Method for detecting Br element in single cell based on mass spectrum flow type

Abstract

The invention discloses a method for detecting a Br element in a single cell based on a mass spectrum flow type. The detection method comprises the following steps: collecting a biological sample exposed by a brominated compound; preparing a single-cell suspension from a biological sample; labeling cells in the single-cell suspension with metal isotopes A and B respectively; the metal isotope A can be used for simultaneously marking survival and dead cells; the metal isotope B can mark dead cells; and performing single cell mass spectrometry flow detection on the single cell suspension, firstly separating single cells according to the signal of the metal isotope A, recording the single cells as a set S, then obtaining individuals with the signal of A but not with the signal of B from the set S, recording the individuals as a set P, and obtaining the single cell mass spectrometry flow detection result through a standard curve according to the detected signal intensity value of the Br element in the cells. According to the method, the Br content in a single cell can be detected, a single-cell inductively coupled plasma time-of-flight mass spectrometer is used for detection, the flux is high, and the analysis speed can reach 600 cells per second.

Description

Technical Field

[0001] The present invention relates to a method for detecting Br element in a single cell based on mass spectrometry. Background Art

[0002] Brominated flame retardants (BFRs) are a class of bromine-containing compounds, including aliphatic, alicyclic, aromatic, and aromatic-aliphatic compounds. They are currently the most produced and used flame retardants. At high temperatures, they release bromide ions, which react with free radicals to form flame-retardant compounds, effectively preventing the occurrence and spread of fires. Due to their significant flame-retardant properties, BFRs are widely used in industries such as plastics, electronics, and building materials.

[0003] In recent years, studies have shown that brominated compounds gradually accumulate in organisms and may have certain toxic effects on human health, affecting the nervous, endocrine, and immune systems. Therefore, the toxicity research of these compounds has become a hot topic.

[0004] Currently, bromine in biological samples is primarily detected using inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS is a highly sensitive, high-resolution analytical technique capable of measuring bromine isotopes of varying masses. It is widely used in elemental and isotopic analysis in the fields of biology, environment, and medicine. Its high sensitivity and precision enable accurate determination of bromine content in organisms, providing powerful technical support for toxicity studies of brominated compounds. However, ICP-MS requires converting the sample into a gaseous or plasma state for analysis. During this process, the physical structure of the cell is often disrupted, allowing the elements or compounds within it to be released and detected. Because this pretreatment disrupts the cell structure, ICP-MS is more suitable for measuring the total or average level of an element in a batch of cells or tissue samples and is generally unable to directly measure the content of an element or compound within a single cell. ICP-MS can only detect the total bromine content of a sample, which limits its ability to measure bromine content in individual cells.

[0005] Cytometry by time-of-flight (CyTOF) is a single-cell detection technology that has developed rapidly in recent years. Based on the principle of mass spectrometry detection, it uses the metal elements iridium (Ir) or rhodium (Rh) to label cell nuclei for the screening and identification of single cells. This method offers precise signals, multiple detection channels, and high stability. Because it does not require correction for spectral overlap, it can simultaneously detect information on up to 135 elements in a single cell, compared to traditional mass spectrometers. Compared to ICP-MS, CyTOF can measure the Br content in individual cells. Using CyTOF technology, the heterogeneity of different cell types can be studied at the single-cell level by measuring differences in Br content in different cell types, providing more accurate and comprehensive information for a better understanding of the toxic effects of brominated compounds on organisms.

[0006] Therefore, establishing a single-cell-based Br element detection method is of great significance for exploring the transport, transformation or toxic effects of brominated compounds at the cellular level of organisms. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for detecting the Br element in a single cell, which is specifically obtained through single-cell mass spectrometry flow detection, with high throughput and fast analysis speed.

[0008] The present invention provides a method for detecting Br in a single cell, comprising the following steps:

[0009] S1. Collect biological samples exposed to brominated compounds;

[0010] S2. preparing a single cell suspension using the biological sample;

[0011] S3, labeling cells in the single cell suspension with metal isotope A and metal isotope B respectively;

[0012] The metal isotope A can simultaneously label living and dead cells;

[0013] The metal isotope B can label the dead cells;

[0014] S4. The single-cell suspension is subjected to single-cell mass spectrometry flow cytometry detection. First, single cells are separated according to the signal of the metal isotope A, and recorded as set S (a person skilled in the art can determine the range based on the single-cell mass spectrometry flow cytometry detection results. Signals smaller than this range are signals of some cell fragments, and signals larger than this range may be signals of two or more cell adhesions). Then, individuals with the signal of the metal isotope A but not with the signal of the metal isotope B are obtained from the set S, and recorded as set P. The Br content in the single cell can be obtained by calculation based on the detected intracellular Br element signal intensity value using a standard curve.

[0015] In the detection method of the present invention, in step S1, the biological sample includes cells cultured in vitro and organs of model animals;

[0016] The cells cultured in vitro include mouse liver parenchymal cells (such as AML12) and mouse liver macrophages (RAW264.7);

[0017] The organs of the model animal include the liver, spleen and bone marrow of mice.

[0018] In the detection method of the present invention, in step S1, the brominated compound includes tetrabromobisphenol A (TBBPA) and octabromoether (TBBPA-BDBPE).

[0019] In the detection method of the present invention, in step S3, the metal isotope A is 193 Ir, 103 Rh or lanthanide metal isotopes;

[0020] The metal isotope B is 19 5Pt.

[0021] According to research requirements, cells in the single cell suspension may be labeled with antibodies.

[0022] In the detection method of the present invention, in step S4, the single-cell mass spectrometry flow detection is performed in a single-cell inductively coupled plasma time-of-flight mass spectrometer.

[0023] In the detection method of the present invention, in step S4, after the single-cell mass spectrometry flow cytometry detection, 50,000 to 100,000 events (the number of signals collected by Cytof) are collected for each group of samples. The number of events collected can be appropriately increased according to the increase in the number of cell subpopulations to be analyzed.

[0024] In the detection method of the present invention, in step S4, the data obtained are collated and analyzed on a mass spectrometry flow cytometry data processing website (www.cytobank.org), and software including but not limited to flowjo 10.0 can be used.

[0025] In the detection method of the present invention, in step S4, the conditions for the single-cell mass spectrometry flow cytometry detection are as follows:

[0026] Cell suspension injection flow rate: 30-31 μl / min;

[0027] Event collection rate: <500 events / s;

[0028] EQbeads concentration: 10%;

[0029] EQbeads monitors elements: 140 / 142 Ce (cerium), 151 / 153 Eu(Europium), 165 Ho (holmium), 175 / 176 Lu(lutetium);

[0030] Elements to be tested: 81 Br, 191 / 193 Ir, 195 Pt.

[0031] In the detection method of the present invention, in step S4, the Br element signal intensity value of the cell is obtained according to the single-cell mass spectrometry flow cytometry detection, and then the Br content in the sample to be tested is calculated.

[0032] Preferably, the determination of the Br content is achieved by a standard curve method;

[0033] Preferably, the standard curve can be obtained according to a method comprising the following steps:

[0034] (a) Using cells to absorb and enrich Br in Br ion solutions with different concentration gradients; the Br ion solutions with different concentration gradients are prepared by diluting a Br ion standard solution (a solution with a known Br ion concentration) with a concentration gradient;

[0035] (b) labeling the cells treated in step (a) with the metal isotope A (e.g., labeling nucleic acids);

[0036] (c) Performing single-cell mass spectrometry on the cells treated in step (b) to obtain a standard curve between the Br element signal intensity value and the Br element concentration of the cells.

[0037] In the process of obtaining the standard curve and detecting the sample to be tested, except for the difference in the sample used for interaction with cells, other conditions remain the same.

[0038] Among them, the signal of the metal isotope A (the metal isotope A has only cell signal but no signal in the solution) and the signal of the Br element to be detected (the Br element to be detected has both cell signal and may have signal in the solution) that can be detected simultaneously is a cell signal, otherwise it is a signal in the solution.

[0039] The present invention has the following beneficial effects:

[0040] (1) The detection method of the present invention can detect the Br content in a single cell.

[0041] (2) The present invention uses a single-cell inductively coupled plasma time-of-flight mass spectrometer for detection, which has a high throughput and an analysis speed of up to 600 cells per second.

[0042] (3) The present invention is beneficial to scholars in studying the health hazards of brominated compounds to organisms. By statistically analyzing the Br element intensity in different cell subpopulations at the cellular level, the heterogeneity between different cell populations can be studied, thereby assisting in exploring the reaction mechanism after brominated compounds enter the organism. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The mouse liver parenchymal cells AML12 and mouse liver macrophages RAW264.7 in Example 1 of the present invention were exposed to TBBPA. 81 Comparison of Br content, among which, Figure 1 a) Mouse liver parenchymal cells AML12 were exposed to 1, 5, 10, and 50 μM TBBPA. 81 Br content diagram; Figure 1 b shows the intracellular expression of TBBPA in mouse liver macrophages RAW264.7 after exposure to 1, 5, 10, and 50 μM TBBPA. 81 Br content diagram.

[0044] Figure 2 This is the gating strategy for the data on the Cytof machine after the mice were exposed to TBBPA-BDBPE by tail vein injection in Example 2 of the present invention.

[0045] Figure 3 The single cell in the liver of mice exposed to TBBPA-BDBPE by tail vein injection in Example 2 of the present invention 81 Br response intensity results. DETAILED DESCRIPTION

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0047] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0048] The present invention provides a method for detecting Br element in a single cell, comprising the following steps:

[0049] 1. Compound Exposure and Single Cell Preparation

[0050] In vitro cells:

[0051] 1) Cultivate cells to an appropriate density to meet the needs of subsequent steps.

[0052] 2) Add a certain concentration of bromine-containing compounds to the cell culture dish and expose for 24 hours.

[0053] Cells in the body:

[0054] 1) Expose C57BL / 6J male mice to a certain concentration of bromine-containing compounds (select oral gavage, intravenous injection, lung exposure, etc. according to experimental needs) for 6 hours, then dissect and remove the liver.

[0055] 2) Preparation of single-cell suspension from the liver.

[0056] 2. Single cell nucleic acid labeling and surface protein labeling

[0057] Use other metal isotope labels other than the element to be tested to label other biological molecules used (such as cisplatin ( 195 Pt) to label the nucleic acids of dead cells, and iridium ( 193 Ir) Cellular nucleic acids are labeled, and leukocytes in single cells obtained from organs are labeled using CD45 antibodies.

[0058] 3. Sample testing

[0059] The density of the labeled samples was adjusted to 1.5-1.8×10 6 The samples were detected by mass cytometry, and 50,000 events were collected for each sample for data analysis.

[0060] 4. Data Analysis

[0061] FlowJo 10.0 software was used to analyze the obtained data and calculate the Br content.

[0062] Furthermore, those skilled in the art can make simple changes or substitutions to the above implementation methods, for example

[0063] In step 1, the in vitro cells include but are not limited to mouse liver parenchymal cells, macrophages, etc., and the in vivo cells include but are not limited to mouse liver cells;

[0064] The mice described in step 1 include but are not limited to C57BL / 6J male mice;

[0065] The bromine-containing compound in step 1 includes but is not limited to TBBPA and TBBPA-BDBPE;

[0066] The exposure time and concentration of the exposed compound in step 1 can be appropriately adjusted according to the physicochemical properties of the exposed compound;

[0067] The organ in step 1 includes but is not limited to the liver, and the preparation of the single cell suspension includes but is not limited to using a dissociation kit corresponding to the organ;

[0068] In step 2, other metal isotope markers other than the element to be measured include but are not limited to lanthanide metal isotopes, etc. 193 Ir can also be used, but is not limited to rhodium ( 103 The replacement of Rh) can be adjusted according to the cell type to be analyzed, such as using CD19 to mark B cells, CD4, CD8a to mark T cells, etc.

[0069] The number of events collected in step 3 can be appropriately increased as the number of cell types to be analyzed increases;

[0070] The data processing software described in step 4 can be replaced by using, but not limited to, a mass spectrometry flow cytometry data processing website (www.cytobank.org);

[0071] In the following examples, C57BL / 6J male mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0072] CyTOF Reagents: Metal Isotope Labels 193 / 191 Ir, 195 Pt, cell staining buffer (CSB), fix and perm buffer and EQTM four-element calibration magnetic bead solution were purchased from Fluidigm (USA).

[0073] Example 1: Comparison of the differences in Br content in individual cells of mouse liver parenchymal cells AML12 and mouse liver macrophages RAW264.7 exposed to TBBPA (tetrabromobisphenol A)

[0074] The specific implementation process is as follows:

[0075] 1) Prepare liquid culture medium containing TBBPA at concentrations of 1, 5, 10, and 50 μM, respectively.

[0076] 2) Mouse liver parenchymal cells AML12 and mouse liver macrophages RAW264.7 were cultured in the above culture medium and exposed for 24 hours.

[0077] 3) Each sample was treated with cisplatin at a final concentration of 0.5 μM (if the number of cells is greater than 10 7 , the cell density needs to be adjusted to 1×10 7 Incubate at room temperature for 2 minutes (< 100 μg / mL). Terminate the labeling reaction with cell staining buffer (CSB). Wash and resuspend the cells in PBS.

[0078] 4) Add 1 ml of Ir or 500 nM Rh to the fix and perm buffer to a final concentration of 125 nM, or 500 nM, for each sample. Mix gently and immediately, and incubate at 4°C overnight.

[0079] 5) Centrifuge at room temperature (800g, 5min, 4°C) and discard the supernatant. Wash the cells twice with CSB (800g, 5min, 4°C) and discard the supernatant. Add 1ml of CAS and count. Resuspend the cells in CAS solution to a concentration of 1.5-1.8×10 based on the count results. 6 / mL suspension.

[0080] 6) Take 900 μl of the suspension and filter it through a flow cytometer with a 35 μm filter. Add 100 μl of EQ Beads and collect data.

[0081] The detection parameters of the mass spectrometry flow cytometer in this embodiment are as follows:

[0082] Cell suspension injection flow rate: 30-31 μl / min;

[0083] Event collection rate < 500 events / s;

[0084] EQbeads concentration: about 10%;

[0085] EQbeads monitors elements: 140 / 142 Ce (cerium), 151 / 153 Eu(Europium), 165 Ho (holmium), 175 / 176 Lu(lutetium);

[0086] Elements to be tested: 81 Br, 191 / 193 Ir, 195 Pt.

[0087] Test results such as Figure 1 shown.

[0088] The results showed that the intracellular Br content of different types of cells increased significantly after being exposed to different concentrations of TBBPA.

[0089] Figure 1 a shows the changes in the intracellular contents of mouse liver parenchymal cells AML12 after exposure to different concentrations of TBBPA. 81 Br content. It can be seen that after TBBPA exposure at 10 and 50 μM, the 81 The Br content increased significantly with the increase of exposure concentration.

[0090] Figure 1b shows the changes in the intracellular contents of mouse liver macrophages RAW264.7 after exposure to different concentrations of TBBPA. 81 Br content. It can be seen that after TBBPA exposure, except for the concentration of 5μM, the 81 The Br content increased significantly with the increase of exposure concentration;

[0091] From this experiment, we can infer that TBBBPA enters cells through a certain mechanism, and whether it has entered cells can be determined by the intracellular Br content.

[0092] Example 2: Comparison of the response intensity of Br in the liver of C57BL / 6J mice after exposure to TBBPA-BDBPE via tail vein injection

[0093] The specific implementation process is as follows:

[0094] 1) Prepare a 57 μM TBBPA-BDBPE solution.

[0095] 2) Ten 6-8 week old C57BL / 6J male mice were divided into two groups and exposed to PBS or 57 μM TBBPA-BDBPE solution via tail vein injection, respectively.

[0096] 3) After 5 hours of exposure via tail vein injection, mice were killed by cardiac perfusion, and their livers (and gallbladders) were removed for single cell production.

[0097] 4) Single-cell preparation: A single-cell suspension was prepared from the liver using a liver dissociation kit. The supernatant was removed by centrifugation and the cells were resuspended in PBS.

[0098] 5) Cell Lysis: Add 3 volumes of cell lysis buffer to the single-cell suspension and incubate at room temperature for 10 minutes. Add 2 ml of PBS to stop the reaction and centrifuge. Wash once with PBS, centrifuge, and discard the supernatant. Reserve 0.5 ml of the solution and mix thoroughly.

[0099] 6) Cisplatin labeling: Add cisplatin at a final concentration of 0.5 μM (if there are more than 10 cells 7 , the cell density needs to be adjusted to 1×10 7 1 mL of room-temperature CSB was added, mixed, and the cells were incubated at room temperature for 2 minutes. The labeling reaction was terminated by adding 1 mL of room-temperature CSB and mixing. The cells were centrifuged. The cells were washed once with PBS and resuspended in 0.5 mL of PBS.

[0100] 7) Cell Fixation: Homogenize the cell suspension by pipetting or shaking in a centrifuge tube. Add an equal volume of 2X fixation solution (3.2% PFA in PBS) to the cell suspension, resulting in a final PFA concentration of 1.6%. Immediately pipette up and down and vortex to mix thoroughly to reduce cell dimerization. Incubate at room temperature for 10 minutes. Add CSB to slow the PFA fixation reaction, centrifuge, discard the supernatant, and wash once with PBS.

[0101] 8) Cryopreservation: Wash or carefully pour off some of the supernatant, leaving approximately 50 μL of residual liquid to resuspend the cells to a single-cell suspension. In the remaining liquid, flick the pellet with your fingers. Vortex to thoroughly mix the cell pellet. Add 1 mL of FBS containing 10% (vol / vol) DMSO to resuspend the cells. Aliquot and store in a -80°C freezer.

[0102] 9) Remove the frozen cells, thaw them naturally at room temperature, count them, centrifuge them, discard the supernatant, and wash them once with CSB.

[0103] 10) The cells were suspended in 50 μl of CSB, and 50 μl of CSB containing 1 μl of anti-mouse CD45-147Sm antibody was added. The mixture was mixed well and allowed to stand at room temperature for 30 min.

[0104] 11) Wash the cells twice by centrifugation with CSB and discard the supernatant.

[0105] 12) Add I or Rh to a final concentration of 125 nM or 500 nM to the fix and perm buffer, using 1 ml for each sample. Mix gently and immediately, and incubate at 4°C overnight.

[0106] 13) Centrifuge at room temperature (800g, 5min, 4°C) and discard the supernatant. Wash the cells twice with CSB (800g, 5min, 4°C) and discard the supernatant. Add 1ml of CAS and count. Resuspend the cells in CAS solution to a concentration of 1.5-1.8×10 6 / mL suspension.

[0107] 14) Take 900 μl of the suspension and filter it through a flow cytometer with a 35 μm filter. Add 100 μl of EQ Beads and collect data.

[0108] Mass cytometry detection parameters:

[0109] Cell suspension injection flow rate: 30-31 μl / min;

[0110] Event collection rate < 500 events / s;

[0111] EQbeads concentration: about 10%;

[0112] EQbeads monitors elements: 140 / 142 Ce (cerium), 151 / 153 Eu(Europium), 165 Ho (holmium), 175 / 176 Lu(lutetium);

[0113] Elements to be tested: 147 Sm(CD45), 81 Br, 191 / 193 Ir, 195 Pt.

[0114] use 140 Ce beads and event length and 191 Ir and 193 Ir was gated hierarchically to identify intact single cells from debris and cell aggregates. 195 Pt identifies living cells and reuses them 147 Sm(CD45) divides live cells into CD45-positive and CD45-negative cells. The gating process is as follows Figure 2 Analysis of live liver cells, CD45+ and CD45- cells 81 The response intensity of Br is as follows Figure 3 shown.

[0115] The results show that: Figure 3 It can be seen that the mice exposed to TBBPA-BDBPE by tail vein injection had 81 The Br response intensity increased significantly. Comparison of different cell types revealed that the Br response intensity varied among different cell types, suggesting that different cells have different capacities for TBBPA-BDBPE uptake.

[0116] This example demonstrates that by detecting Br in single cells, it is possible to explore the differences in Br uptake by different types of single cells in the liver of mice after exposure to bromine-containing compounds, thereby providing a basis for inferring the possible mechanism by which Br enters different types of liver cells.

Claims

1. A method for detecting Br in a single cell, comprising the following steps: S1. Collect biological samples exposed to brominated compounds; S2. preparing a single cell suspension using the biological sample; S3, labeling cells in the single cell suspension with metal isotope A and metal isotope B respectively; The metal isotope A can simultaneously label living and dead cells; The metal isotope B can label the dead cells; S4. The single-cell suspension is subjected to single-cell mass spectrometry flow cytometry detection. First, single cells are separated according to the signal of the metal isotope A, which is recorded as set S. Then, individuals with the signal of the metal isotope A but without the signal of the metal isotope B are obtained from the set S, which is recorded as set P. According to the detected intracellular Br element signal intensity value, the Br content in the single cell can be obtained through the standard curve.

2. The detection method according to claim 1, wherein: In step S1, the biological samples include cells cultured in vitro and organs of model animals.

3. The detection method according to claim 2, wherein: The cells cultured in vitro include mouse liver parenchymal cells and mouse liver macrophages; The organs of the model animal include the liver, spleen and bone marrow of mice.

4. The detection method according to any one of claims 1 to 3, characterized in that: In step S1, the brominated compound includes tetrabromobisphenol A and octabromoether.

5. The detection method according to any one of claims 1 to 4, characterized in that: In step S3, the metal isotope A is 193 Ir, 103 Rh or lanthanide metal isotopes; The metal isotope B is 19 5Pt.

6. The detection method according to any one of claims 1 to 5, characterized in that: Before step S4, the detection method further includes a step of labeling the cells in the single cell suspension with antibodies.

7. The detection method according to any one of claims 1 to 6, characterized in that: In step S4, the single-cell mass spectrometry flow detection is performed in a single-cell inductively coupled plasma time-of-flight mass spectrometer.

8. The detection method according to any one of claims 1 to 7, characterized in that: In step S4, after the single-cell mass spectrometry flow cytometry detection, 50,000 to 100,000 events are collected for each group of samples.

9. The detection method according to any one of claims 1 to 8, characterized in that: In step S4, the obtained data is collated and analyzed on a mass spectrometry flow cytometry data processing website.

10. The detection method according to any one of claims 1 to 9, characterized in that: In step S4, the conditions for the single-cell mass spectrometry flow cytometry detection are as follows: Cell suspension injection flow rate: 30-31 μl / min; Event collection rate: <500 events / s; EQbeads concentration: 10%; EQbeads monitors elements: 140 / 142 Ce, 151 / 153 Eu, 165 Ho, 175 / 176 Lu; Elements to be tested: 81 Br, 191 / 193 Ir, 195 Pt.

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