Method for simultaneous detection of multiple chemical elements on a single cell

Abstract

The application discloses a method for simultaneously detecting multiple chemical elements on a single cell, which comprises the following steps: performing first cleaning treatment on the cell to be detected by using isotonic buffer, and fixing the cell subjected to the first cleaning treatment by using isotonic fixing solution, wherein the isotonic buffer and the isotonic fixing solution do not contain endogenous marker elements and chemical elements to be detected; performing second cleaning treatment on the fixed cell by using ultrapure water, counting the cell, diluting the cell according to the counting result by using ultrapure water, and obtaining a diluted cell suspension as a sample to be detected; performing single-cell multi-element detection on the sample to be detected by using an inductively coupled plasma-time of flight mass spectrometer, obtaining cell detection data; screening effective cell detection data from the cell detection data based on endogenous marker elements of the cell, and performing quantitative analysis on the effective cell detection data, thereby obtaining quantitative results of multiple elements on the cell. The application can effectively and high-throughput detect multiple chemical elements in a single cell.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis technology for biological samples, and in particular to a method for simultaneously detecting multiple chemical elements on a single cell based on single-cell inductively coupled plasma mass spectrometry (ICP-MS). Background Technology

[0002] The chemical composition of cells is highly complex, and these elements often have important physiological functions. Taking human sperm cells as an example, studies have shown that macroelements (such as Na and K) and microelements (such as Zn, Cu, Se, and Mn) in human semen play crucial roles in the entire reproductive process from spermatogenesis to fertilization, thus affecting semen quality. Currently, conventional multi-element analysis often uses methods to determine the total elemental content after digestion. This method only provides the average level of elemental content within cells and cannot reflect the elemental content of individual cells or the heterogeneity of elemental distribution among different cells.

[0003] ICP-MS is an advanced chemical elemental analysis technique characterized by high sensitivity, a wide dynamic linear range, and fast detection speed. Single-cell ICP-MS (scICP-MS) can detect elemental signals generated by a single cell, overcoming the limitations of total elemental analysis. Typically, traditional quadrupole ICP-MS can only detect one element per single cell. Although different elements can be measured in different batches, most cell samples are small, making multi-batch and long-term analysis difficult. More importantly, multi-element information from multiple batch measurements cannot be matched to the same cell. Therefore, there is an urgent need to develop a multi-element detection method at the single-cell level. Summary of the Invention

[0004] In view of this, the main objective of the present invention is to provide a method for simultaneously detecting multiple chemical elements on a single cell, in order to at least solve some of the aforementioned technical problems.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A method for simultaneously detecting multiple chemical elements on a single cell includes the following steps: First, washing the cells to be tested using an isotonic buffer solution, wherein the isotonic buffer solution does not contain endogenous cell marker elements or the chemical elements to be tested; fixing the cells after the first washing treatment using an isotonic fixative solution to maintain the cell structure and morphology, wherein the isotonic fixative solution does not contain endogenous cell marker elements or the chemical elements to be tested; second, washing the fixed cells using ultrapure water, counting the cells, and diluting the cells with ultrapure water based on the counting results to obtain a diluted cell suspension as the sample to be tested; performing single-cell multi-element detection on the sample to be tested using inductively coupled plasma-time-of-flight mass spectrometry (ICP-TOF-MS) to obtain cell detection data related to multiple chemical elements to be tested within a single cell; selecting effective cell detection data from the cell detection data based on the endogenous cell marker elements, and then performing quantitative analysis on the effective cell detection data to obtain quantitative results of multiple elements on the cell.

[0007] As can be seen from the above technical solution, the method for simultaneously detecting multiple chemical elements on a single cell in this invention has at least one or a portion of the following beneficial effects:

[0008] This invention uses ICP-TOF-MS to simultaneously detect all elements on a single cell, which can reflect the total elements (…). 7 Li- 238 The analysis process for cell samples was optimized by taking into account the characteristics of both cell samples and the ICP-TOF-MS instrument. Specifically, a treatment reagent free of endogenous cell markers and the main analyte chemical elements was selected to treat the cell samples, allowing the cells to be completely dispersed in ultrapure water. This avoided interference from the biological matrix on the ICP-TOF-MS instrument. Furthermore, endogenous cell markers were used to effectively screen cell information during the analysis, avoiding inaccuracies in the detection results caused by false positive peaks due to cell debris, particulate impurities, and signal fluctuations.

[0009] Through the above-mentioned technical means, the method for simultaneous detection of multiple chemical elements on a single cell provided by the present invention achieves high-throughput detection of multiple chemical elements on a single cell within an analytical batch. Based on endogenous marker elements, it can accurately and effectively screen cell signals. It is applicable to different types of cells, such as animal cells and lower single-celled organism cells. It has the advantages of low sample consumption, fast detection speed, no matrix effect interference and minimal instrument damage, and has great application prospects and significance. Attached Figure Description

[0010] Figure 1This is a flowchart of a method for simultaneously detecting multiple chemical elements on a single cell according to an embodiment of the present invention;

[0011] Figure 2 This is a flowchart of the multi-element quantitative detection and analysis of human sperm cells in Embodiment 1 of the present invention;

[0012] Figure 3 This is a schematic diagram illustrating the screening of effective cell data from cell detection data related to individual cells in Embodiment 1 of the present invention. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0014] Traditional single-cell ICP-MS typically detects only one element on a single cell within an analytical batch, providing limited information. Furthermore, false positives caused by cell debris, particulate impurities, and signal fluctuations can easily affect the accuracy of the results. In developing this invention, it was discovered that the emerging ICP-MS equipped with a time-of-flight (TOF) mass analyzer (i.e., ICP-TOF-MS) can simultaneously monitor all elements on a single nanoparticle or cell. 7 Li- 238 ICP-TOF-MS (U), which can meet the analytical needs of simultaneous multi-element detection in single cells, is also highly sensitive to interfering elements in the matrix. Therefore, it is necessary to remove interfering matrix as much as possible while ensuring cell integrity. This invention proposes a method for simultaneously detecting multiple chemical elements on a single cell. It utilizes endogenous elements within the cell as marker elements to accurately identify cell signals, while also taking into account the characteristics of both cell samples and the ICP-TOF-MS instrument. The sample processing steps and analytical workflow are optimized. After washing, fixing, counting, and dilution, the cells can be completely dispersed in ultrapure water, avoiding the influence of the biological matrix on the instrument.

[0015] Specifically, according to some embodiments of the present invention, a method for simultaneously detecting multiple chemical elements on a single cell is provided. Figure 1 This is a flowchart of a method for simultaneously detecting multiple chemical elements on a single cell, according to an embodiment of the present invention. Figure 1 As shown, the method includes operations S101 to S105.

[0016] In operation S101, the cells to be tested are first washed using isotonic buffer, which does not contain endogenous cell markers or the chemical elements to be tested.

[0017] In operation S102, isotonic fixative is used to fix the cells after the first washing treatment to maintain the cell structure and morphology. The isotonic fixative does not contain endogenous cell marker elements or the chemical elements to be tested.

[0018] In operation S103, after the fixed cells are washed a second time with ultrapure water, cell counting is performed, and the cells are diluted with ultrapure water according to the counting results to obtain a diluted cell suspension as a sample to be tested.

[0019] In operation S104, ICP-TOF-MS was used to perform single-cell multi-element detection on the test sample to obtain cell detection data on multiple chemical elements to be tested in a single cell.

[0020] In operation S105, effective cell detection data are screened from the cell detection data based on endogenous cell marker elements, and then the effective cell detection data are quantitatively analyzed to obtain multi-element quantitative results on the cells.

[0021] According to embodiments of the present invention, taking into account both the characteristics of cell samples and the instrument features of ICP-TOF-MS, a complete analytical workflow from sample pretreatment and sample detection to detection data processing has been established. This workflow can accurately identify and screen cell signals, enabling high-throughput non-targeted detection of multiple chemical elements on a single cell. It eliminates the need to predetermine the content range of target elements and to perform multiple batch measurements. It features low sample consumption (only a few hundred microliters of cell sample are required), fast detection speed (the detection time for one sample is only a few minutes), minimal matrix interference (cells are dispersed in ultrapure water), and minimal damage to the instrument. It can be applied to various animal cells, cells of lower single-celled organisms, etc., to study the distribution patterns of elements and the correlation characteristics between elements and cell physiological states.

[0022] According to an embodiment of the present invention, before operation S101, the method further includes obtaining cells, specifically including: preserving and transporting cell samples in a frozen state; thawing and resuscitating the frozen cell samples; adding the above-mentioned isotonic buffer to the resuscitated cell samples for dilution and centrifugation to obtain the cells to be tested.

[0023] According to embodiments of the present invention, specifically, optionally, an appropriate amount of cryoprotectant can be added to the cell sample, and it can be frozen and stored in liquid nitrogen or a -80°C freezer. Specifically, optionally, the frozen cell sample can be placed in dry ice to maintain a low temperature for transfer and transportation. Specifically, optionally, the frozen cell sample can be thawed and revived at room temperature for about 30 minutes, and then thoroughly mixed using a wide-bore pipette. Specifically, optionally, isotonic buffer is added to the mixed cell sample for dilution, followed by centrifugation to remove the supernatant, yielding the cells to be tested.

[0024] According to embodiments of the present invention, phosphorus is selected as an endogenous marker element for cell detection data screening. Since phosphorus is highly abundant in cells, it is beneficial for effectively screening cell signals, and because cell membranes all contain phospholipids, it is universally applicable to different cell types, especially animal cells. In other embodiments, taking algal cells as an example, magnesium can also be selected as an endogenous marker element; however, magnesium is generally not suitable for screening animal cells.

[0025] According to embodiments of the present invention, in the operation of S101 or the cell acquisition operation, the isotonic buffer used is an organic buffer to maintain osmotic pressure and cell integrity, so that the cells can be completely dispersed in ultrapure water after fixation. Examples include 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer, 3-morpholine propanesulfonic acid (MOPS), etc. The isotonic buffer used in this invention differs from conventional phosphate buffers; it is an organic buffer. When phosphorus is used as an endogenous marker element in cells, it avoids the interference caused by conventional phosphate buffers in the detection of the target element.

[0026] According to embodiments of the present invention, the isotonic buffer may include an organic buffer with a concentration of 0.01 to 0.025 M, NaCl with a mass-volume concentration of 0.9%, and a pH of 7.2 to 7.4.

[0027] More specifically, taking the organic buffer HEPES as an example, the preparation method of isotonic buffer includes: dissolving an appropriate amount of HEPES reagent in ultrapure water to obtain a HEPES stock solution concentration of 0.1–1 M. Diluting the HEPES stock solution with ultrapure water to 0.01–0.025 M, adding 0.9% (w / v) NaCl, and obtaining a 0.01–0.025 M HEPES isotonic buffer. Then adjusting the pH to 7.2–7.4 using a NaOH solution of appropriate concentration.

[0028] According to an embodiment of the present invention, in operation S102, the isotonic fixative used is an aldehyde fixative to fix the cells, maintain their structure and morphology, and allow them to be completely dispersed in ultrapure water. For example, paraformaldehyde (PFA) fixative or glutaraldehyde fixative can be selected, but the method is not limited to these; other types of fixatives can also be used. The isotonic fixative used in this invention also does not contain phosphorus, thus avoiding interference with ICP-TOF-MS detection.

[0029] According to embodiments of the present invention, the isotonic fixative may include a fixative with a mass-volume concentration of 2-5%, an organic buffer with a concentration of 0.01-0.025M, NaCl with a mass-volume concentration of 0.9%, and a pH of 7.2-7.4.

[0030] More specifically, taking PFA as a fixative and HEPES as an organic buffer, the preparation method of the isotonic fixative includes: placing an appropriate amount of PFA reagent in ultrapure water and stirring in a water bath at 50-60°C until completely dissolved to obtain a PFA stock solution. Adding an appropriate amount of HEPES stock solution and NaCl reagent to the PFA stock solution cooled to room temperature, so that the PFA concentration in the obtained fixative is 2-5% (w / v), the HEPES concentration is 0.01-0.025M, and the NaCl concentration is 0.9% (w / v). Then, adjusting the pH to 7.2-7.4 using a suitable concentration of NaOH solution.

[0031] According to an embodiment of the present invention, in operation S103, ultrapure water is used to wash, count, and dilute the fixed cells to avoid interference during full-element detection by ICP-TOF-MS, and to prevent excessively high element concentrations in the matrix from saturating the detector and damaging the ICP-TOF-MS instrument. The concentration of the diluted cell suspension is 4 × 10⁻⁶. 5 If the cell concentration is too high, it will affect the detection of single cells. Therefore, an appropriate cell suspension concentration can ensure the detection of single cells by ICP-TOF-MS.

[0032] According to an embodiment of the present invention, in operation S104, the instrument parameters of the ICP-TOF-MS include: sample introduction flow rate of 0.4 mL / min, radio frequency power of 1550 W, nebulizer gas flow rate of 1.06 L / min, auxiliary gas flow rate of 0.8 L / min, TOF mass analyzer acquisition frequency of 21.7 kHz, shielding mass-to-charge ratio of 31.5, 35.7, 40.2, and 80.0, and residence time of 0.003 s. By setting the instrument parameters within appropriate ranges, especially controlling the sample introduction flow rate and residence time, single-cell detection can be ensured, and the shielding mass-to-charge ratio can remove strong background interference from the gas or solution matrix in the instrument.

[0033] According to an embodiment of the present invention, in operation S105, when a phosphorus signal is detected in the cell detection data of a single cell, the cell detection data of that single cell is determined to be valid cell detection data; otherwise, it is invalid cell detection data. These invalid cell detection data are usually caused by cell debris, particulate impurities, and signal fluctuations.

[0034] According to an embodiment of the present invention, the method further includes operations S106 and S107.

[0035] In operation S106, ICP-TOF-MS was used to simultaneously determine multiple elements in a series of standard solutions containing multiple analyte chemical elements using the same detection method as the analyte sample, and the response-mass correction coefficients of multiple analyte chemical elements were determined.

[0036] In operation S107, the standard solutions of gold nanoparticles and chloroauric acid were measured using ICP-TOF-MS to obtain the atomization efficiency of ICP-TOF-MS.

[0037] According to an embodiment of the present invention, further, the quantitative analysis of effective cell detection data in operation S105 includes: using the response-mass correction coefficients and atomization efficiency of multiple analyte chemical elements to correct the effective cell detection data, thereby obtaining multi-element quantitative results on the cells.

[0038] According to an embodiment of the present invention, the instrument response data is converted into elemental concentration results based on the correction of the response-mass correction coefficient and the nebulization efficiency, thereby completing the quantitative analysis. Repeated determination of the response-mass correction coefficient and nebulization efficiency during different batches of testing ensures the consistency of data as much as possible when the instrument is in a state of inconsistency.

[0039] Specifically, the mass of the chemical element to be tested in a single cell is calculated using the following formula (1):

[0040]

[0041] Where m is the mass of an element in a single cell (ng), I is the element signal intensity of the cell event (counts or cps), s is the correction coefficient of the element obtained from the determination of a series of multi-element standard solutions (counts / (μg / L) or cps / (μg / L)), η is the nebulization efficiency, g is the injection flow rate (mL / min), and t d The dwell time is in seconds (s).

[0042] According to embodiments of the present invention, in order to minimize interference from elements in the environment and containers, clean and uncontaminated polypropylene centrifuge tubes and glass containers conditioned with 30% (v / v) nitric acid for more than 24 hours are used to hold samples or reagents; and chemical reagents of analytical grade or higher are used.

[0043] According to embodiments of the present invention, the cells to be detected can be human or animal sperm cells, but are not limited to such cells, including other human or animal somatic cells and separable cells in body fluids, as well as single-celled organisms such as algae and yeast cells. The method provided by the present invention has good universality.

[0044] The technical solution of the present invention will be further illustrated below through specific embodiments. It should be noted that the specific embodiments described below are merely illustrative examples and are not intended to limit the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials, reagents, instruments, etc., used are commercially available unless otherwise specified.

[0045] Example 1: Quantitative Detection and Analysis of Multiple Elements in Single-Cell Human Sperm Cells

[0046] This embodiment provides a method for multi-element quantitative detection of human sperm cells using scICP-TOF-MS. Figure 2 This is a flowchart of the multi-element quantitative detection and analysis of human sperm cells in Embodiment 1 of the present invention, as shown below. Figure 2 As shown, it includes the following steps:

[0047] (1) Preservation and transportation of sperm cell samples: Take about 800 μL of liquefied semen and place it in a cryopreservation tube. Add an equal amount of cryoprotectant and freeze it in liquid nitrogen. Then transfer it to a -80°C freezer in dry ice for storage.

[0048] (2) Sperm cell thawing and separation: The frozen semen was thawed at room temperature for 30 minutes. The sperm cell sample was thoroughly mixed by pipetting ten times with a wide-bore pipette. 1 mL of the mixed sperm cell sample was placed in a polypropylene centrifuge tube, and 1 mL of 0.01 M HEPES isotonic buffer was added and mixed. The sample was centrifuged at 550 g for 7 minutes at room temperature, and the supernatant was discarded.

[0049] (3) Wash sperm cells: Resuspend the sperm cells obtained in step (2) with 0.01M HEPES isotonic buffer, centrifuge again at 550g for 7min at room temperature, and discard the supernatant. Repeat this step twice.

[0050] The HEPES isotonic buffer solution used in steps (2) and (3) is prepared as follows: Dissolve an appropriate amount of 4-hydroxyethylpiperazine ethanesulfonic acid reagent powder in ultrapure water to obtain a stock solution concentration of 0.2M. Dilute the stock solution to 0.01M with ultrapure water, add 0.9% (w / v) NaCl to obtain 0.01M HEPES isotonic buffer solution. Then adjust the pH to 7.2-7.4 using a 0.1M NaOH solution.

[0051] (4) Cell fixation: Resuspend the sperm cells washed in step (3) in 4% (w / v) PFA fixative and fix the cells at room temperature for 15 min. After fixation, centrifuge at 750g for 7 min at room temperature and discard the supernatant.

[0052] The PFA fixative used in step (4) is prepared as follows: An appropriate amount of PFA reagent powder is placed in ultrapure water and stirred in a 60°C water bath until completely dissolved. An appropriate amount of 0.2M HEPES stock solution and NaCl reagent are added to the PFA stock solution cooled to room temperature, so that the PFA concentration in the resulting fixative is 4% (w / v), the HEPES concentration is 0.01M, and the NaCl concentration is 0.9% (w / v). The pH is then adjusted to 7.2-7.4 using a 0.1M NaOH solution.

[0053] (5) Wash sperm cells: Resuspend the sperm cells fixed in step (4) in ultrapure water, centrifuge again at 750g for 7min at room temperature, and discard the supernatant. Repeat this step twice.

[0054] (6) Sperm cell counting and dilution: Resuspend the sperm cells washed in step (5) in ultrapure water and count the concentration of the cell suspension using a hemocytometer. Dilute the sperm cell suspension with ultrapure water to a concentration of approximately 4 × 10⁵ cells / mL.

[0055] (7) Single-cell multi-element detection: ICP-TOF-MS was used to perform single-cell multi-element detection on the sperm cell suspension diluted in step (6). The instrument parameters used for the determination are shown in Table 1.

[0056] Table 1. Operating parameters of the ICP-TOF-MS instrument

[0057]

[0058]

[0059] (8) Standard Curve and Nebulization Efficiency Determination: The quantitative method used was the standard curve method. A series of standard solutions containing the same concentrations of Ag, Al, As, Ba, Be, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, K, Li, Mg, Mn, Ni, P, Pt, Rb, Se, Sr, Ti, U, V, and Zn were prepared, with a concentration gradient range of 0.1-5 μg / L. The standard solutions were measured using the same method as for cell detection to obtain the response-mass correction coefficients for each element. The nebulization efficiency was determined using a standard solution of 50 μg / L 40 nm gold nanoparticles and HAuCl4 containing 1 μg / L Au.

[0060] (9) Data Processing and Analysis: Cell signals are identified using software, and the mass of each element in a single cell is calculated based on the standard curve and atomization efficiency obtained in step (8). Further analysis is conducted on element distribution patterns and the correlation between elements and cell physiological states. Specifically, the following operations are included:

[0061] First, the software automatically identifies cell signals, and calculates the mass of each element in a single cell according to formula (1) based on the correction coefficient and atomization efficiency obtained in step (8). Then, valid cell data are screened using P as the cell marker element. Figure 3 This is a schematic diagram illustrating the screening of effective cell data from cell detection data related to individual cells in Embodiment 1 of the present invention. Figure 3As shown, when a P signal is detected in a cell event automatically identified by the software, it is considered a valid cell event; conversely, if no P signal is detected but other element signals are detected, it is considered an invalid false positive cell event. Finally, based on the screened valid cell data, further analysis can be performed on the element distribution patterns and the correlation between elements and cell physiological states.

[0062] Analysis of the mass distribution of various elements in a single sperm cell reveals that essential elements (P, Zn, Cu, K, Ca, Mg, etc.) and non-essential elements (Cr, Ag, Au, Sr, Ba, Pb, etc.) may exhibit different distribution patterns or physiological functions. P and Zn show high similarity in their distribution patterns or physiological functions within sperm cells, and statistical analysis indicates a high correlation between these two elements and certain sperm quality parameters.

[0063] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for simultaneously detecting multiple chemical elements on a single cell, comprising the following steps: The cells to be tested were first washed using an isotonic buffer solution, wherein the isotonic buffer solution does not contain endogenous cell marker elements or the chemical elements to be tested; Cells after the first washing process are fixed using an isotonic fixative to maintain their structural morphology, wherein the isotonic fixative does not contain the cell's endogenous marker elements or the chemical elements to be tested; After the fixed cells were washed with ultrapure water for the second time, the cells were counted, and the cells were diluted with ultrapure water according to the counting results to obtain a diluted cell suspension as a sample to be tested. The sample to be tested was subjected to single-cell multi-element detection using inductively coupled plasma time-of-flight mass spectrometry to obtain cell detection data on multiple chemical elements to be tested in a single cell. Based on the endogenous marker elements in the cells, valid cell detection data are screened from the cell detection data, and then the valid cell detection data are quantitatively analyzed to obtain multi-element quantitative results on the cells.

2. The method according to claim 1, characterized in that, The isotonic buffer is an organic buffer, and the endogenous marker element of the cell is phosphorus.

3. The method according to claim 2, characterized in that, The isotonic buffer solution comprises an organic buffer with a concentration of 0.01–0.025 M, NaCl with a mass-volume ratio of 0.9%, and a pH of 7.2–7.

4.

4. The method according to claim 1, characterized in that, The isotonic fixative comprises a fixative with a mass-volume concentration of 2-5%, an organic buffer with a concentration of 0.01-0.025M, and NaCl with a mass-volume concentration of 0.9%, and a pH of 7.2-7.

4.

5. The method according to claim 1, characterized in that, The concentration of the diluted cell suspension was 4 × 10⁻⁶. 5 or less per mL.

6. The method according to claim 1, characterized in that, The method further includes: Using the inductively coupled plasma-time-of-flight mass spectrometer, a series of standard solutions containing the various analyte chemical elements were simultaneously measured using the same detection method as the analyte sample, and the response-mass correction coefficients of the various analyte chemical elements were determined.

7. The method according to claim 6, characterized in that, Quantitative analysis of the effective cell detection data includes: The effective cell detection data are corrected based on the response-mass correction coefficients and atomization efficiency of the various chemical elements to be tested, so as to obtain the quantitative results of multiple elements on the cells.

8. The method according to claim 1, characterized in that, The process of screening effective cell detection data from the cell detection data based on the endogenous cell marker elements includes: If a phosphorus signal is detected in the cell detection data of a single cell, the cell detection data of that single cell is determined to be valid cell detection data; otherwise, it is invalid cell detection data.

9. The method according to claim 1, characterized in that, The cells are sperm cells, somatic cells, cells that can be isolated from body fluids, algal cells, or yeast cells.

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