Method for detecting content of metal elements in single cell

Abstract

The invention provides a method for detecting the content of metal elements in a single cell, belongs to the field of cell analysis methods, and adopts a single cell inductively coupled plasma mass spectrometry technology to detect the content of metal elements in the single cell. Wherein the cells are respectively cleaned and fixed by cysteine and glutaraldehyde solutions, then the atomization efficiency of the cells is detected by utilizing endogenous magnesium elements of the cells, and finally the metal content in the single cells is calculated. The method disclosed by the invention can be used for measuring the real atomization efficiency of the cells in SC-ICP-MS and quantifying the content of the metal elements in the single cells, has the advantages of good accuracy, high detection sensitivity, good repeatability and the like, and can be widely applied to quantitative analysis of various metal elements in different types of cells.

Description

Technical Field

[0001] The present invention relates to the field of cell analysis methods, and in particular to a method for detecting the content of metal elements in a single cell. Background Art

[0002] Cells can use the physicochemical properties of metal ions to control a series of physiological processes, such as cell respiration, signal transduction, metabolic function, etc. In conventional cell metal content analysis, it is generally necessary to digest the cell sample and then detect the total amount of metal elements. However, due to the heterogeneity of cells, conventional detection methods cannot reflect the differences in metal elements between single cells, resulting in information loss. Therefore, in order to understand the heterogeneity of the distribution of metal elements between single cells, it is necessary to detect the metal content in single cells, which is of great significance for the study of metal-related biological processes.

[0003] At present, there are related methods and literature reports on the analysis of metal elements in single cells. Among them, SC-ICP-MS technology, based on the original ICP-MS technology, realizes the detection of metal element content in single cells by improving the sample introduction and data acquisition mode. In SC-ICP-MS analysis, accurate determination of cell atomization efficiency is the key to accurate quantification of metal elements in single cells. Due to the lack of universal cell standard reference materials, metal nanoparticles are generally used as reference materials to determine the atomization efficiency in SC-ICP-MS analysis. However, since cells and metal nanoparticles have different properties, there is a difference between the atomization efficiency measured using metal nanoparticles as reference materials and the actual atomization efficiency of cells in SC-ICP-MS, which leads to errors in the calculation results of metal content in single cells. Therefore, the existing analysis methods cannot achieve accurate quantitative analysis of metal content in single cells.

[0004] The inventors of this application proposed a method for detecting the metal content in a single cell, using the endogenous magnesium element in the cell to detect the true atomization efficiency of the cell, avoiding the error caused by using metal nanoparticles as reference materials, and providing a powerful tool for studying the transmission process, physiological functions, and toxic effects of metals in cells. Summary of the invention

[0005] The embodiment of the present application provides a method for detecting the content of metal elements in a single cell, which aims to solve the problem that the existing SC-ICP-MS analysis method cannot accurately measure the metal content. The endogenous magnesium element in the cell is used to measure the true atomization efficiency of the cell, thereby achieving accurate detection of the atomization efficiency and the metal content in a single cell.

[0006] In order to solve the above technical problems, the technical solution proposed in this application is:

[0007] The present invention provides a method for detecting the content of metal elements in a single cell, comprising the following steps:

[0008] (a), initially washing the cells with a solution containing a specific concentration of cysteine;

[0009] (b), fixing the cells after the initial washing using a fixative containing a specific concentration, and then performing a secondary washing on the fixed cells using ultrapure water;

[0010] (c), counting the cell sample after the secondary washing, and diluting it with ultrapure water according to the cell density;

[0011] (d) using SC-ICP-MS in single particle mode to analyze the diluted cell sample online, using the endogenous magnesium signal of the cell to detect the atomization efficiency of the cell in the SC-ICP-MS, and determining the metal element content in the single cell through a specific calculation formula.

[0012] Preferably, in step (a), the concentration of the cysteine ​​solution is 10 mmol / L.

[0013] Preferably, in step (a), the method further comprises adding a phosphate buffer consisting of 1 mmol / L potassium dihydrogen phosphate, 6 mmol / L disodium hydrogen phosphate and 154 mmol / L sodium chloride to the cysteine ​​solution, wherein the pH of the buffer is 7.0 to 7.2.

[0014] Preferably, the fixative in step (b) is a 2.5% (v / v) glutaraldehyde solution.

[0015] Preferably, in step (b), the method further comprises adding a phosphate buffer consisting of 1 mmol / L potassium dihydrogen phosphate, 6 mmol / L disodium hydrogen phosphate and 154 mmol / L sodium chloride to the fixative, wherein the pH of the buffer is 7.0 to 7.2.

[0016] Preferably, in step (c), the cell density after sample dilution is 1×10 5 Pieces / mL.

[0017] Preferably, in step (d), the detection mode of SC-ICP-MS is single particle mode, the incident power is 1550W, the sample injection method is peristaltic pump injection, the inner diameter of the injection tube is 0.025mm, the injection rate is 16μL / min, the carrier gas flow rate is 1.0L / min, the nebulizer pressure is 270kPa, the integration time is 3ms, and the data acquisition time is 60s.

[0018] Preferably, in step (d), the atomization efficiency of the cells in SC-ICP-MS is calculated according to the number of cellular magnesium signals, and the atomization efficiency (η) of the cells can be calculated by the following formula (1):

[0019]

[0020] Where N is the number of cellular magnesium signals (cells), C is the cell density of the injection (cells / mL), Q is the injection rate (mL / min), and t is the data acquisition time (s).

[0021] Preferably, the step (b) specifically comprises: resuspending the cell sample obtained in step (a) in a glutaraldehyde solution for fixation, standing at room temperature for 1 hour, and then repeatedly washing the cells twice with ultrapure water to remove residual glutaraldehyde solution.

[0022] Compared with the prior art, the method for detecting the content of metal elements in a single cell of the present invention has achieved the following beneficial technical effects: it can realize the accurate determination of the real atomization efficiency of cells in SC-ICP-MS analysis, and then realize the accurate quantitative analysis of metal elements in a single cell.

[0023] The method for detecting the metal element content of a single cell of the present invention can detect the real atomization efficiency of cells with unknown metal element content in SC-ICP-MS analysis, thereby realizing accurate analysis of the metal element content of a single cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. 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:

[0025] Figure 1 This is a main flow chart of a method for detecting the metal element content in a single cell according to the present invention.

[0026] Figure 2 This is the frequency distribution of the metal element content of Synechocystis PCC 6803 according to an embodiment of the method for detecting the metal element content in a single cell of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] This application uses single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) technology to measure the transmission efficiency of cells with endogenous elements, and then conducts quantitative analysis of metal elements in single cells, such as Figure 1 As shown in the flowchart, a method for detecting the metal element content of a single cell in an embodiment of the present application uses cleaning and fixation pretreatment to remove metals adsorbed on the cell surface and improve the stability of the cell; then, the cell sample enters the SC-ICP-MS detector, and its true atomization efficiency is determined using endogenous magnesium in single particle mode, and then its metal content is quantitatively analyzed. The specific steps are as follows:

[0029] A method for detecting the content of metal elements in a single cell, using SC-ICP-MS technology to quantitatively analyze the content of metal elements in a single cell, the method comprising:

[0030] (a) Initial sample cleaning: The collected cell samples were cleaned with cysteine ​​solution to remove the extracellular adsorbed metals;

[0031] (b) Sample fixation and secondary washing: The cell sample obtained in step (a) was resuspended in a glutaraldehyde solution for fixation, allowed to stand at room temperature for 1 hour, and then the cells were repeatedly washed twice with ultrapure water to remove the residual glutaraldehyde solution;

[0032] (c) Sample counting and dilution: The cell sample obtained in step (c) is counted, and the sample is diluted with ultrapure water according to the counting result.

[0033] (d) SC-ICP-MS quantitative analysis: The diluted cell sample in step (c) is used to enter the SC-ICP-MS and is analyzed online using the single particle mode to obtain the pulse signal peak of a single cell. The signal of the magnesium element in the cell is used to detect the atomization efficiency, and the quantitative analysis of the metal elements in the single cell is achieved through calculation.

[0034] The present invention is further described in detail below in conjunction with embodiments, but the scope of the present invention is not limited thereto.

[0035] Example:

[0036] Samples and reagents: 50% (v / v) glutaraldehyde solution was purchased from Shanghai Yien Chemical Technology Co., Ltd. L-cysteine ​​hydrochloride monohydrate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Phosphate buffer (1mmol / L potassium dihydrogen phosphate, 6mmol / L sodium hydrogen phosphate and 154mmol / L sodium chloride) was purchased from Cytiva, USA. 1000μg / mL magnesium, mercury, cadmium, and cobalt ion standard stock solutions were purchased from National Standard (Beijing) Testing and Certification Co., Ltd. Ultrapure water (DDW, 18MΩ.cm) was from Milli-Q Advantage A10 system (Millipore, USA).

[0037] Sample initial washing operation conditions: The collected cell samples were centrifuged at 1500g for 5 minutes and the supernatant was removed. The cells were then resuspended in 10mmol / L cysteine ​​solution and pipetted evenly. Finally, the samples were centrifuged at 1500g for 5 minutes and the supernatant was removed.

[0038] Selection and optimization of cell fixation conditions: Fixatives and treatment time affect the fixation effect of cells. The experiment investigated the differences in the fixation effects of 4% (v / v) formaldehyde, 4% (v / v) paraformaldehyde, and 2.5% (v / v) glutaraldehyde solutions on cells at different treatment times (0.5, 1, and 2h). When 2.5 (v / v) glutaraldehyde solution was selected for treatment for 1h, the number of cells and the recovery rate of the metal content in the cells were higher, and the fixation effect was better, so it was selected as the cell fixation condition.

[0039] SC-ICP-MS system and operating conditions: 8800 ICP-MS (Agilent, USA). The sample injection method is peristaltic pump injection, the inner diameter of the injection tube is 0.025 mm, the injection rate is 16 μL / min, the carrier gas flow rate is 1.0 L / min, the nebulizer pressure is 270 kPa, the integration time is 3 milliseconds, and the data acquisition time is 60 seconds.

[0040] Screening of endogenous elements in cells: The selection of endogenous elements in cells affects the accuracy of cell atomization efficiency determination. The experiment investigated the effects of magnesium, phosphorus, iron, calcium, zinc, and copper as representatives of endogenous elements in cells for determining cell atomization efficiency. When magnesium was selected, the signal-to-noise ratio of the SC-ICP-MS spectrum was relatively high, so it was selected as the monitoring element for cell atomization efficiency determination.

[0041] The method of the present invention is used to detect the content of mercury, cadmium and cobalt in Synechocystis PCC 6803. The specific implementation process is as follows: first, Synechocystis cells are exposed to culture media containing 100 μg / L mercury, cadmium and cobalt ions for 24 hours respectively; the exposed cell samples are collected, centrifuged at 1500g for 5 minutes, the supernatant is removed, the cells are resuspended in 10 mmol / L cysteine ​​solution, and the samples are blown evenly and centrifuged at 1500g for 5 minutes and the supernatant is removed; the cell samples are resuspended in 2.5% (v / v) glutaraldehyde solution, and after standing for 1 hour, they are centrifuged at 1500g for 5 minutes and the supernatant is removed, and then they are repeatedly washed with ultrapure water twice; the treated cells are resuspended in ultrapure water and counted, and the counting results are used to dilute the sample with ultrapure water so that the cell density in the sample to be tested is 1×10 5 / mL. The sample to be tested was injected by a peristaltic pump, wherein the inner diameter of the injection tube was 0.025mm, the injection rate was 16μL / min, the carrier gas flow rate was 1.0L / min, the nebulizer pressure was 270kPa, the integration time was 3 milliseconds, and the data acquisition time was 60 seconds. The magnesium, mercury, cadmium, and cobalt elements in the single cell were detected in turn, and the test results were recorded by the signal acquisition device to obtain the original time-intensity data. The above data were then calculated and statistically analyzed to obtain the frequency distribution of the cell metal content ( Figure 2 ).

[0042] It can be seen from the above examples that the method of the present invention can quantitatively analyze the content of metal elements in single cells.

[0043] The present invention proposes a method for detecting the content of metal elements in single cells. Taking Synechocystis exposed to different metals as an example, the content of metal elements in Synechocystis is detected using single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS). SC-ICP-MS is a rapid and sensitive method for detecting the metal content in single cells. By using the endogenous magnesium element in the cells to measure the true atomization efficiency of the cells, the error caused by using metal nanoparticles as reference materials to measure the atomization efficiency of the cells can be avoided, and the accurate detection of the atomization efficiency of the cells can be achieved, thereby achieving accurate quantification of the content of metal elements in single cells. Through condition optimization, the stability of the cells can be enhanced by fixation treatment, and the accuracy of the results of the cell atomization efficiency determination and metal element quantitative analysis can be improved by using the endogenous magnesium element in the cells. This method is also applicable to other cells. Therefore, the present application establishes a new method for accurately detecting the content of metal elements in single cells using SC-ICP-MS technology.

[0044] The present invention has been described in detail above, but the present invention is not limited to the specific embodiments described herein. It is understood by those skilled in the art that other changes and modifications may be made to the technical solution without departing from the scope of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A method for detecting the content of metal elements in a single cell, characterized in that: The following steps are involved: (a), initially washing the cells with a solution containing a specific concentration of cysteine; (b), fixing the cells after the initial washing using a fixative containing a specific concentration, and then performing a secondary washing on the fixed cells using ultrapure water; (c), counting the cell sample after the secondary washing, and diluting it with ultrapure water according to the cell density; (d) using SC-ICP-MS in single particle mode to analyze the diluted cell sample online, using the endogenous magnesium signal of the cell to detect the atomization efficiency of the cell in the SC-ICP-MS, and determining the metal element content in the single cell through a specific calculation formula.

2. The method according to claim 1, characterized in that In step (a), the concentration of the cysteine ​​solution is 10 mmol / L.

3. The method according to claim 2, characterized in that In step (a), the method further comprises adding a phosphate buffer consisting of 1 mmol / L potassium dihydrogen phosphate, 6 mmol / L disodium hydrogen phosphate and 154 mmol / L sodium chloride to the cysteine ​​solution, wherein the pH of the buffer is 7.0 to 7.

2.

4. The method according to claim 1, characterized in that: The fixative in step (b) is a 2.5% (v / v) glutaraldehyde solution.

5. The method according to claim 4, characterized in that In step (b), the method further comprises adding a phosphate buffer composed of 1 mmol / L potassium dihydrogen phosphate, 6 mmol / L disodium hydrogen phosphate and 154 mmol / L sodium chloride to the fixing agent, wherein the pH of the buffer is 7.0 to 7.

2.

6. The method according to claim 1, characterized in that In step (c), the cell density after sample dilution is 1×10 5 Pieces / mL.

7. The method according to claim 1, characterized in that In step (d), the detection mode of SC-ICP-MS is single particle mode, the incident power is 1550 W, the sample injection method is peristaltic pump injection, the inner diameter of the injection tube is 0.025 mm, the injection rate is 16 μL / min, the carrier gas flow rate is 1.0 L / min, the nebulizer pressure is 270 kPa, the integration time is 3 ms, and the data acquisition time is 60 s.

8. The method according to claim 1, characterized in that In step (d), the nebulization efficiency of the cells in SC-ICP-MS is calculated according to the number of cellular magnesium signals. The nebulization efficiency (η) of the cells can be calculated by the following formula (1): Where N is the number of cellular magnesium signals (cells), C is the cell density of the injection (cells / mL), Q is the injection rate (mL / min), and t is the data acquisition time (s).

9. The method according to claim 4, characterized in that The step (b) specifically includes: resuspending the cell sample obtained in step (a) in a glutaraldehyde solution for fixation, standing at room temperature for 1 hour, and then repeatedly washing the cells twice with ultrapure water to remove residual glutaraldehyde solution.

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