A mass spectrometry imaging method for detecting metrafenone on the surface of wheat leaves

Abstract

The present invention relates to a mass spectrometry imaging detection method for metrafenone on the surface of wheat leaves, which comprises the following steps: S1, providing a wheat leaf with metrafenone attached to the surface; S2, applying a matrix to the surface of the wheat leaf by a sublimation method; S3, setting the spraying thickness to 0.7 μm-1.5 μm, the laser spot diameter and the laser intensity to (40 μm-75 μm, 56.4%-71.0%) and the imaging step length to (30 μm-50 μm, 30 μm-50 μm) for MALDI-MSI analysis. According to the mass spectrometry imaging detection method for metrafenone on the surface of wheat leaves of the present invention, the interference of the matrix to the target object can be effectively avoided by limiting the coating method and the instrument parameters, and the imaging heat map is made clear and intuitive by optimizing the instrument parameters.

Description

Technical Field

[0001] The present invention relates to the field of molecular imaging technology, and more particularly to a mass spectrometry imaging detection method for metrafenone on the surface of wheat leaves. Background Art

[0002] Mefenoxam (MF) is a benzophenone fungicide primarily used to control powdery mildew and eye spot diseases in cereal crops, tomatoes, cucumbers, and grapes. Due to its excellent preventive and therapeutic effects, it is currently widely used on a variety of crops. This pesticide is typically applied by diluting it with water and spraying it on the surface of crop leaves. However, there is currently no analytical instrumentation that can directly detect the substance type and concentration on the actual leaf surface.

[0003] Mass spectrometry imaging (MSI) is a new molecular imaging technique developed from mass spectrometry. It simultaneously obtains qualitative, quantitative, and localization information on biomolecules by directly scanning biological tissue sections. It has the advantages of being label-free, high-throughput, and high-resolution, and has broad application prospects in various research fields such as drug analysis, cancer research, and environmental toxicology. However, for pesticide molecules (especially mefenacet), there is a lack of practical examples of using mass spectrometry imaging to accurately obtain their spatial distribution on actual leaf surfaces and directly detect their concentrations. Furthermore, when using MSI to analyze pesticide molecules, a matrix must be added, and the matrices commonly used in existing technologies tend to significantly interfere with the target. Summary of the Invention

[0004] In order to solve the problem in the prior art that pesticides are difficult to analyze directly on the surface of plant leaves, the present invention provides a mass spectrometry imaging method for detecting metrafenone on the surface of wheat leaves.

[0005] According to the present invention, the mass spectrometry imaging detection method for metrafenone on the surface of wheat leaves includes the following steps: S1, providing a wheat leaf with metrafenone attached to the surface; S2, coating a matrix onto the surface of the wheat leaf by a sublimation method; S3, setting the spraying thickness range to 0.7 μm-1.5 μm, the laser spot diameter and laser intensity range to (40 μm-75 μm, 56.4%-71.0%), and the imaging step size to (30 μm-50 μm, 30 μm-50 μm) for MALDI-MSI analysis.

[0006] Preferably, in step S1, the amount of metrafenone deposited on the surface of the wheat leaf is ≥2.0 ng. In a preferred embodiment, the concentration of metrafenone deposited on the surface of the wheat leaf is 100 μM, and the amount of metrafenone deposited is 40.0 ng.

[0007] Preferably, in step S2, the substrate is CHCA or DHB.

[0008] Preferably, step S2 specifically includes: loading matrix powder into the slit cavity of the sublimator for coating.

[0009] Preferably, the sublimator is set to coat the substrate at a thickness of 1.2 μm.

[0010] Preferably, the mass spectrometry imaging detection method further comprises observing the longitudinal cross-sectional distribution of metrafenone in wheat leaves in combination with a frozen section technique.

[0011] Preferably, the mass spectrometry imaging detection method further comprises preparing metrafenone solutions with different concentration gradients, and using a mass spectrometry imaging instrument to establish an external standard method standard curve to perform quantitative analysis of metrafenone.

[0012] Preferably, the concentration gradient of the metrafenone solution for determining the concentration detection limit is 1 μM, 5 μM, 10 μM, 15 μM, and 20 μM.

[0013] Preferably, the concentrations of metrafenone in the external standard method standard curve are 20 μM, 50 μM, 80 μM and 100 μM, respectively.

[0014] According to the mass spectrometry imaging detection method for metrafenone on the surface of wheat leaves of the present invention, by screening the type of matrix, further optimizing the coating method and instrument parameters, the interference of the matrix on the target object can be effectively avoided, and the imaging heat map is made clear and intuitive by optimizing the instrument parameters. This method is an in-situ analysis method. Compared with traditional technical methods (such as liquid chromatography, high-resolution mass spectrometry, etc.), to perform qualitative and quantitative analysis of pesticides on the surface of leaves, the pesticides must be eluted. The elution method also needs to be explored, verified and optimized, and the existing pre-treatment steps will reduce the accuracy of the detection (such as quantitative analysis). The present invention proposes for the first time to conduct qualitative and quantitative research on metrafenone on the surface of leaves by mass spectrometry imaging, especially in terms of quantitative analysis. The external standard method is used for the quantitative research on the concentration of metrafenone on the surface of leaves in the mass spectrometry imaging instrument, which has the advantages of being fast and simple, requiring no pre-treatment, having a low detection limit, high sensitivity, and intuitive and clear material spatial distribution. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1a is a mass spectrum signal diagram of MF according to an embodiment of the present invention, wherein C MF =500 μM, the horizontal axis is the mass-to-charge ratio (m / z), the vertical axis is the average intensity of the mass spectrum peak, the m / z ([M+H] + ) are 409.06 and 411.06;

[0016] Figure 1b is a spatial distribution diagram of MF on the holes of the metal target plate according to an embodiment of the present invention;

[0017] Figure 2a3 is a mass spectrometric signal diagram of MF in a positive ion mode and with a CHCA matrix according to an embodiment of the present invention;

[0018] Figure 2b 3 is a mass spectrometry signal diagram of MF in positive ion mode and with DHB as the matrix according to an embodiment of the present invention;

[0019] Figure 2c 3 is a mass spectrometry signal diagram of MF in negative ion mode and with a matrix of 9AA according to an embodiment of the present invention;

[0020] Figure 3a is a mass spectrometry signal using a DHB matrix under an airbrush method according to an embodiment of the present invention;

[0021] Figure 3b The spatial distribution of the DHB matrix using the airbrush method according to an embodiment of the present invention;

[0022] Figure 3c is a mass spectrometry signal using a DHB matrix under a matrix sublimation method according to an embodiment of the present invention;

[0023] Figure 3d The spatial distribution of the DHB matrix under the matrix sublimation method according to an embodiment of the present invention;

[0024] Figure 4 is a spatial distribution diagram of MF under different spraying thicknesses by the matrix sublimation method according to an embodiment of the present invention;

[0025] Figure 5a is the ion mass spectrum peak of MF under different laser spot diameters and laser intensities according to an embodiment of the present invention;

[0026] Figure 5b is a spatial distribution diagram of MF under different laser spot diameters and laser intensities according to an embodiment of the present invention;

[0027] Figure 6a is the ion mass spectrum peak of MF under different imaging step sizes according to an embodiment of the present invention;

[0028] Figure 6b is a spatial distribution diagram of MF under different imaging step sizes according to an embodiment of the present invention;

[0029] Figure 7a is C according to an embodiment of the present invention MF = 1 μM (deposition amount 0.4 ng) in positive ion mode and MF mass spectrometry signal diagram with CHCA as matrix;

[0030] Figure 7b is C according to an embodiment of the present invention MF= 5 μM (deposition amount 2.0 ng) in positive ion mode and MF mass spectrometry signal diagram using CHCA as matrix;

[0031] Figure 8 7 is an external standard method standard curve diagram according to an embodiment of the present invention;

[0032] Figure 9 is the distribution characteristics of MF on the surface of wheat leaves according to an embodiment of the present invention;

[0033] Figure 10 MF is the longitudinal cross-sectional distribution of wheat leaves according to an embodiment of the present invention. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present invention are given below in conjunction with the accompanying drawings and described in detail.

[0035] The mass spectrometer used in the present invention was purchased from Shimadzu Corporation (iMScope TRIO TM ).

[0036] Example 1

[0037] This embodiment can be used for in-situ qualitative and quantitative analysis of metrafenone on plant leaf surfaces. This embodiment is a specific implementation method for in-situ qualitative and quantitative analysis of metrafenone on actual leaf surfaces.

[0038] (1) Wheat cultivation

[0039] The wheat used in the experiment was obtained by soil culture in an artificial climate incubator. The specific procedure was as follows: the wheat seeds were cleaned, soaked, and then spread onto seedling paper for germination in the dark. When the sprouts were approximately 1 cm long, they were cultured in an artificial climate incubator with a set light-dark cycle. The light cycle was 14 hours, 20°C temperature, 75% humidity, and 25,000 lux; the dark cycle was 10 hours, 15°C temperature, 65% humidity, and 0.05 lux. Wheat was used in the experiment when it reached the 5-leaf stage.

[0040] (2) Preliminary exploration of the method

[0041] A certain amount of MF was weighed and dissolved in acetonitrile (ACN) to prepare a 500 μM MF standard solution. CHCA was dissolved in ACN / H2O (1:1, V:V) to prepare a 10 mg·mL -1 Solution (containing 0.1% trifluoroacetic acid). Mix the MF standard solution with the CHCA matrix solution at a 1:1 (V:V) ratio. Pipette 0.5 μL of the MF / CHCA mixture onto a well on a metal target plate, dry it, and perform MALDI-MSI analysis in positive ion mode. Figure 1a The mass spectrometry signals of MF are given: the two bromine isotope structures of MF in positive ion mode ([M+H] +=409.06 / 411.06), and Figure 1a The signal was clearly detected in Figure 1b The spatial distribution of MF on the metal target plate is shown: the optical image at the target point is shown on the left, the MF signal and intensity distribution is shown in the middle, and the composite image of the optical image and MF signal is shown on the right. This clearly demonstrates the feasibility of this method for in-situ qualitative analysis of MF. This preliminary spot coating experiment detected the distribution of MF on the metal target plate, demonstrating the feasibility of MALDI-MSI for the analysis and detection of pesticide molecules.

[0042] (3) Matrix selection and optimization

[0043] Wheat leaves were fixed to a conductive glass slide coated with indium tin oxide using double-sided conductive adhesive. MF standard solution was diluted to a concentration of 100 μM and then added dropwise to the surface of the experimental leaves (3 μL) and allowed to dry naturally. α-Cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 9-aminoacridine (9AA) were dissolved in ACN / H2O (1:1, V:V) to form 10 mg·mL -1 The three matrix solutions were added to the leaf surface samples using the dry drop method, with a drop volume of 3 μL. The samples were dried again before testing. Figure 2a The mass spectrometric signals of MF in positive ion mode with CHCA as matrix are given. Figure 2b The mass spectrometric signal of MF in positive ion mode with DHB as matrix is ​​given. Figure 2c The mass spectrometry signals of MF in negative ion mode using the 9AA matrix are shown. This experiment revealed that CHCA and DHB matrices have significant advantages in positive ion mode. MF's mass spectrometry peaks are very strong, while DHB's matrix peaks have minimal interference. Although CHCA matrix peaks are numerous and strong, they do not interfere with MF's mass spectrometry detection.

[0044] (4) Selection and optimization of substrate coating method

[0045] The preparation of the MF leaf sample was the same as (3), and DHB was selected as the matrix. The coating was carried out by airbrush method and matrix sublimation method. In the airbrush method, DHB was dissolved in ACN / H2O (1:1, V:V) to form 10 mg·mL -1 4 ml of the solution was taken and loaded into the gun cup of a commercial spray gun (Platinum 0.2, Chiyoda, Japan). The muzzle air flow valve and the tail liquid valve were adjusted to the lowest spray speed. Each layer was sprayed for 10 seconds and rested for 10 minutes. A total of 10 layers were applied. The sample was tested after it was completely dried. The matrix sublimation method was used to directly load an appropriate amount of DHB powder into the slit cavity. The sublimator (iMLayer TM , Shimadzu, Japan) program set the matrix thickness to 1.2 μm. Figure 3aThe mass spectrometry signal under the spray gun method is given. Figure 3b The spatial distribution under the airbrush method is given. Figure 3c The mass spectrometry signal under matrix sublimation method is given. Figure 3d The spatial distribution under the matrix sublimation method is given, where Figure 3b and Figure 3d The left image shows the MF signal and intensity distribution, while the right image is a composite of the optical image of the blade surface and the MF signal. Comparing the two matrix coating methods, the MF mass spectrometry signal response is excellent, but the airbrush method exhibits excessive matrix artifacts. The MF spatial distribution diagram shows that the original spatial distribution of the substance is disrupted, primarily due to airflow and droplet erosion. The matrix sublimation method is simple to use, requiring no matrix preparation, time-consuming spraying and drying, and controllable matrix thickness, making experiments easily reproducible and producing fewer matrix artifacts. Because the matrix sublimation process is gentle, the original MF spatial distribution is maintained.

[0046] (5) Optimization of spray thickness by matrix sublimation method

[0047] The preparation of the MF leaf sample was the same as in (3), and the matrix selection and coating method were the same as in (4) matrix sublimation method. The sublimator program set the matrix thickness to 0.7μm, 1.0μm, 1.2μm and 1.5μm respectively. Figure 4 The spatial distribution of MF at different substrate thicknesses is shown. The MF images show that the four spray thicknesses are all clear, so the optimal spray thickness range is 0.7μm-1.5μm, with 1.2μm being the optimal thickness for the matrix sublimation spray method.

[0048] (6) Imaging mass spectrometer parameter optimization

[0049] The leaf surface was dripped with MF at a concentration of 100 μM and a volume of 3 μL. The matrix was DHB, and the matrix thickness was set to 1.2 μm in the matrix sublimation method. First, the laser spot diameter of the DHB matrix and the optimal laser intensity that matched it were simultaneously optimized. Figure 5a The ion mass spectrum peaks of MF under different laser spot diameters and laser intensities are given. Figure 5b The spatial distribution of MF for different laser spot diameters and laser intensities is shown. The MF ion mass spectrum peaks show that due to the high laser intensity and degree of ionization, the MF peak intensities of the (150μm, 87.1%) and (180μm, 89.9%) groups are the highest. However, the peak intensities of the matrix and impurities are also high. In the same ion mass spectrum, the MF peaks are not obvious, while the MF peaks of the (40μm, 56.4%) and (75μm, 71.0%) combinations are more prominent in the mass spectrum. Therefore, a laser diameter range of 20-60μm and a laser intensity range of 56.4-71.0% are sufficient. The MF image of the (40μm, 56.4%) combination is the clearest and most complete.

[0050] Then, six step gradients were selected for experiments, aiming to select the optimal imaging step range. Figure 6a The ion mass spectrum peaks of MF under different imaging step sizes are given. Figure 6b The spatial distribution of MF under different imaging step sizes is given. It can be seen from the MF imaging diagram that the imaging step size of (30μm, 30μm) Figure 10 The analysis is clear, with (50μm, 50μm) being the next best. Since the optimal laser spot diameter is 40μm, selecting an imaging step size of (30μm, 30μm) allows for overlap between adjacent spots during laser scanning, ensuring comprehensive analysis. However, (50μm, 50μm) indicates a 10μm unanalyzed region between the laser spots, but the analysis time is 0.3h shorter than (30μm, 30μm). Therefore, the optimal step size range can be determined to be between (30μm, 30μm) and (50μm, 50μm).

[0051] (7) Determination of the minimum detection limit of the method and determination of the standard curve of the external standard method

[0052] After optimizing the matrix, coating method, and parameters, the method's minimum instrument detection limit (MLD) was determined to test its sensitivity. MF standard solutions were diluted to concentrations of 1 μM, 5 μM, 10 μM, 15 μM, and 20 μM, with a 1 μL droplet volume. The resulting MF deposits after natural drying were 0.4 ng, 2.0 ng, 4.0 ng, 6.0 ng, and 8.0 ng, respectively. CHCA was used as the matrix, and the matrix sublimator was set to a thickness of 1.2 μm. The MALDI-MSI laser spot diameter was 40 μm, the laser intensity was 56.4%, and the imaging step size was 30 μm × 30 μm. At an MF deposit of 0.4 ng, the presence and distribution of MF were virtually undetectable in the image. Even at a detection peak height of 0.10%, no peak corresponding to 0.4 ng of MF was visible in the mass spectrum. However, at a deposit of 2.0 ng, MF distribution and isotopic mass spectrum peaks were visible. Figure 7a C MF =1μM (deposition amount 0.4ng) in positive ion mode and MF mass spectrometry signal diagram in CHCA matrix, Figure 7b C MF =5μM (deposition amount 2.0ng) in positive ion mode and with CHCA as the matrix. It can be seen from the figure that when C MF = 1 μM, no MF mass spectrometry signal was detected, while when C MF=5μM, both bromine isotope structures of MF can be detected. Based on the results of this experiment, the minimum detection limit of MF is 2.0ng. Annangudi et al. established a MALDI-MSI detection method for the fungicide pyraclostrobin in wheat leaves with a minimum detection limit of 60.0ng. In comparison, the detection method of the present invention has a 30-fold increase in sensitivity.

[0053] The standard curve of the external standard method was determined to quantitatively analyze MF on the leaf surface. The MF mother solution was diluted to obtain a concentration gradient of 20μM, 50μM, 80μM, and 100μM standard products, which were added dropwise to the surface of wheat leaves in a volume of 3μL. The samples were dried naturally and then measured. The matrix was CHCA, the coating thickness was 1.2μm, and the MALDI-MSI parameters were the same as those for the determination of the minimum detection limit. Figure 8 As shown in the figure, the average peak value is positively correlated with the molar concentration of MF, and the correlation coefficient of the external standard curve of this method is 0.9946, which has a good correlation and can be used for the quantitative analysis of MF on the leaf surface.

[0054] (7) Distribution characteristics of MF on the surface and longitudinal section of wheat leaves

[0055] To understand the distribution characteristics of MF on the surface of wheat leaves, scanning electron microscopy (SEM) analysis was first performed on the wheat leaf surface. Then, 8 μL of MF (500 μM) standard solution was dripped onto the wheat leaf surface, and the sample was subjected to MALDI-MSI analysis. The matrix was CHCA, the coating thickness was 1.2 μm, and the MALDI-MSI parameters were set as (6). SEM analysis showed that the epidermis of the wheat leaf showed rows of orderly corrugated structures with wrinkles on the surface. The structures such as leaf hairs, stomata, and leaf slits were clearly visible.

[0056] Figure 9 The distribution characteristics of MF on the surface of wheat leaves are shown in the figure on the left, where the MF signal and intensity distribution are shown, and the figure on the right is a composite image of the optical image and MF signal on the leaf surface. It can be seen that the distribution of fungicides on the crop after application is closely related to the crop morphology. Based on the above experiments, combined with the freezing section technology, the longitudinal cross-section distribution of MF after infiltration of wheat leaves was directly observed. 8 μL of MF (500 μM) standard solution was added dropwise until the leaves were dry. After gelatin: H2O = 1:9 (m:m) was melted at 40°C, the leaf sample containing MF was frozen and embedded in a mold. The embedded sample was fixed in a sample plate at -20°C using tissue freezing medium and sliced ​​using a freezing microtome with a thickness set to 60 μm. The matrix CHCA had a coating thickness of 1.2 μm. The MALDI-MSI parameter settings were the same as (6). Figure 10The left figure shows the distribution of MF in the longitudinal section of wheat leaves. The right figure shows the composite image of the optical image of the leaf surface and the MF signal. The results show that this method combined with the cryosection technology can obtain the longitudinal section distribution of MF in wheat leaves after infiltration, which is intuitive and clear.

[0057] (8) In situ detection of commercial MF preparations in agricultural production

[0058] The concentration of a commercial MF preparation (BASF Inteng, suspension concentrate, 42%) was determined to be 0.8 M by HPLC external standard method. In this experiment, the commercial MF preparation was diluted to 100 μM and the amount added to the wheat leaves was 3 μL. The matrix DHB was used with a coating thickness of 1.2 μm, and the MALDI-MSI parameters were set the same as (6). The experimental results show that the in situ analysis method of MF on the leaf surface constructed based on MALDI-MSI is suitable for the detection of commercial MF preparations in actual use and has practical application significance.

[0059] Thus, the present invention compares the mass spectrometry signal intensity of mefenacet (MF) and the interference of matrix mass spectrometry peaks under coating with three different organic matrices to screen out the optimal matrix; selects different coating methods and determines the optimal coating method based on the mass spectra and spatial distribution diagram of MF; sets different spray thicknesses and determines the optimal spray thickness range based on the MF spatial distribution diagram; sets different laser intensities and imaging steps to optimize instrument parameters; sets five MF low-mass gradients to determine the minimum detection limit of the method; configures MF solutions with different concentration gradients and uses a mass spectrometer to establish a standard curve for quantitative analysis of MF; based on MALDI-MSI technology and combined with frozen section technology, directly observes the longitudinal cross-section distribution of MF in wheat leaves after infiltration; finally, uses this method to detect MF commercial preparations produced in actual agricultural production. The present invention provides a mass spectrometry imaging method for detecting MF on the surface of wheat leaves, which is fast and simple, does not require pretreatment, can intuitively and clearly observe the spatial distribution of MF and its photolysis products on the actual leaf surface, and can also accurately quantify MF, which can be well applied in practice.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various modifications are possible. In other words, any simple, equivalent changes and modifications made in accordance with the claims and description of the present invention are within the scope of protection of the patent claims. Anything not fully described in this invention constitutes conventional technology.

Claims

1. A mass spectrometry imaging method for detecting metrafenone on the surface of wheat leaves, characterized in that: The mass spectrometry imaging detection method comprises the following steps: S1, providing wheat leaves with metrafenone attached to the surface, wherein the deposition amount of metrafenone attached to the surface of the wheat leaves is ≥2.0 ng; S2, loading the matrix powder into the slit cavity of the sublimator, and coating the matrix onto the surface of the wheat leaf by sublimation, wherein the matrix is ​​CHCA or DHB, and the sublimator is set to a coating thickness of 1.2 µm; S3, set the spray thickness range to 0.7 µm-1.5 µm, the laser spot diameter to 40 µm-75 µm, the laser intensity range to 56.4%-71.0% and the imaging step size to (30 µm-50 µm, 30 µm-50 µm) for MALDI-MSI analysis.

2. The mass spectrometry imaging detection method according to claim 1, characterized in that: The mass spectrometry imaging detection method also includes combining frozen section technology to observe the longitudinal cross-sectional distribution of mefenamic acid in wheat leaves.

3. The mass spectrometry imaging detection method according to claim 1, characterized in that: The mass spectrometry imaging detection method also includes preparing metrafenone solutions with different concentration gradients, and using a mass spectrometry imaging instrument to establish an external standard method standard curve to perform quantitative analysis on metrafenone.

4. The mass spectrometry imaging detection method according to claim 1, characterized in that: The concentration gradient of the metrafenone solution for determining the concentration detection limit was 1 µM, 5 µM, 10 µM, 15 µM, and 20 µM.

5. The mass spectrometry imaging detection method according to claim 1, characterized in that: The concentrations of mefenamic acid in the standard curve of the external standard method were 20 µM, 50 µM, 80 µM, and 100 µM, respectively.

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