Inter-molecular interaction detection method based on single-molecule fluorescence orientation

Abstract

The invention provides an intermolecular interaction detection method based on single-molecule fluorescence orientation. The detection method comprises the following steps: a first compound molecule and a second compound molecule can form intermolecular interaction; fixing first compound molecules on the surface of the substrate to form a molecular recognition interface; fluorescence labeling the first compound molecule or the second compound molecule with a fluorescence probe molecule; and applying the second molecule to a molecular recognition interface, detecting a single fluorescent molecule signal by using optical imaging equipment, and judging the type and strength of the intermolecular interaction according to the spatial orientation of the single fluorescent molecule. The detection method can visually and dynamically display the influence of external conditions on intermolecular interaction, and has the advantages of simplicity and convenience in operation, high sensitivity, high flux, excellent temporal-spatial resolution and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry and relates to a method for detecting intermolecular interactions based on single-molecule fluorescence orientation. Background Art

[0002] Intermolecular interactions are the core basis of life activities and are widely involved in and regulate various biological processes, including the specific recognition of antibodies and antigens, the transcription and translation of chromatin DNA, and intercellular signal transduction. Imbalances in intermolecular interactions are closely related to the occurrence of many major diseases. For example, prions cause misfolding of normal proteins through abnormal conformational changes, and molecular recognition dysfunction caused by gene mutations. Traditional methods have limitations in detecting changes in the strength of intermolecular interactions and are difficult to achieve high-sensitivity and high-resolution quantitative analysis. Therefore, developing a low-cost and high-sensitivity intermolecular interaction detection technology is of great significance for deeply revealing molecular mechanisms and disease mechanisms.

[0003] Traditional methods for studying intermolecular interactions include techniques such as nuclear magnetic resonance (NMR) and X-ray crystallography. Although these methods can provide complex structure information with angstrom-level resolution, they are time-consuming and cannot detect the dynamic change process of intermolecular interactions. In addition, methods such as electrochemical sensing, quartz crystal microbalance (QCM), and surface-enhanced Raman (SERS) can also be used to detect intermolecular interactions, but these methods are all based on the average signals of macroscopic molecular populations and are difficult to reflect the interaction change characteristics of individual molecules in a specific microenvironment, thus masking the dynamic information at the single-molecule level.

[0004] Single-molecule techniques provide a new strategy for studying intermolecular interactions, and their core lies in observing the dynamic behavior of individual molecules after interacting with target molecules. For example, single-molecule mechanical detection techniques represented by optical tweezers have been widely used in the study of DNA-protein interactions. This method indirectly reflects the conformational changes of complexes through stepwise changes in mechanical signals, but its limitation is that it is difficult to capture the dynamic information of local interactions. On the other hand, although the single-molecule fluorescence resonance energy transfer (smFRET) technique based on optical imaging can reflect the distance changes caused by local intermolecular interactions, the high sensitivity of the smFRET technique requires controlling the distance between two fluorescent labels within the range of 2-8 nm, which poses a great challenge to experimental design. Currently, the analysis of local subtle intermolecular interactions relies more on molecular dynamics simulation calculation methods, and there is still a lack of an efficient method for directly detecting the dynamic changes of local interactions in complexes at the experimental level.

[0005] In view of the need to analyze intermolecular interactions in the process of molecular binding, the existing analysis methods have limitations, mainly manifested as long experimental periods, difficult cost control, high system complexity, and inability to intuitively reflect the dynamic change process of intermolecular interactions, which restricts the in-depth understanding of molecular recognition mechanisms and their kinetic behaviors. Summary of the Invention

[0006] To solve the technical problems existing in the prior art, the present invention provides a method for detecting intermolecular interactions based on single-molecule fluorescence orientation. This detection method can intuitively and dynamically display the influence of external conditions on intermolecular interactions, and has advantages such as simple operation, high sensitivity, high throughput, and excellent spatio-temporal resolution.

[0007] To achieve the above technical effects, the present invention adopts the following technical solutions:

[0008] The present invention provides a method for detecting intermolecular interactions based on single-molecule fluorescence orientation, and its process is as Figure 8 shown. This detection method includes:

[0009] A first compound molecule and a second compound molecule can form an intermolecular interaction;

[0010] Fix the first compound molecule on the surface of the substrate to form a molecular recognition interface;

[0011] Use a fluorescent probe molecule to fluorescently label the first compound molecule or the second compound molecule;

[0012] Apply the second molecule to the molecular recognition interface, use an optical imaging device to detect the signal of a single fluorescent molecule, and judge the type and strength of the intermolecular interaction through the spatial orientation of the single fluorescent molecule.

[0013] As a preferred technical solution of the present invention, the first compound molecule and the second compound molecule independently include any one or at least two combinations of protein molecules, nucleic acid molecules, or ligand small molecules.

[0014] As a preferred technical solution of the present invention, the intermolecular interaction includes any one or at least two combinations of electrostatic interaction, hydrogen bond interaction, π-π stacking interaction, or van der Waals force interaction.

[0015] As a preferred technical solution of the present invention, the surface of the substrate has a functional group for fixing the first compound molecule.

[0016] As a preferred technical solution of the present invention, the functional group includes any one or at least two combinations of amino group, hydroxyl group, sulfonic acid group, epoxy group, biotin, mercapto group, carboxyl group, aldehyde group, cyanate ester, maleimide, N-hydroxysuccinimide, streptavidin, or avidin.

[0017] As a preferred technical solution of the present invention, determining the type and strength of intermolecular interactions through the spatial orientation of a single fluorescent molecule includes analyzing the spatial orientation and dynamic behavior of a single fluorescent molecule based on fluorescence polarization characteristics, and determining the type and strength of intermolecular interactions from the degree of restriction of the movement of the fluorescent molecule.

[0018] As a preferred technical solution of the present invention, the physical quantities describing the spatial orientation and dynamic behavior of a single fluorescent molecule include: the polar angle of the inclination angle between the single fluorescent molecule and the vertical direction of the glass substrate, the azimuth angle after the single fluorescent molecule is projected onto the plane of the glass substrate, and the cone opening angle (swing angle) of the cone model describing the degree of single molecule wobbling.

[0019] As a preferred technical solution of the present invention, the optical imaging device includes a single molecule fluorescence imaging device, and the single molecule fluorescence imaging device has a module for analyzing the polarization of the fluorescence signal.

[0020] As a preferred technical solution of the present invention, the intermolecular interactions are in-situ dynamically regulated and monitored by changing external conditions.

[0021] As a preferred technical solution of the present invention, the external conditions include any one or a combination of at least two of pH, ionic strength, temperature, or potential.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects:

[0023] (1) The present invention provides a method for detecting intermolecular interactions based on single molecule fluorescence orientation. The experimental system of this detection method is flexibly designed and does not need to rely on complex intermolecular distance regulation.

[0024] (2) The present invention provides a method for detecting intermolecular interactions based on single molecule fluorescence orientation. This detection method retains the high spatio-temporal resolution characteristics of single molecule fluorescence imaging technology and can observe the dynamic behavior of molecular binding complexes with a time resolution of milliseconds.

[0025] (3) The present invention provides a method for detecting intermolecular interactions based on single molecule fluorescence orientation. This detection method can monitor in real time the dynamic regulation effect of changes in the external environment on the interactions during the molecular recognition process.

[0026] (4) The present invention provides a method for detecting intermolecular interactions based on single molecule fluorescence orientation. This detection method has the advantages of simple operation, high sensitivity, high throughput, excellent spatio-temporal resolution, etc., and has important innovation and practical value in the field of biochemical analysis. Description of the Drawings

[0027] Figure 1Schematic diagram of physical parameters for describing the spatial movement of single molecules.

[0028] Figure 2 Schematic diagram of the imaging system used in Examples 1 and 2.

[0029] Figure 3 Distribution diagram of the wobbling angle of fluorescent molecules at different pH values detected by using the orientation of fluorescent molecules in Example 1.

[0030] Figure 4 For Example 1, using the orientation of fluorescent molecules to detect the 2+ distribution diagram of the wobbling angle of fluorescent molecules at different concentrations.

[0031] Figure 5 For Example 2, using the orientation of fluorescent molecules to detect the 2+ distribution diagram of the wobbling angle of fluorescent molecules at different concentrations.

[0032] Figure 6 For Example 2, using the orientation of fluorescent molecules to detect the + distribution diagram of the wobbling angle of fluorescent molecules at different Na concentrations.

[0033] Figure 7 Schematic diagram of the modification method of Cy3 fluorescent molecules on ssDNA in Example 1.

[0034] Figure 8 Schematic flow diagram of the method for detecting intermolecular interactions based on single-molecule fluorescence orientation provided by the specific implementation manner of the present invention.

[0035] The present invention will be further described in detail below. However, the following examples are merely simple examples of the present invention and do not represent or limit the scope of the protection of the present invention. The scope of protection of the present invention shall be subject to the claims. Specific implementation manner

[0036] The technical solution of the present application will be further described below through specific implementation manners.

[0037] The specific implementation manner of the present invention provides a method for detecting intermolecular interactions based on single-molecule fluorescence orientation. The detection method includes:

[0038] The first compound molecule and the second compound molecule can form intermolecular interactions;

[0039] Fix the first compound molecule on the surface of the substrate to form a molecular recognition interface;

[0040] Use a fluorescent probe molecule to fluorescently label the first compound molecule or the second compound molecule;

[0041] Apply a second molecule to the molecular recognition interface, use an optical imaging device to detect the signal of a single fluorescent molecule, and judge the type and strength of intermolecular interaction by the spatial orientation of a single fluorescent molecule.

[0042] In the present invention, this detection method can intuitively and dynamically display the influence of external conditions on intermolecular interaction. Its core principle lies in that: the conjugated structure of the fluorescent molecule emits anisotropic fluorescent signals when excited. By analyzing the polarization information of the fluorescent signals and their variation law with time, the spatial motion behavior characteristics of a single fluorescent molecule equivalent to a single dipole are obtained, reflecting the intermolecular interaction strength and dynamic change.

[0043] In a specific embodiment of the present invention, the first compound molecule and the second compound molecule independently include any one or at least two combinations of protein molecules, nucleic acid molecules or ligand small molecules.

[0044] In a specific embodiment of the present invention, the combination of the first compound molecule and the second compound molecule can be protein-protein, protein-nucleic acid, nucleic acid-nucleic acid, nucleic acid or protein and the functionalized group on the glass substrate surface (molecular recognition interface), protein or nucleic acid and small molecule ligand.

[0045] In a specific embodiment of the present invention, the intermolecular interaction includes any one or at least two combinations of electrostatic interaction, hydrogen bond interaction, π-π stacking interaction or van der Waals force interaction. However, it is not limited to the above-mentioned intermolecular interactions, and other intermolecular interactions can also be detected by the method of the present application.

[0046] In a specific embodiment of the present invention, the substrate surface has a functionalized group for fixing the first compound molecule.

[0047] In a specific embodiment of the present invention, the substrate can be a glass substrate, but it is not limited to the glass substrate. Other substrates that do not interfere with fluorescence detection and can fix the first compound molecule are applicable to this detection method.

[0048] In a specific embodiment of the present invention, the functionalized group includes any one or at least two combinations of amino group, hydroxyl group, sulfonic acid group, epoxy group, biotin, mercapto group, carboxyl group, aldehyde group, cyanate ester, maleimide, N-hydroxysuccinimide, streptavidin or avidin. The functionalized group can form a chemical bond with the group in the first compound molecule, thereby fixing the first compound molecule on the substrate surface.

[0049] In a specific embodiment of the present invention, the fluorescent probe molecule can be Cy3 or Cy5. The fluorescent probe molecule can be modified in the non-binding region of the second compound molecule to ensure that it does not interfere with the specific binding process between molecules. When the second compound is a small molecule compound, the fluorescent probe molecule can also be modified on the first compound molecule immobilized on the surface of the substrate.

[0050] In a specific embodiment of the present invention, the optical imaging device includes a single-molecule fluorescence imaging device, and the single-molecule fluorescence imaging device has a module for analyzing the polarization of the fluorescence signal.

[0051] In a specific embodiment of the present invention, the wavelength of the laser of the optical imaging device can be 405nm, 488nm, 561nm or 637nm, etc., the exposure time for single-molecule data acquisition can be 5 - 100ms, and the power of the laser can be 1 - 100mW, so as to ensure that the single-molecule imaging system can obtain single-molecule fluorescence signals with sufficient signal-to-noise ratio.

[0052] In a specific embodiment of the present invention, during fluorescence detection, every 100μm 2 the density of fluorescent molecules within the imaging field of view should be between 10 and 20 molecules.

[0053] In a specific embodiment of the present invention, determining the type and strength of intermolecular interactions through the spatial orientation of a single fluorescent molecule includes analyzing the spatial orientation and dynamic behavior of a single fluorescent molecule based on fluorescence polarization characteristics, and determining the type and strength of intermolecular interactions from the degree of motion restriction of the fluorescent molecule.

[0054] In a specific embodiment of the present invention, the physical quantities describing the spatial orientation and dynamic behavior of a single fluorescent molecule include: the polar angle (θ) of the inclination angle between a single fluorescent molecule and the vertical direction of the glass substrate, and the azimuth angle after a single fluorescent molecule is projected onto the plane of the glass substrate and the cone half-angle swing angle (α, between 0° and 90°) of the cone model describing the degree of single-molecule wobbling.

[0055] In a specific embodiment of the present invention, in-situ dynamic regulation and monitoring of intermolecular interactions are carried out by changing external conditions.

[0056] In a specific embodiment of the present invention, the external conditions include any one or a combination of at least two of pH, ionic strength, temperature or potential. However, it is not limited to the above conditions, and other conditions that can cause changes in the interaction between the first molecule and the second molecule are also applicable to this detection method.

[0057] To better illustrate the present invention and facilitate understanding of its technical solutions, typical but non-limiting embodiments of the present invention are as follows:

[0058] Example 1

[0059] Taking the electrostatic adsorption of single-stranded DNA (ssDNA) on an amino-modified substrate as an example. The ssDNA modified with Cy3 fluorescent molecules was electrostatically adsorbed onto the amino-modified substrate under neutral conditions, and the intermolecular interactions were observed by changing the pH and ionic concentration in the solution.

[0060] (1) Preparation of amino-functionalized glass substrate

[0061] Under neutral conditions, the phosphate backbone of ssDNA is negatively charged due to ionization. Therefore, it is necessary to construct a glass substrate with a positively charged surface under neutral conditions to achieve the electrostatic adsorption of ssDNA. A transparent glass substrate (22 mm × 22 mm × 0.17 mm) was used in the experiment, and the following cleaning steps were carried out in sequence: ultrasonic cleaning with 5% hydrochloric acid solution for 15 minutes, and then ultrasonic cleaning with 1 M KOH for 15 minutes to eliminate the fluorescence background interference caused by impurities on the glass substrate surface. The cleaned glass substrate was placed in a clean petri dish and dried in an oven at 75 °C for 30 minutes to remove the residual moisture. Subsequently, the dried glass substrate was transferred to a vacuum desiccator containing 3-aminopropyltriethoxysilane (APTES), and the vacuum state was maintained at room temperature for 2 days. APTES was modified on the glass substrate surface by chemical vapor deposition method, and finally an amino-functionalized glass substrate was obtained for the electrostatic adsorption of ssDNA.

[0062] (2) Electrostatic adsorption of ssDNA and amino-functionalized glass substrate

[0063] The DNA sequence from the 5' end to the 3' end is: AAAAAAA / iCy3 / AAAAAAAA, and the Cy3 molecule is modified on the phosphate backbone between the seventh and eighth adenines. For the modification details, see Figure 7 . Subsequently, the ssDNA was dissolved in 200 μL of 10 mM Tris (pH 7.70) buffer at a concentration of 1 μM, and then it was dropped onto the surface of the amino-modified glass substrate and incubated at room temperature for 1 hour to ensure that the ssDNA was fully adsorbed onto the substrate surface through electrostatic interaction. Subsequently, the glass substrate was repeatedly washed with 10 mM Tris (pH 7.70) buffer to remove the unadsorbed ssDNA.

[0064] (3) Single-molecule fluorescence imaging

[0065] The single-molecule fluorescence imaging system includes: an excitation optical path, a sample stage, and a fluorescence emission and imaging optical path. Among them, a phase mask is added in the Fourier plane of the fluorescence emission and imaging optical path to regulate the point spread function and generate different imaging patterns for the single-molecule orientation.

[0066] For the specific schematic diagram of the imaging optical path, seeFigure 2 。

[0067] (4) Single-molecule fluorescence signal analysis

[0068] Using techniques such as joint sparse basis deconvolution, parameter information such as the brightness, position, and three-dimensional orientation of fluorescent molecules is recovered from the raw single-molecule fluorescence imaging images that have been regulated by the point spread function and contain noise.

[0069] (5) Real-time observation of the response of the orientation of fluorescent molecules to pH changes

[0070] The adsorption of ssDNA on the substrate surface mainly depends on the electrostatic interaction between its phosphate backbone and the amino-modified glass substrate. Under neutral conditions, the amino group is positively charged due to protonation. However, as the pH value of the solution increases, the degree of protonation of the amino group gradually weakens, resulting in a decrease in the positive charge carried on its surface. In this example, 10 mM Tris buffer solutions with pH values of 7.70, 9.15, and 10.00 are selected as the imaging solutions. Within this pH range, the DNA phosphate backbone with a lower pKa value can maintain a fully ionized state and stably carry a negative charge. By gradually increasing the pH value of the solution, the spatial orientation changes of the Cy3 fluorescent molecules on ssDNA are observed, thereby analyzing the change process of intermolecular interactions.

[0071] During the experiment, after ssDNA was successfully adsorbed on the glass substrate surface, 10 mM Tris (pH 7.70) buffer solution was used to gently pipette the ssDNA sample, and then the solution was completely aspirated. This process was repeated 4 times. Finally, 200 μL of 10 mM Tris (pH 7.70) buffer solution was retained to reduce the interference of free ssDNA on the experimental results. After the data collection (excited by a 20 mW 561 nm laser, 50 ms exposure time) was completed, using the same experimental steps, 10 mM Tris (pH 9.15) and 10 mM Tris (pH 10.00) buffer solutions were selected, and the pH value was increased in sequence to obtain the experimental result diagrams as Figure 3 shown.

[0072] Through Figure 3 It can be seen that as the pH increases, especially under the condition of pH 10.00, the shaking degree of the fluorescent molecules increases significantly, indicating that the adsorption ability of ssDNA on the amino-modified glass substrate surface weakens. This phenomenon corresponds to the weakening of the protonation ability and the decrease in the charge carried on the glass slide surface.

[0073] (6) Real-time observation of the response of the orientation of fluorescent molecules to Mg 2+ concentration

[0074] As described above, since the phosphate backbone of ssDNA is negatively charged and can undergo electrostatic interaction with the positively charged amino group, it is adsorbed on the surface of the glass substrate. According to the charge screening effect in the electrolyte solution, the greater the ionic strength in the solution, the stronger the screening effect. The Debye length is usually used to describe the action distance of the charge screening effect. If the distance is greater than this length, it can be considered that there is no electrostatic interaction. Its definition formula is as follows:

[0075]

[0076] Among them, λ D is the Debye length, which represents the distance at which the electric potential around a charged particle is significantly screened by the surrounding charges. ε r is the relative permittivity, ε0 is the vacuum permittivity, K B is the Boltzmann constant, T is the temperature (Kelvin), N A is the Avogadro constant, and I is the ionic strength in the solution.

[0077]

[0078] Among them, c i is the ion concentration, and z i is the charge number.

[0079] During the experiment, different volumes of 1M MgCl2 solution were added to 200 μL of the imaging solution (10 mM Tris, pH 7.70) to obtain Tris solutions (10 mM, pH 7.70) with MgCl2 concentrations of 0 mM, 100 mM, and 200 mM, respectively. The corresponding Debye lengths are approximately 3.30 nm, 0.55 nm, and 0.39 nm. Under the condition that the ionic strength of the solution gradually increases, the dynamic change of the spatial orientation of the Cy3 molecules in ssDNA is observed to reflect the change of the adsorption behavior. The data acquisition parameters are: excited by a 20 mW 561 nm laser, with an exposure time of 50 ms. The experimental results are as Figure 4 shown.

[0080] From Figure 4 it can be seen that as the concentration of Mg 2+ gradually increases, the electrostatic interaction between ssDNA and the amino interface of the glass substrate weakens, resulting in a significant increase in the degree of fluorescence molecule shaking.

[0081] Example 2

[0082] Take the adsorption of ssDNA on the sulfonic acid-modified substrate as an example. In a high-pH solution environment, the single-stranded DNA modified with Cy3 fluorescent molecules is adsorbed on the surface of the sulfonic acid-modified substrate, and the types and concentrations of the solution ions are changed to observe the intermolecular hydrogen bond interaction.

[0083] Due to its extremely low pKa (-2 to 0), the sulfonic acid group can be completely ionized and carry a negative charge at pH > 7. Although the phosphate backbone of ssDNA also carries a negative charge under this condition, the electrostatic repulsion between it and the sulfonic acid group can, to a certain extent, inhibit the adsorption of the sulfonic acid group to ssDNA. However, the completely ionized sulfonic acid group contains three strongly electronegative oxygen atoms, which can form stable hydrogen bonds with the amino hydrogen atoms on the adenine bases in ssDNA. Therefore, ssDNA can still be effectively adsorbed on the surface of the sulfonic acid-modified substrate through hydrogen bond interactions.

[0084] (1) Preparation of sulfonic acid group-modified glass imaging substrate

[0085] In the experiment, a transparent glass (22 mm × 22 mm × 0.17 mm) substrate was cleaned successively through the following steps: ultrasonically cleaned with 5% hydrochloric acid solution for 15 minutes, and then ultrasonically cleaned with 1 M KOH for 15 minutes to eliminate the fluorescence background interference that may be generated by impurities on the glass substrate. The cleaned glass substrate was placed in a clean petri dish and dried in an oven at 75 °C for 30 minutes to remove residual moisture. The glass substrate was placed in a vacuum desiccator containing 3-glycidoxypropyltrimethoxysilane (GOPTS), and the vacuum state was maintained at room temperature for 2 days. Finally, GOPTS was modified on the surface of the glass substrate by vapor deposition to obtain an epoxy group-functionalized glass substrate. Subsequently, 400 μL of 20 mM 3-aminopropanesulfonic acid (dissolved in 10 mM sodium carbonate buffer at pH 9.20) was dropped onto the surface of the epoxy group-modified glass substrate and reacted at room temperature for 18 hours, enabling 3-aminopropanesulfonic acid to covalently bind to the epoxy group through a nucleophilic ring-opening reaction, thereby introducing the sulfonic acid group onto the surface of the glass substrate. After the reaction, the glass substrate was thoroughly cleaned with 10 mM sodium carbonate buffer at pH 9.20 to remove unreacted 3-aminopropanesulfonic acid, and finally, a sulfonic acid group-functionalized glass substrate was obtained for subsequent ssDNA adsorption experiments.

[0086] (2) Hydrogen bond adsorption of ssDNA to sulfonic acid group-functionalized glass substrate

[0087] The DNA sequence from the 5'-end to the 3'-end is: AAAAAAA / iCy3 / AAAAAAAA, and the Cy3 molecule is modified on the phosphate backbone between the seventh and eighth adenines. For modification details, see Figure 7 . Subsequently, ssDNA was dissolved at a concentration of 1 μM in 200 μL of 10 mM (pH 9.20) sodium carbonate buffer, and then it was dropped onto the surface of the sulfonic acid-modified glass substrate and incubated at room temperature for 1 hour to ensure that ssDNA was fully adsorbed onto the substrate surface through hydrogen bond interactions. Subsequently, the glass substrate was repeatedly cleaned with 10 mM (pH 9.20) sodium carbonate buffer to remove unadsorbed free ssDNA.

[0088] (3) Single-molecule fluorescence imaging

[0089] The single-molecule fluorescence imaging system includes: an excitation optical path, a sample stage, and a fluorescence emission and imaging optical path. Among them, a phase mask is added in the Fourier plane of the fluorescence emission and imaging optical path to regulate the point spread function, so as to generate different imaging patterns for the single-molecule orientation.

[0090] For the specific schematic diagram of the imaging optical path, see Figure 2 .

[0091] (4) Single-molecule fluorescence signal analysis

[0092] Using techniques such as joint sparse basis deconvolution, parameter information such as the brightness, position, and three-dimensional orientation of fluorescent molecules is recovered from the original single-molecule fluorescence imaging images that have been regulated by the point spread function and contain noise.

[0093] (5) Real-time observation of the response of the orientation of fluorescent molecules to the types and concentrations of solution ions

[0094] Since ssDNA is mainly adsorbed on the surface of the sulfonic acid group-modified glass substrate through hydrogen bonding, changing the ionic environment of the solution will significantly affect the ssDNA adsorption mechanism. This study focuses on exploring the effects of Mg 2+ and Na + on the adsorption behavior of ssDNA: Mg 2+ has a higher affinity for the phosphate backbone of DNA. Compared with nitrogenous bases, it preferentially binds to phosphate groups. In the presence of low-concentration Mg 2+ , on the one hand, Mg 2+ can neutralize the electrostatic repulsion between the ssDNA phosphate backbone and the sulfonic acid group substrate; on the other hand, Mg 2+ can form an ion-mediated connection between the ssDNA phosphate backbone and the sulfonic acid group substrate through bridging, resulting in the transformation of the intermolecular interaction from hydrogen bond interaction to Mg 2+ -mediated electrostatic interaction. After washing away Mg 2+ , by adjusting the concentration of Na + and observing the fluorescence molecule orientation response, the influence of the ionic environment on the ssDNA adsorption mechanism is further verified.

[0095] After the ssDNA adsorption is completed, use 10 mM (pH 9.20) sodium carbonate buffer solution to gently pipette the ssDNA sample, and then aspirate all the solution. Repeat this process 4 times, and finally retain 200 μL of 10 mM (pH 9.20) sodium carbonate buffer solution to reduce the interference of free ssDNA on the experimental results. After the data acquisition (excited by a 20 mW 561 nm laser, 50 ms exposure time) is completed, use the same experimental steps to increase Mg2+ The concentration was increased to 5 mM, and data were collected. Subsequently, the sample was washed 4 times with 10 mM sodium carbonate buffer (pH 9.20) to remove Mg 2+ completely, and data were collected with the same parameters; then the NaCl concentration in the imaging solution was increased to 333 mM, and data were collected. The experimental results are as Figure 5 、 6 shown.

[0096] As Figure 5 shown, high concentration of Mg 2+ significantly restricted the spatial movement of fluorescent molecules. Since Mg 2+ has a higher affinity for the phosphate backbone, this phenomenon reflects the change of the intermolecular interaction force between ssDNA and sulfonic acid groups from being dominated by hydrogen bonds to being mediated by Mg 2+ -mediated ion bridging. To verify this mechanism, after removing Mg 2+ by washing, a significant enhancement of the spatial movement of fluorescent molecules was observed under the condition of 10 mM Na + (10 mM sodium carbonate buffer with pH 9.20) ( Figure 6 ). This result indicates that Na + cannot effectively restrict the spatial movement of Cy3 fluorescent molecules, which is attributed to the fact that the interaction between ssDNA and sulfonic acid groups is mainly maintained by hydrogen bonds between nitrogen-containing bases and sulfonic acid groups, and the movement of the phosphate backbone is relatively unrestricted. In addition, it can be seen from Figure 6 that even if the concentration of Na + is further increased, the spatial movement of fluorescent molecules is still not significantly restricted, further confirming the weak regulatory effect of Na+ on the adsorption behavior of ssDNA.

[0097] The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must rely on the above detailed structural features to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the components selected by the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0098] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0099] In addition, it should be noted that, among the various specific technical features described in the above specific embodiments, they can be combined in any appropriate manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0100] In addition, any combination can be made among various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A method for detecting intermolecular interactions based on single-molecule fluorescence orientation, characterized in that, The detection method includes: The first compound molecule and the second compound molecule can form intermolecular interactions; Fix the first compound molecule on the surface of the substrate to form a molecular recognition interface; Use a fluorescent probe molecule to fluorescently label the first compound molecule or the second compound molecule; Apply the second molecule to the molecular recognition interface, use an optical imaging device to detect the signal of a single fluorescent molecule, and judge the type and strength of the intermolecular interaction by the spatial orientation of the single fluorescent molecule.

2. The detection method according to claim 1, wherein The first compound molecule and the second compound molecule independently include any one or a combination of at least two of protein molecules, nucleic acid molecules or ligand small molecules.

3. The detection method according to claim 1, characterized in that The intermolecular interaction includes any one or a combination of at least two of electrostatic interaction, hydrogen bond interaction, π-π stacking interaction or van der Waals force interaction.

4. The detection method according to claim 1, characterized in that The surface of the substrate has a functional group for fixing the first compound molecule.

5. The detection method according to claim 4, wherein, The functional group includes any one or a combination of at least two of amino group, hydroxyl group, sulfonic acid group, epoxy group, biotin, mercapto group, carboxyl group, aldehyde group, cyanate ester, maleimide, N-hydroxysuccinimide, streptavidin or avidin.

6. The detection method according to claim 1, characterized in that, Judging the type and strength of the intermolecular interaction by the spatial orientation of a single fluorescent molecule includes analyzing the spatial orientation and dynamic behavior of a single fluorescent molecule based on the fluorescence polarization characteristics, and judging the type and strength of the intermolecular interaction by the degree of movement restriction of the fluorescent molecule.

7. The detection method according to claim 6, wherein The physical quantities describing the spatial orientation and dynamic behavior of the single fluorescent molecule include: the polar angle of the tilt angle between the single fluorescent molecule and the vertical direction of the glass substrate, the azimuth angle after the single fluorescent molecule is projected onto the plane of the glass substrate, and the cone opening angle swing angle of the cone model describing the degree of single molecule wobbling.

8. The detection method according to claim 1, wherein The optical imaging device includes a single molecule fluorescence imaging device, and the single molecule fluorescence imaging device has a module for analyzing the polarization of the fluorescence signal.

9. The detection method according to claim 1, wherein In-situ dynamic regulation and monitoring of the intermolecular interaction are carried out by changing external conditions.

10. The detection method according to claim 9, characterized in that, The external conditions include any one or a combination of at least two of pH, ionic strength, temperature or potential.

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