Method for rapid localization and semi-quantification of novel organic pollutants in zebrafish in vivo by mass spectrometry imaging

Abstract

The application relates to a method for rapidly locating and semi-quantitatively analyzing new organic pollutants in zebrafish in vivo by mass spectrometry imaging. The method comprises the following steps: obtaining a whole zebrafish sample after exposure and water flushing; preparing a whole fish frozen section after embedding and freezing by sodium carboxymethyl cellulose (CMC); analyzing the tissue section by using desorption electrospray ionization mass spectrometry imaging (DESI-MSI); simultaneously adding standard samples with a series of concentrations on a blank glass slide, establishing a standard curve based on the average ion intensity; extracting the average ion intensity of a region of interest (ROI) in the tissue section, substituting the average ion intensity into the standard curve to calculate the concentration of the detected substance in the region, and completing semi-quantitative analysis. The application can rapidly and directly locate the distribution of new organic pollutants in different tissues of zebrafish, realize semi-quantitative evaluation of the local concentration of the tissues, and is suitable for ecological toxicology research and risk prediction of new organic pollutants.

Description

Technical Field

[0001] This invention relates to the field of novel organic pollutant detection and risk analysis technology, specifically to a method for rapid localization and semi-quantitative analysis of novel organic pollutants in zebrafish using mass spectrometry imaging. Background Technology

[0002] Persistent organic pollutants (POPs) have long been a focus of attention due to their high toxicity, persistence, bioaccumulation, and long-distance migration capabilities, posing significant threats to the environment and human health. Since 2001, over 100 countries and regions, including China, have signed and acceded to the Stockholm Convention on Persistent Organic Pollutants. With industrial development and the widespread use of chemicals, a large number of novel synthetic chemicals are being released into the environment, exhibiting higher concentrations and greater ecological and health hazards compared to traditional POPs. Sixteen novel organic pollutants have been included in the Stockholm Convention's controlled list, and my country's Ministry of Ecology and Environment published a "List of Key Controlled New Pollutants" in 2022, with perfluorinated compounds (PFOCs) among the first batch of controlled pollutants. However, the bioaccumulation and toxicity of novel organic pollutants are not yet fully understood. Therefore, developing technologies for rapidly locating their distribution in organisms is crucial for in-depth research into their toxicological mechanisms and for assessing the health hazards of exposure. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a method for rapid localization and semi-quantitative analysis of novel organic pollutants in zebrafish using mass spectrometry imaging. This method is rapid, intuitive, and semi-quantitative, and can be used to locate the tissue distribution of novel organic pollutants in zebrafish and estimate local concentrations.

[0004] This invention is achieved through the following technical solution: A method for rapidly locating and semi-quantitatively analyzing the distribution of novel organic pollutants in zebrafish using mass spectrometry imaging technology includes the following steps: (1) Obtain whole zebrafish samples after exposure to novel organic pollutants and rinse the fish with running water; (2) The whole zebrafish was embedded in sodium carboxymethyl cellulose solution, frozen, and then frozen sectioned to obtain tissue sections; (3) The tissue sections were analyzed using desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to obtain mass spectrometry imaging data; (4) Add a series of standard solutions of the analyte at different concentrations to a blank slide and perform imaging acquisition under the same DESI-MSI conditions as in step (3). Establish a standard curve based on the average ion intensity of the imaging area of ​​each concentration standard and the corresponding concentration. (5) In the mass spectrometry imaging image obtained in step (3), select the region of interest (ROI), extract the average ion intensity of the region, substitute it into the standard curve established in step (4), calculate the concentration of the analyte in the region, and complete the semi-quantitative analysis.

[0005] Further specifying, the water rinsing time in step (1) is 10-12 seconds.

[0006] Further specifying, the sodium carboxymethyl cellulose solution in step (2) has a mass fraction of 5%, and is frozen at -80°C after encapsulation.

[0007] Further specified, the thickness of the frozen section in step (2) is 25 to 50 μm.

[0008] To further specify, in step (2), an optical microscope is used to select tissue sections with intact tissue for mass spectrometry imaging analysis in step (3).

[0009] Further specified, the DESI-MSI in step (3) adopts positive ion mode, the spray solvent is methanol:water = 98:2 (volume ratio), and 0.01% formic acid is added, with a flow rate of 3 μL / min.

[0010] Further, the parameters of the DESI source in step (3) are set as follows: spray voltage 1 kV, angle between spray needle and sample surface 75°, distance from sprayer to inlet 4 mm, distance from sprayer to sample surface 1.5 mm, and nitrogen pressure 0.05 MPa.

[0011] Further specifying, the standard curve described in step (4) uses the average ionic strength as the abscissa and the standard concentration per unit area (ng / cm²) as the ordinate. 2 ) is used as the vertical axis.

[0012] Further specifying, the region of interest (ROI) in step (5) is selected as a region on the mass spectrometry imaging image where the ion intensity and color are uniform.

[0013] The beneficial effects of this invention are as follows: This method for rapid localization and semi-quantitative analysis of novel organic pollutants in zebrafish using mass spectrometry imaging involves the following steps: rinsing whole zebrafish after exposure; CMC embedding and frozen sectioning; DESI-MSI mass spectrometry imaging analysis; establishing an average ion intensity-concentration standard curve using blank slide standards; and substituting the average ion intensity of the ROI into the standard curve to achieve semi-quantitative analysis. It requires no complex pretreatment and can simultaneously obtain spatial distribution information and semi-quantitative concentration data of pollutants within the organism. The method is rapid, reproducible, and suitable for toxicological screening and risk pre-assessment of novel organic pollutants.

[0014] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0015] Figure 1 Optical microscope image of a frozen section of zebrafish stained with hematoxylin and eosin (H&E); Figure 2 Mass spectrometry images of zebrafish slices exposed to different concentrations of 6PPDQ; Figure 3 Mass spectrometry images of a series of concentrations of 6PPDQ standards on a blank glass slide; Figure 4 Based on Figure 3 Standard curves were established by comparing the average ion intensity of the imaging region of each concentration standard with the corresponding concentration. Figure 5 The results of 6PPDQ semi-quantitative analysis of different regions of interest (ROI) in zebrafish slices are shown in the figure. Figure 6 This is a flowchart illustrating the operation of the present invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. It should be understood that instrument parameters can be appropriately adjusted for different target pollutants, including but not limited to acquisition mode, resolution, acquisition rate, and ion pair parameters.

[0017] This invention provides a technical solution: a method for rapidly locating and semi-quantitatively analyzing the distribution of novel organic pollutants in zebrafish using mass spectrometry imaging technology, comprising the following steps: (1) Obtain whole zebrafish samples after exposure to novel organic pollutants and rinse the fish with running water; (2) The whole zebrafish was embedded in sodium carboxymethyl cellulose solution, frozen, and then frozen sectioned to obtain tissue sections; (3) The tissue sections were analyzed using desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to obtain mass spectrometry imaging data; (4) Add a series of standard solutions of the analyte at different concentrations to a blank slide and perform imaging acquisition under the same DESI-MSI conditions as in step (3). Establish a standard curve based on the average ion intensity of the imaging area of ​​each concentration standard and the corresponding concentration. (5) In the mass spectrometry imaging image obtained in step (3), select the region of interest (ROI), extract the average ion intensity of the region, substitute it into the standard curve established in step (4), calculate the concentration of the analyte in the region, and complete the semi-quantitative analysis.

[0018] Specifically, the water rinsing time in step (1) is 10-12 seconds.

[0019] Specifically, the sodium carboxymethyl cellulose solution in step (2) has a mass fraction of 5%, and is frozen at -80°C after encapsulation.

[0020] Specifically, the thickness of the frozen section in step (2) is 25–50 μm.

[0021] Specifically, in step (2), an optical microscope is used to select intact tissue sections for mass spectrometry imaging analysis in step (3).

[0022] Specifically, in step (3), the DESI-MSI adopts positive ion mode, the spray solvent is methanol:water = 98:2 (volume ratio), and 0.01% formic acid is added, with a flow rate of 3 μL / min.

[0023] Specifically, in step (3), the parameters of the DESI source are set as follows: spray voltage 1 kV, angle between spray needle and sample surface 75°, distance from sprayer to inlet 4 mm, distance from sprayer to sample surface 1.5 mm, and nitrogen pressure 0.05 MPa.

[0024] Specifically, the standard curve described in step (4) uses the average ionic strength as the abscissa and the concentration of the standard per unit area (ng / cm²) as the ordinate. 2 ) is used as the vertical axis.

[0025] Specifically, in step (5), the region of interest (ROI) is selected from the area on the mass spectrometry imaging image where the ion intensity and color are uniform.

[0026] The specific implementation steps using the above method include: S1: Obtain zebrafish samples after exposure After domestication, zebrafish were randomly placed in glass jars containing 6 PPDQ experimental solution (30 zebrafish / jar, 25 L solution). Half the initial concentration solution was replaced daily to maintain a stable concentration. After 30 days of exposure, the whole zebrafish were rinsed under running water without 6 PPDQ for 10 seconds to reduce the impact of contaminants adhering to the fish surface.

[0027] S2: Preparation of frozen zebrafish slices Whole zebrafish were embedded in a 5% sodium carboxymethyl cellulose (CMC) solution and frozen at -80°C. Whole fish sections were prepared using a Leica CM3600 cryostat, with section thicknesses ranging from 25 to 50 μm. Sections were transferred to glass slides, observed under an optical microscope, and sections with intact tissue were selected for subsequent mass spectrometry imaging analysis. Figure 1 H&E stained optical microscope images of frozen zebrafish sections, used to identify tissue morphology.

[0028] S3: Mass spectrometry imaging analysis (DESI-MSI) Analysis was performed using a desorption / electrospray ionization mass spectrometry (DESI-MSI, Xevo TQ-XS, Waters) system. DESI source parameters were set as follows: positive ion mode; spray solvent: methanol:water = 98:2 (containing 0.01% formic acid); flow rate: 3 μL / min; spray voltage: 1 kV; spray needle angle to sample surface: 75°; distance from sprayer to inlet: 4 mm; distance from sprayer to sample surface: 1.5 mm; nitrogen pressure: 0.05 MPa. The mass spectrometry scan range was set according to the target compound. Data acquisition and image reconstruction were performed using HDI software.

[0029] Figure 2 Mass spectrometry images of zebrafish slices exposed to different concentrations of 6PPDQ. In the images, A and B represent the high-concentration (200 μg / L) treatment groups (A: male, B: female); C and D represent the low-concentration (2 μg / L) treatment groups (C: male, D: female). The color intensity in the images reflects the relative content of 6PPDQ.

[0030] S4: Establishing the Standard Curve A series of 6PPDQ standard solutions with concentrations (0.08, 0.16, 0.41, 0.82, 1.64, 4.10, 8.20 ng / cm²) were added to blank glass slides. Imaging was performed under the same DESI-MSI conditions as in S3. Figure 3 The images show mass spectrometry images of the above-mentioned series of 6PPDQ standards on blank slides. In HDI software, the ROI tool was used to sequentially select the imaging regions for each concentration of standard, and the average ion intensity of each region was recorded. The average ion intensity was plotted on the x-axis, and the concentration per unit area of ​​the standard (ng / cm²) was plotted on the y-axis. 2 Using y as the ordinate, establish a standard curve, such as... Figure 4 As shown.

[0031] S5: Semi-quantitative analysis of samples In the zebrafish tissue mass spectrometry image obtained in S3 (e.g.) Figure 2As shown in the image, use the ROI tool to select a region of interest with uniform color (i.e., uniform ion intensity), such as abdominal or muscle tissue. Extract the average ion intensity of this region and substitute it into the standard curve established in S4. Figure 4 The concentration of 6PPDQ in the region was calculated, and a semi-quantitative analysis was completed. Figure 5 The results of 6PPDQ semi-quantitative analysis of different regions of interest (ROIs) in zebrafish slices are presented, where red dots represent concentration values ​​in the abdominal region and green dots represent concentration values ​​in the muscle region.

[0032] Results Explanation Through the above steps, the distribution of 6PPDQ in different tissues of zebrafish can be visually located. Figure 2 ), and based on the standard curve ( Figure 4 ) for specific organizational regions ( Figure 5 This method allows for concentration calculations, enabling semi-quantitative analysis at the tissue level. It is rapid, intuitive, and suitable for ecotoxicological studies and risk pre-assessment of emerging organic pollutants.

[0033] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for rapidly locating and semi-quantitatively analyzing the distribution of novel organic pollutants in zebrafish using mass spectrometry imaging technology, characterized in that, Includes the following steps: (1) Obtain whole zebrafish samples after exposure to novel organic pollutants and rinse the fish with running water; (2) The whole zebrafish was embedded in sodium carboxymethyl cellulose solution, frozen, and then frozen sectioned to obtain tissue sections; (3) The tissue sections were analyzed using desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to obtain mass spectrometry imaging data; (4) Add a series of standard solutions of the analyte at different concentrations to a blank slide and perform imaging acquisition under the same DESI-MSI conditions as in step (3). Establish a standard curve based on the average ion intensity of the imaging area of ​​each concentration standard and the corresponding concentration. (5) In the mass spectrometry imaging image obtained in step (3), select the region of interest (ROI), extract the average ion intensity of the region, substitute it into the standard curve established in step (4), calculate the concentration of the analyte in the region, and complete the semi-quantitative analysis.

2. The method for rapidly locating and semi-quantitatively analyzing the distribution of novel organic pollutants in zebrafish using mass spectrometry imaging technology according to claim 1, characterized in that, The rinsing time in step (1) is 10-12 seconds.

3. The method for rapidly locating and semi-quantitatively analyzing the distribution of novel organic pollutants in zebrafish using mass spectrometry imaging technology according to claim 1, characterized in that... The sodium carboxymethyl cellulose solution in step (2) has a mass fraction of 5%, and is frozen at -80°C after encapsulation.

4. The method for rapidly locating and semi-quantitatively analyzing the distribution of novel organic pollutants in zebrafish using mass spectrometry imaging technology according to claim 1, characterized in that, The thickness of the frozen section in step (2) is 25-50 μm.

5. The method for rapidly locating and semi-quantitatively analyzing the distribution of novel organic pollutants in zebrafish using mass spectrometry imaging technology according to claim 1, characterized in that, Use an optical microscope to select intact tissue sections for mass spectrometry imaging analysis in step (3).

6. The method for rapidly locating and semi-quantitatively analyzing the distribution of novel organic pollutants in zebrafish using mass spectrometry imaging technology according to claim 1, characterized in that, In step (3), the DESI-MSI uses positive ion mode, the spray solvent is methanol:water = 98:2 (volume ratio), and 0.01% formic acid is added, with a flow rate of 3 μL / min.

7. The method for rapidly locating and semi-quantitatively analyzing the distribution of novel organic pollutants in zebrafish using mass spectrometry imaging technology according to claim 1, characterized in that, In step (3), the parameters of the DESI source are set as follows: spray voltage 1 kV, angle between spray needle and sample surface 75°, distance from sprayer to inlet 4 mm, distance from sprayer to sample surface 1.5 mm, and nitrogen pressure 0.05 MPa.

8. The method for rapidly locating and semi-quantitatively analyzing the distribution of novel organic pollutants in zebrafish using mass spectrometry imaging technology according to claim 1, characterized in that, The standard curve described in step (4) uses the average ionic strength as the abscissa and the concentration of the standard per unit area (ng / cm²) as the ordinate. 2 ) is used as the vertical axis.

9. The method for rapidly locating and semi-quantitatively analyzing the distribution of novel organic pollutants in zebrafish using mass spectrometry imaging technology according to claim 1, characterized in that, The region of interest (ROI) mentioned in step (5) is a region on the mass spectrometry imaging image where the ion intensity and color are uniform.

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