Insecticide active ingredient crystal form analysis method based on three-dimensional electron diffraction

Abstract

The invention provides a method for analyzing crystal forms of effective components of an insecticide based on three-dimensional electron diffraction. The method comprises the steps of sample preparation, cooling sample introduction, data observation and collection and data processing. The sample preparation comprises spraying a to-be-detected solution on the substrate, so that microcrystals are adhered to the carrier net; the cooling sample introduction is to transfer the carrier net to a freezing rod for cooling and then feed the carrier net into a transmission electron microscope for vacuumizing; observing the acquired data includes observing the sample and acquiring three-dimensional electron diffraction data. The data processing comprises format conversion, diffraction point integral calculation, analysis file generation, structure analysis refinement and the like. The method can rapidly and accurately identify the crystal form of the effective component of the insecticide under actual working conditions, and provides technical support for design of insecticide preparations.

Description

Technical Field

[0001] The invention relates to the field of testing technology development, in particular to a method for analyzing the crystal form of an active ingredient of an insecticide based on three-dimensional electron diffraction. Background Art

[0002] Pesticides usually contain one or more organic synthetic active ingredients, which usually have complex molecular structures and physical and chemical properties. Recent studies have shown that different crystal forms of a pesticide can have performance differences of more than ten times in actual use. Therefore, accurate identification and characterization of the crystal form characteristics of pesticides in actual use are of great significance for optimizing pesticide formulations and improving product performance and stability.

[0003] Unlike drug crystal forms, pesticides often need to go through processes such as dissolution, dispersion, spraying, and evaporation during actual use, so the crystal form of its preparation can only be considered as an "intermediate crystal form" rather than the final crystal form that works. However, due to the low dosage of pesticides and unsatisfactory crystallization conditions during actual use, the effective crystal form in use cannot be captured instantaneously, and it is difficult to detect its diffraction signal using traditional methods such as PXRD and single crystal XRD.

[0004] In summary, the existing technology has deficiencies in identifying the crystal forms of active ingredients in the actual use of pesticides. There is an urgent need to develop highly sensitive, rapid, efficient, stable and direct methods to quickly characterize and identify the effective crystal forms of active ingredients in new pesticides. Summary of the invention

[0005] The embodiment of the present invention provides a method for analyzing the crystal form of an active ingredient of an insecticide based on three-dimensional electron diffraction, which aims to solve the problems existing in the current technology, such as the crystal form in actual use is an intermediate state, the dosage is low, the crystallization conditions are not ideal, the diffraction signal is difficult to detect using traditional methods, and the crystal form of the active ingredient cannot be effectively identified.

[0006] The core technology of the present invention is mainly to use three-dimensional electron microcrystal diffraction technology to develop a test method for rapid characterization and identification of the active crystal form of the pesticide under actual working conditions, including the steps of sample preparation, cooling and sampling, observation and collection of data, and data processing.

[0007] In a first aspect, the present invention provides a method for analyzing the crystal form of an active ingredient of an insecticide based on three-dimensional electron diffraction, the method comprising the following steps: S00, Sample preparation: Spray the solution to be tested on a flat and clean substrate, and after the solution to be tested evaporates and dries, a thin layer of microcrystalline crystals will form on the surface of the substrate; Use tweezers to place the front of the grid on the substrate and press and move it so that the insecticide microcrystals in the test solution adhere to the grid. The concentration of the crystals can be controlled by adjusting the pressure and movement during sample preparation. S10, sample cooling and injection: The grid with the insecticide was quickly transferred to a cryostat, the sample was cooled with liquid nitrogen, and then the cryostat was transported into the TEM and evacuated; S20, sample observation and data collection: Observe the sample morphology, distribution and crystal quality at low magnification, select appropriate microcrystalline samples for marking, and make fine adjustments in height and level. Then collect three-dimensional electron diffraction data, tilt ±60° along the inclination direction of the frozen sample rod, and collect three-dimensional electron diffraction spots with high resolution and high diffraction intensity. S30, data processing: Select diffraction data of appropriate intensity to integrate the diffraction intensity, determine the sample unit cell parameters, further perform absorption correction, and analyze the crystal structure.

[0008] Furthermore, in step S00, the substrate includes one of glass, silicon wafer, mica and blade.

[0009] Furthermore, in step S30, the specific data processing steps include: Converting the raw image format from a format suitable for electron diffraction data to a format suitable for subsequent software processing; Use a software tool that can select diffraction points for unit cell calculation to perform index unit cell calculation. When the calculation results meet the preset target parameters, further perform intensity integration operation on the diffraction points. Using a software that can be used to process and generate files required for solving crystal structures to generate corresponding files; The crystal structure was solved and refined using a software package that includes methods capable of structure solution and refinement.

[0010] Furthermore, in step S30, the specific data processing steps include: Convert the original image format from mrc format to SMV format; Use XDS or Pets2 software to select diffraction points for index cell calculation. If the target parameters are met, further perform diffraction point intensity integration. The ins files required for crystal structure analysis were processed using XPREP software, and the structures were analyzed and refined using ShelxT or ShelxD methods in the Olex2 software package.

[0011] Furthermore, the original image format is converted from mrc format to SMV format through Python script.

[0012] Furthermore, in step S10, after the copper mesh with the insecticide is quickly transferred to the cryogenic freezing rod, liquid nitrogen is first added to immerse the tip of the cryogenic freezing rod and then naturally cool it. After cooling to the set temperature, the cryogenic freezing rod is partially inserted into the transmission electron microscope and evacuated. After the evacuation is completed, the cryogenic freezing rod is completely sent into the transmission electron microscope.

[0013] Furthermore, the temperature was set to 98K.

[0014] The main contributions and innovations of the present invention are as follows: 1. Achieve rapid and accurate identification The present invention can realize the rapid characterization and identification of the crystal form of the active ingredient of the pesticide under actual working conditions. No post-processing is required for the crystal form produced under actual working conditions, which ensures that the crystal form of the sample will not change during the preparation of the test, and accurate crystal form information can be directly obtained.

[0015] 2. Highly sensitive detection of trace crystals By utilizing the trace detection characteristics of microcrystal electron diffraction technology, very small amounts of crystals can be detected. This is of great significance for situations where the pesticide dosage is low and the crystallization conditions are not ideal in actual use, and can effectively detect crystal forms that are difficult to detect with traditional methods.

[0016] 3. Acquisition of strong diffraction signals By utilizing the strong interaction between electrons and crystals, even under actual working conditions, when the insecticide itself is poorly crystallized, a strong diffraction signal can be obtained, thus improving the reliability and accuracy of detection.

[0017] 4. Simultaneous detection of multiple information It can be combined with the imaging technology of electron microscopy to simultaneously detect the crystal form, crystal morphology and size under actual working conditions, providing more information for a comprehensive understanding of the crystal characteristics of pesticides.

[0018] 5. Accurately determine the crystal form This method can directly obtain the unit cell parameters of the sample, determine the crystal form more accurately, and if there are microcrystals with good diffraction, the structure can be directly solved, further improving the accuracy of crystal form analysis.

[0019] 6. Breakthrough of technical bottlenecks It can effectively solve the technical bottleneck of the difficulty in analyzing the effective crystal forms of existing insecticides under actual working conditions, bring important breakthroughs in improving the accuracy, efficiency and data support capabilities of crystal form analysis, and provide strong technical support for the design of insecticide formulations.

[0020] The details of one or more embodiments of the invention are set forth in the following drawings and description so that other features, objects, and advantages of the invention are more readily apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 is a flow chart of a method for analyzing the crystal form of an active ingredient of an insecticide based on three-dimensional electron diffraction according to an embodiment of the present invention; Figure 2 is a microcrystalline sample morphology image of a sample under an electron microscope according to an embodiment of the present invention; Figure 3 is an electron diffraction diagram of a sample microcrystal according to an embodiment of the present invention; Figure 4 is the atomic structure of the aphidamine insecticide according to an embodiment of the present invention. DETAILED DESCRIPTION

[0022] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The implementations described in the following exemplary embodiments do not represent all implementations consistent with one or more embodiments of this specification. Instead, they are merely examples of devices and methods consistent with some aspects of one or more embodiments of this specification as detailed in the appended claims.

[0023] It should be noted that: in other embodiments, the steps of the corresponding method are not necessarily performed in the order shown and described in this specification. In some other embodiments, the steps included in the method may be more or less than those described in this specification. In addition, a single step described in this specification may be decomposed into multiple steps for description in other embodiments; and multiple steps described in this specification may be combined into a single step for description in other embodiments.

[0024] Embodiment 1 The present invention aims to propose a method for analyzing the crystal form of an active ingredient of an insecticide based on three-dimensional electron diffraction. Figure 1 , the method comprising: S00. Sample preparation: Spray the aqueous solution of dinotefuran on any flat and clean substrate, such as glass, silicon wafers, mica, or even leaves. After the solution evaporates, a thin film of organic analytical crystals can be observed on the surface of the substrate. Place the front of the copper mesh (grid) and gently press it with tweezers and move it back and forth several times. The insecticide microcrystals can be successfully adhered to the grid. The concentration of the crystals on the grid can be adjusted by adjusting the strength during sample preparation.

[0025] In this embodiment, the light source of the transmission electron microscope adopts an electron beam with a wavelength of 0.0251Å, and the detector is Ceta-S. Take the aqueous solution of dinotefuran as an example.

[0026] The cryogenic freezing rod is also called the 3D reconstruction cryotransfer sample rod: a cryotransfer and 3D reconstruction electron microscope sample rod for preparing glassy samples by rapid freezing method. Function of 3D reconstruction cryotransfer sample rod: by tilting the sample rod in the cryogenic environment of transmission electron microscope, a series of diffraction spots or images at different angles are obtained, and then the atomic structure of microcrystals is analyzed by 3D reciprocal space information and electron diffraction crystallography technology, or the 3D reconstruction technology is used to obtain the 3D space information of the sample, which is particularly suitable for protein crystals, small molecule compound crystals, crystal materials, etc. It is mainly suitable for various types of electron microscopes of ThermoFisher / JEOL / ELDICO on the market.

[0027] S10. Use tweezers to quickly transfer the copper mesh with insecticide to a cryogenic freezing rod and cool the sample with liquid nitrogen; then transport the cryogenic freezing rod into the transmission electron microscope and evacuate it.

[0028] In this embodiment, the sample copper mesh is transferred to the sample rod (low temperature freezing rod) in advance, and liquid nitrogen is added to allow the tip of the rod to be immersed in the liquid nitrogen for natural cooling. After 6-8 minutes, it is cooled to 98K (about -175.15°C in Celsius). The sample rod is inserted into the transmission electron microscope for vacuuming. After the vacuuming is completed, the sample rod is completely inserted into the transmission electron microscope, and the injection is completed.

[0029] S20. On the basis of cooling the single crystal sample, observe the sample morphology at low magnification, browse the sample distribution and observe the sample crystal quality. Then select several suitable sample areas for marking, make fine adjustments in height and level, and then start collecting three-dimensional electron diffraction data. And along the direction of the frozen sample rod inclination, perform ±60° tilt, step length, exposure time, and collect as many high-resolution and high-diffraction intensity three-dimensional electron diffraction spots as possible for later data analysis and processing.

[0030] In this embodiment, the 200kV electron microscope sample stage and the frozen 3D reconstruction sample rod are designed for continuous sampling of nanoscale samples during the rotation process, which can ensure that the sample is basically kept at the center position of the electron beam irradiation during the rotation process. Before sampling, the sample position needs to be calibrated to the concentric height of the sample stage rotation, which is achieved by adjusting the size of Z. This is the basic operation and principle of the transmission electron microscope, which will not be repeated here.

[0031] S30, perform post-processing of data, such as selecting diffraction points of appropriate intensity to integrate the diffraction intensity and determine the unit cell parameters of the sample; structural diagram after analysis (to determine the crystal type, only indexing is required. The analysis of the crystal structure can be performed by cultivating single crystals under different conditions and comparing the obtained structure with the unit cell).

[0032] The "index" here refers to the process of indexing or calibrating the diffraction points. After selecting the diffraction points using relevant software (such as XDS software or Pets2 software), the index operation is performed to calculate the unit cell parameters. This process involves analyzing and processing information such as the position and intensity of each diffraction point in the diffraction pattern to determine the geometric structure and related parameters of the unit cell, which is an important step in crystal structure analysis.

[0033] In this embodiment, the data processing steps are: 1-Convert the original image format from mrc format to SMV format through Python script; 2-Use XDS or Pets2 software to select diffraction points for index cell calculation. At this time, you can see whether the indexed cell parameters are the target parameters; if so, further perform diffraction point intensity integration; 3-Use XPREP software to process and write the ins file required for crystal structure analysis. Use ShelxT or ShelxD method in Olex2 software package to analyze the structure and refine it.

[0034] like Figure 2 This is the sample microcrystal morphology obtained during the analysis process.

[0035] like Figure 3 The diffraction pattern of the sample obtained during the analysis.

[0036] like Figure 4 This is a schematic diagram of the existing structure of cypermethrin. The unit cell parameters of cypermethrin are: a=5.450(1), b=16.81(3), c=10.060(2), α=90 β=90.77(3), γ=90.

[0037] Example 2 Based on Example 1, this example provides an example of the data processing steps: 1. Original image format conversion Purpose: Convert the original mrc format image to SMV format so that the subsequent software can better process the data.

[0038] Steps: Use Python scripts to convert the format. Python libraries such as numpy and h5py may be used to read the mrc format data, and then reorganize and write the data according to the SMV format specification. The following is a simple sample code framework of a Python script (for reference only, it may need to be adjusted according to the specific data structure and requirements): import numpy as np import h5py def mrc_to_smv(mrc_file_path, smv_file_path): with h5py.File(mrc_file_path, 'r') as mrc_file: data = mrc_file['data'][()]# Read data from mrc file # Process and write data according to the SMV format specification # Here you need to understand the specific requirements of the SMV format, such as data arrangement, header information, etc. with open(smv_file_path, 'wb') as smv_file: # Write header information in SMV format smv_file.write(b'SMV header information...') # Write data smv_file.write(data.tobytes()) 2. Diffraction point selection and unit cell calculation Purpose: To select appropriate diffraction points and perform unit cell calculations using XDS software or Pets2 software.

[0039] Steps: For XDS software, prepare the image data files converted to SMV format and the relevant experimental parameter files (such as detector parameters, geometric relationship between crystal and detector, etc.).

[0040] Run the XDS software, which will identify and select the diffraction points in the image based on the internal algorithm. Usually, relevant parameters such as integration radius, resolution range, etc. are set in the software configuration file to ensure that the appropriate diffraction points are selected.

[0041] The software will perform unit cell calculations based on the selected diffraction points and output the calculation results, including unit cell parameters and other information.

[0042] For Pets2 software, similarly, the SMV format data file is imported into the Pets2 software.

[0043] Set relevant parameters in the software interface, such as background subtraction method, peak search threshold, etc., in order to accurately select diffraction points.

[0044] The Pets2 software will perform unit cell calculations and output the results.

[0045] 3. Diffraction point intensity integration Purpose: When the unit cell calculation results meet the target parameters, perform intensity integration on the diffraction points.

[0046] Steps: If you use XDS software to perform the previous steps, there is usually a corresponding module in the XDS software to automatically perform the diffraction point intensity integration operation when the conditions are met. The integrated intensity data can be viewed in the output results of the software.

[0047] If you use the Pets2 software, you may need to export the calculation results of Pets2 and use other tools (possibly self-written scripts combined with relevant mathematical libraries) to perform intensity integration. For example, according to the coordinates and intensity values ​​of the diffraction points, use numerical integration methods (such as the trapezoidal method, Simpson's method, etc.) to integrate the intensity.

[0048] 4. Generate the files needed to analyze the crystal structure Purpose: To use XPREP software to process and generate the ins file required for crystal structure analysis.

[0049] Steps: Prepare the intensity-integrated diffraction data and related experimental information (such as crystal symmetry information, elemental composition, and other possible known information).

[0050] Run the XPREP software and input relevant information according to the software's operating procedures.

[0051] The XPREP software will generate the ins file required for analyzing the crystal structure based on the input information and internal algorithms. This file contains some initial settings and constraints for crystal structure analysis.

[0052] 5. Crystal structure analysis and refinement Objective: To solve and refine the crystal structure using ShelxT or ShelxD method in Olex2 software package.

[0053] Steps: Import the generated ins file and the original diffraction data file into the Olex2 software package.

[0054] Select ShelxT or ShelxD method in Olex2 software package for crystal structure analysis. The software will perform iterative calculations based on the input information and the selected method, continuously optimizing the crystal structure model to better match the experimental data.

[0055] After multiple iterations, the final crystal structure analysis results are obtained, including detailed information such as atomic coordinates, occupancy, thermal parameters, etc.

[0056] Those skilled in the art should understand that the technical features of the above embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0057] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for analyzing the crystal form of active ingredients of pesticides based on three-dimensional electron diffraction, characterized in that: The following steps are involved: S00, Sample preparation: Spray the solution to be tested on a flat and clean substrate, and after the solution to be tested evaporates and dries, a thin layer of microcrystalline crystals will form on the surface of the substrate; Use tweezers to place the front of the grid on the substrate and press and move it so that the insecticide microcrystals in the test solution adhere to the grid. The concentration of the crystals can be controlled by adjusting the pressure and movement during sample preparation. S10, sample cooling and injection: The grid with the insecticide was quickly transferred to a cryostat, the sample was cooled with liquid nitrogen, and then the cryostat was transported into the TEM and evacuated; S20, sample observation and data collection: Observe the sample morphology, distribution and crystal quality at low magnification, select appropriate microcrystalline samples for marking, and make fine adjustments in height and level. Then collect three-dimensional electron diffraction data, tilt ±60° along the inclination direction of the frozen sample rod, and collect three-dimensional electron diffraction spots with high resolution and high diffraction intensity. S30, data processing: Select diffraction data of appropriate intensity to integrate the diffraction intensity, determine the sample unit cell parameters, further perform absorption correction, and analyze the crystal structure.

2. A method for analyzing the crystal form of an active ingredient of an insecticide based on three-dimensional electron diffraction according to claim 1, characterized in that: In step S00, the substrate includes one of glass, silicon wafer, mica and blade.

3. The method for analyzing the crystal form of active ingredients of pesticides based on three-dimensional electron diffraction according to claim 1, characterized in that: In step S30, the specific data processing steps include: Converting the raw image format from a format suitable for electron diffraction data to a format suitable for subsequent software processing; Use a software tool that can select diffraction points for unit cell calculation to perform index unit cell calculation. When the calculation results meet the preset target parameters, further perform intensity integration operation on the diffraction points. Using a software that can be used to process and generate files required for solving crystal structures to generate corresponding files; The crystal structure was solved and refined using a software package that includes methods capable of structure solution and refinement.

4. A method for analyzing the crystal form of an active ingredient of an insecticide based on three-dimensional electron diffraction according to claim 3, characterized in that: In step S30, the specific data processing steps include: Convert the original image format from mrc format to SMV format; Use XDS or Pets2 software to select diffraction points for index cell calculation. If the target parameters are met, further perform diffraction point intensity integration. The ins files required for crystal structure analysis were processed using XPREP software, and the structures were analyzed and refined using ShelxT or ShelxD methods in the Olex2 software package.

5. A method for analyzing the crystal form of an active ingredient of an insecticide based on three-dimensional electron diffraction according to claim 4, characterized in that: Convert the original image format from mrc format to SMV format through Python script.

6. A method for analyzing the crystal form of an active ingredient of an insecticide based on three-dimensional electron diffraction according to any one of claims 1 to 5, characterized in that: In step S10, after the copper mesh with the insecticide is quickly transferred to the cryogenic freezing rod, liquid nitrogen is first added to immerse the tip of the cryogenic freezing rod and then naturally cool it. After cooling to the set temperature, the cryogenic freezing rod is partially inserted into the transmission electron microscope and vacuumed. After the vacuuming is completed, the cryogenic freezing rod is completely sent into the transmission electron microscope.

7. A method for analyzing the crystal form of an active ingredient of an insecticide based on three-dimensional electron diffraction according to claim 6, characterized in that: The set temperature is 98K.

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