 300 nm as an electrode.

[0057] The surface of the gold nanoprobe was cleaned to remove impurities and wrapped with high-density polyethylene (HDPE) to ensure the stability and repeatability of the gold nanoelectrode in the experiment.

[0058] S2. Modify the bridging molecules on the gold nanoprobe electrode substrate: The gold nanoprobe electrode was immersed in a 10mM cysteine ​​solution, dissolved using 40kHz ultrasound, and left to stand for 12 hours. Cysteine ​​molecules can be regularly modified on the surface of the gold nanoprobe electrode through thiol groups, forming a well-defined molecular film.

[0059] S3. Modification of the surface of gold nanoparticles with bridging molecules: 2µL of a 10mM cysteine ​​solution was added dropwise to 1mL of a 150pM gold nanoparticle solution. The solution was allowed to stand for 12 hours to allow the molecules to fully adhere to the gold nanoparticle surface. To purify the modified solution, the supernatant was removed and fresh deionized water was added. This process was repeated three times to fully remove any residual molecules.

[0060] S4. Construction of plasmon-molecule pairs based on intermolecular hydrogen bonds: A gold nanoprobe electrode modified with cysteine ​​molecule solution was fixed in a Raman test solution pool, and 200µL of gold nanoparticle solution modified with cysteine ​​molecule solution was injected into the solution pool. Subsequently, a small amount (10µL) of guanine molecule solution (concentration of 10mM) was injected into the solution pool and allowed to stand for about 30 minutes to form a stable plasmon molecular pair structure through hydrogen bond interaction.

[0061] S5. Electrochemical Control and Dynamic Regulation of Molecular Binding: By adjusting the electrode potential, the nanogap between the gold nanoparticles and the gold nanoelectrodes is regulated, thereby affecting the state of the molecular junction.

[0062] S6. Real-time monitoring and identification of SERS signals: A 633nm laser was used as the excitation light source, with a laser power of 40mW and an exposure time of 0.1s. In situ identification and detection of single guanine molecules were achieved through the characteristic peaks of Raman spectroscopy.

[0063] This paper proposes an innovative method for constructing biological single-molecule probes. This method leverages intermolecular hydrogen bonding interactions to precisely control the distance between gold nanoelectrodes and gold nanoparticles, enabling efficient detection of target biomolecules (such as DNA bases or proteins). The tunable nature of hydrogen bonding allows the molecular junction formed between the gold nanoelectrode and gold nanoparticles to be electrochemically controlled and "opened and closed." This unique structure significantly enhances signal sensitivity.

[0064] By precisely controlling the electrochemical potential, the present invention not only adjusts the gap between the gold nanoelectrode and the gold nanoparticles, but also dynamically changes the state of the molecular bonding, thereby achieving highly sensitive, real-time monitoring of target molecules. The innovation of this technology lies in its ability to precisely capture the dynamic changes in intermolecular hydrogen bonds and convert these changes into SERS signals, thus achieving high-precision detection at the molecular level.

[0065] Traditional SERS detection usually uses gold nanoparticles to form nanogaps, and its surface structure is regular, so its signal enhancement effect can only reach the traditional amplification factor (10 7 ~10 8 ), while the present invention uses corroded gold nanoelectrodes as the substrate, whose surface is uneven. Under the action of plasmons, multiple single-atom protrusions are formed. These single-atom protrusions can concentrate the light field around the protruding gold atoms. This concentrated light field greatly enhances the electric field, resulting in a significant increase in the Raman signal amplification factor, which can usually reach 10 9 ~10 10 .

[0066] Compared to existing technologies, this invention significantly improves the efficiency and accuracy of molecular recognition and has broad application prospects in the field of biomolecular detection. By synergistically optimizing the bridging molecule and the molecule being detected, this invention achieves efficient capture and signal amplification of biomolecular hydrogen bond networks, significantly improving detection sensitivity and specificity. It has broad application prospects, particularly in the fields of biomolecular detection, single-molecule analysis, and high-throughput screening.

[0067] The embodiments of the present invention are described in detail above, but the contents are only preferred embodiments of the present invention and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A single molecule in situ recognition and detection method based on intermolecular hydrogen bonds, characterized by: The following steps are involved: S1. Preparation and surface treatment of gold nanoelectrode substrate: Gold nanoelectrodes were prepared by electrochemical etching: gold wire was used as raw material and electrochemical etching was used to treat the gold wire to obtain gold nanoelectrodes with nanostructures; S2. Modify the bridging molecules on the gold nanoelectrode substrate: A bridging molecule solution with a thiol group is used to bind to the surface of the gold nanoelectrode. The bridging molecule provides both a hydrogen bond donor and an acceptor, and can form a molecular pairing with the subsequent target molecule. S3. Modification of the surface of gold nanoparticles with bridging molecules: By mixing the bridging molecule solution of step S2 with gold nanoparticles, the modified gold nanoparticles can bind to the target molecules through hydrogen bonds, thereby providing structural support for the subsequent assembly of molecular pairs; S4. Construction of molecular junctions: placing the gold nanoelectrode modified in step S2 into the gold nanoparticle solution modified in step S3, then adding the target molecule solution into the gold nanoparticle solution, and forming a molecular junction by utilizing the electrostatic adsorption between the gold nanoparticles and the gold nanoelectrode; S5. Electrochemical Control and Dynamic Regulation of Molecular Binding: By adjusting the electrode potential, the nanogap between the gold nanoparticles and the gold nanoelectrode is regulated, thereby affecting the state of the molecular junction; S6. Real-time monitoring of SERS signals and molecular recognition: As the electrode potential is adjusted, the size of the gap between the gold nanoelectrode and the gold nanoparticles changes, resulting in changes in the SERS signal intensity. By analyzing the SERS signal, the dynamic changes of intermolecular hydrogen bond interactions can be captured and the identification information of the target molecules can be obtained in real time.

2. The single-molecule in situ recognition and detection method based on intermolecular hydrogen bonds according to claim 1, characterized in that: In step S1, the gold wire has a purity of 99.9% and a diameter of 0.2 mm.

3. The single-molecule in situ recognition and detection method based on intermolecular hydrogen bonds according to claim 1, characterized in that: In step S1, the surface of the gold nanoelectrode is cleaned to remove impurities and then wrapped with high-density polyethylene.

4. The single-molecule in situ recognition and detection method based on intermolecular hydrogen bonds according to claim 1, characterized in that: In step S2, the bridging molecule is 4-mercaptobenzoic acid or cysteine.

5. The single-molecule in situ recognition and detection method based on intermolecular hydrogen bonds according to claim 1, characterized in that: In step S3, the bridging molecules are modified onto the surface of the gold nanoparticles by centrifugation, standing and extraction.

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