SC-ICP-MS detection method for trace elements in single mammalian cell based on temperature control sampling system

Abstract

The invention provides an SC-ICP-MS detection method for trace elements in a single mammalian cell based on a temperature control sample introduction system. The SC-ICP-MS detection method comprises the following steps: carrying out correction calculation on the transmission efficiency of a cell suspension sample based on an internal reference phosphorus element in a non-reaction gas mode; determining sample introduction conditions according to the calculated transmission efficiency; and detecting the content of trace elements in the single cell according to the determined sample introduction condition and a pre-established element ion standard curve. Absolute quantification of trace elements can be rapidly completed, and the method is suitable for large-scale single-cell omics analysis.

Description

Technical Field

[0001] The invention relates to the fields of biotechnology and element detection, and in particular to a SC-ICP-MS detection method for trace elements in a single mammalian cell based on a temperature-controlled sampling system. Background Art

[0002] In the analysis of single cell behavior in related technologies, the existence of cell heterogeneity has become a consensus. This heterogeneity not only leads to differences in cell phenotypes, but also differences in cell composition including proteins, molecular ions and metabolic compounds, and the uptake of exogenous inorganic metal elements.

[0003] Compared with the traditional total inductively coupled plasma mass spectrometry (ICP-MS) method to analyze the collective information of cell populations, single-cell analysis can be better used to study the differences between cells and is more helpful in clarifying the practical significance of single-cell element analysis in the biological field, such as cell development and differentiation. At present, some studies have revealed the heterogeneous distribution of heavy metal elements in individual microorganisms such as algae, Tetrahymena and bacteria. However, few studies have revealed the element distribution status in single mammalian cells, or the occurrence characteristics of heavy metal elements in single mammalian cells under low-level exposure.

[0004] Single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) has become a powerful tool for single-cell analysis of trace elements and biomacromolecules. However, its sensitivity is extremely limited for complex intracellular matrices and extremely low levels of elements to be analyzed; at the same time, the detection effect of SC-ICP-MS is also significantly affected by the injection transmission efficiency (TE). Summary of the invention

[0005] In view of this, in order to at least partially solve at least one of the above-mentioned technical problems, the present invention provides a SC-ICP-MS detection method for trace elements in single mammalian cells based on a temperature-controlled sampling system.

[0006] According to an embodiment of one aspect of the present invention, a SC-ICP-MS detection method for trace elements in a single mammalian cell based on a temperature-controlled injection system is provided, comprising:

[0007] In the non-reactive gas mode, the transmission efficiency of the cell suspension sample was corrected and calculated based on the internal reference phosphorus element;

[0008] Determine the injection conditions based on the calculated transmission efficiency;

[0009] The content of trace elements in single cells was detected according to the determined injection conditions and the pre-established element ion standard curve.

[0010] According to an embodiment of the present invention, the trace element content in a single cell is corrected based on the internal reference phosphorus element by using the calculation formula shown in the following formula (1):

[0011] m= m det η d + (1-η d ) (1)

[0012] Where m is the trace element content of a single cell, fg cell -1 ;m det is the median mass of the single-cell trace elements detected in the population, fg cell -1 ; η d is the trace element detection rate, specifically referring to the ratio of the number of cells in the population where trace elements are detected to the number of cells in P.

[0013] According to an embodiment of the present invention, the above detection method further includes adjusting the parameters of the inductively coupled plasma mass spectrometer by a preset parameter adjustment method; the preset parameter adjustment method includes:

[0014] Adjust the flow rates of atomizing gas and make-up gas to the preset flow ratio;

[0015] Adjust the extraction lens 1 voltage within a preset range until the signal response reaches a maximum value;

[0016] Perform batch auto-tuning of an inductively coupled plasma mass spectrometer;

[0017] Adjust the RF matching voltage and the octopole RF voltage.

[0018] According to the embodiment of the present invention, the preset adjustment range of the voltage of the extraction lens 1 is -10~-20V; the adjustment range of the RF matching voltage is 0~1V.

[0019] According to an embodiment of the present invention, the octopole radio frequency voltage is adjusted in the range of 120-140V when detecting light elements; the octopole radio frequency voltage is adjusted in the range of 140-180V when detecting heavy elements.

[0020] According to an embodiment of the present invention, the internal reference phosphorus element is used to calculate the number of cells in the cell suspension sample; and the transmission efficiency of the cell suspension sample is calculated according to the number of cells in the cell suspension sample.

[0021] According to the embodiment of the present invention, the preset flow ratio of the atomizing gas and the makeup gas is 0.25~0.65 L min -1 .

[0022] According to an embodiment of the present invention, the above method further comprises transmitting the cell suspension sample to the inductively coupled plasma mass spectrometer via a microfluidic injection workstation via a fully consumable atomization temperature control platform;

[0023] The fully consumable atomization temperature control platform includes an atomizer, a fully consumable atomization chamber and a temperature control device.

[0024] According to an embodiment of the present invention, the injection conditions include injection flow rate, sample density, injection flux, and atomization temperature.

[0025] According to an embodiment of the present invention, the determined injection conditions include:

[0026] The injection flow rate is 15~20 µL min -1 The sample density is 1×10 5 ~2×10 5 cells mL -1 , injection throughput is 1500~4000 min -1 , the atomization temperature is 100~120℃.

[0027] According to the embodiment of the present invention, trace elements (heavy metals and rare earths, etc.) are generally measured in the non-reactive gas mode. Based on the optimized TE and the measurement of P element in the non-reactive gas mode, the mode of measuring P when optimizing TE is consistent with the trace element mode, reducing the deviation of the optimization conditions; during the optimization process, the P element is measured in the non-reactive gas mode to ensure that the TE optimization conditions are consistent with the trace element determination mode. This consistency effectively reduces the detection deviation caused by mode switching, thereby improving the accuracy of quantitative analysis of trace elements (such as heavy metals such as mercury and cadmium and rare earth elements). Higher TE can enable the detector to detect more cell signals within a specified time, and the analysis of the element occurrence characteristics in the later cell population is more accurate. In addition, the temperature-controlled injection system can reduce the baseline drift and signal fluctuation caused by temperature changes, further reduce the detection limit (LOD), and improve the detection ability of trace elements; according to the pre-established element ion standard curve combined with real-time internal reference correction, the absolute quantification of trace elements can be quickly completed, which is suitable for large-scale single-cell omics analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The above and other objects, features and advantages of the present invention will become more apparent through the following description of the embodiments of the present invention with reference to the accompanying drawings, in which:

[0029] Figure 1 A schematic diagram of the sample injection system for the inductively coupled plasma mass spectrometry detection method of trace elements in a single mammalian cell according to an embodiment of the present invention;

[0030] Figure 2This is a comparison diagram of ion signal intensity of a standard mercury ion solution under different extraction lens 1 voltages according to an embodiment of the present invention;

[0031] Figure 3 This is a comparison diagram of ion signal intensity of standard mercury ion solution at different atomization temperatures according to an embodiment of the present invention;

[0032] Figure 4 A schematic diagram showing a reliability comparison of a verification method based on an Ir labeling strategy according to an embodiment of the present invention;

[0033] Figure 5 This is a schematic diagram comparing the results of detecting single-cell mercury content using conventional ICP-MS and SC-ICP-MS methods according to an embodiment of the present invention;

[0034] Figure 6 This is a graph showing changes in mercury content in a single cell of the THP-1 cell line according to an embodiment of the present invention.

[0035] The symbols in the accompanying drawings are as follows:

[0036] 1-Syringe pump; 2-100 µL micro-injector; 3-Capillary connecting tube (inner diameter 0.15 mm); 4-3D printed syringe pump support bracket; 5-Standard glass concentric nebulizer Micro Mist; 6-Stackable nebulization temperature control device; 7-Magnetic bracket. DETAILED DESCRIPTION

[0037] Below, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of embodiments of the present invention. However, it is apparent that one or more embodiments may also be implemented without these specific details. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion of concepts of the present invention.

[0038] The terms used herein are only for describing specific embodiments and are not intended to limit the present invention. The term "comprising" used herein indicates the presence of features, steps, operations, but does not exclude the presence or addition of one or more other features.

[0039] In the case of using expressions such as "at least one of A, B, and C, etc.", it should generally be interpreted in accordance with the meaning of the expression generally understood by those skilled in the art (for example, "a system having at least one of A, B, and C" should include but is not limited to a system having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, C, etc.). In the case of using expressions such as "at least one of A, B, or C, etc.", it should generally be interpreted in accordance with the meaning of the expression generally understood by those skilled in the art (for example, "a system having at least one of A, B, or C" should include but is not limited to a system having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, C, etc.).

[0040] In the process of realizing the concept of the present invention, it was found that based on the traditional standard dual-channel spray chamber, the transmission efficiency (TE) is usually only about 1%. The temperature increase of the atomization environment is conducive to the evaporation process of the cell suspension before entering the plasma, and can effectively improve the TE under the same conditions. Standard nanoparticles are often used as a tool to optimize TE, but due to their significant differences in size and homogeneity from actual cell individuals, the results of the optimized conditions of the injection method often have deviations. Natural internal references (such as Na + , K + ) is suitable for fast and low-cost experiments; artificial internal references (such as In and Re) are suitable for high-precision requirements.

[0041] Therefore, accurate and direct determination of trace inorganic elements in mammalian cells remains a great challenge.

[0042] In some embodiments, it has been found that constant elements such as phosphorus (P) and sulfur (S) inside cells can be used to accurately evaluate the TE of SC-ICP-MS. However, when using SC-ICP-MS to determine low-mass elements such as P and S, a collision reaction cell (CRC) is usually required, which greatly reduces the sensitivity while reducing background interference. In addition, phosphorus mainly exists in the form of phosphate in cells and may participate in a variety of metabolic processes. If cell metabolism changes during the experiment, it may affect the P content, requiring appropriate correction methods.

[0043] Specifically, according to an embodiment of one aspect of the present invention, a SC-ICP-MS detection method for trace elements in a single mammalian cell based on a temperature-controlled sampling system is provided, comprising the following steps S1 to S3:

[0044] Step S1: in a non-reactive gas mode, the transmission efficiency of the cell suspension sample is corrected and calculated based on the internal reference phosphorus element;

[0045] Step S2: Determine the injection conditions according to the calculated transmission efficiency;

[0046] Step S3: Detect the content of trace elements in single cells according to the determined injection conditions and the pre-established element ion standard curve.

[0047] According to the embodiment of the present invention, trace elements (heavy metals and rare earths, etc.) are generally measured in the non-reactive gas mode. Based on the optimized TE and the measurement of P element in the non-reactive gas mode, the mode of measuring P when optimizing TE is consistent with the trace element mode, reducing the deviation of the optimization conditions; During the optimization process, the P element is measured in the non-reactive gas mode to ensure that the TE optimization conditions are consistent with the trace element determination mode. This consistency effectively reduces the detection deviation caused by mode switching, thereby improving the accuracy of quantitative analysis of trace elements (such as heavy metals such as mercury and cadmium and rare earth elements). Higher TE can enable the detector to detect more cell signals within a specified time, and the analysis of the element occurrence characteristics in the later cell population is more accurate. In addition, the temperature-controlled injection system can reduce the baseline drift and signal fluctuation caused by temperature changes, further reduce the detection limit (LOD), and improve the detection ability of trace elements; according to the pre-established element ion standard curve combined with real-time internal reference correction, the absolute quantification of trace elements can be quickly completed, which is suitable for large-scale single-cell omics analysis.

[0048] According to an embodiment of the present invention, the trace element content in a single cell is corrected based on the internal reference phosphorus element and is calculated by the calculation formula shown in the following formula (1):

[0049] m= m det η d + (1-η d ) (1)

[0050] Where m is the trace element content of a single cell, fg cell -1 ;m det is the median mass of the single-cell trace elements detected in the population, fg cell -1 ; η d is the trace element detection rate, specifically referring to the ratio of the number of cells in the population where trace elements are detected to the number of cells in P.

[0051] According to an embodiment of the present invention, formula (1) is based on the median of the detected cell mass (m det ) multiplied by the detection rate (η d ), representing the effective signal actually observed; for undetected cells (accounting for 1-η d ), using the limit of quantification (LOQ m) as a conservative estimate to avoid underestimation or data bias due to missing signals. det ) rather than the mean, which can reduce the interference of extreme values ​​such as highly enriched cells on population distribution and enhance robustness. Replacing the true value of undetected cells not only meets the processing specifications of "data below the detection limit" in trace analysis, but also reduces the risk of false negatives.

[0052] Furthermore, the single cell element quantitative detection limit LOQ that can be achieved by the above method is m As low as 0.006 fg cell -1 , calculated by the mean and 10-fold standard deviation of the trace element signal response in a single cell at low exposure concentration; LOQ m Half of the single-cell mercury content was used to replace the undetected mercury content in the part of the population; the cell density detection limit LOD d As low as 7.18×10 at 80°C 3 cells mL -1 , at 120℃, it is as low as 8.14×10 2 cells mL -1 .

[0053] In some specific embodiments of the present invention, the following steps S101 to S102 are also included before step S1:

[0054] Step S101: The cell suspension sample is transferred to the inductively coupled plasma mass spectrometer through the microfluidic injection workstation via the fully consumable atomization temperature control platform; the fully consumable atomization temperature control platform includes a nebulizer, a fully consumable atomization chamber and a temperature control device.

[0055] Step S102: adjusting the parameters of the inductively coupled plasma mass spectrometer by a preset parameter adjustment method.

[0056] In step S101, the microfluid injection workstation and the fully consumable atomization temperature control platform are used in conjunction with Agilent ICP-MS. The microfluid injection workstation consists of a 3D printed bracket, a syringe pump, a 100 µL microinjector, and a capillary connection tube (inner diameter 0.15 mm), which can provide a stable driving force for the cell suspension sample in the isotropic horizontal direction; the fully consumable atomization temperature control platform consists of a standard glass concentric atomizer Micro Mist, a single-channel fully consumable atomization chamber, a temperature control device that can be stacked, and a magnetic bracket. Among them, the atomization chamber uses a temperature-controlled fully consumable atomization chamber, which promotes the evaporation of cell suspension droplets by heating the atomization environment, reduces the proportion of large droplets, and allows more single cells to stably enter the plasma in the form of aerosols. Through the combination of temperature control and fully consumable design, the core problems of low transmission efficiency, insufficient sensitivity, and easy cell rupture in single-cell trace element detection are solved, providing a highly sensitive and stable technical platform for single-cell heterogeneity research.

[0057] In step S102, further steps S1021 to S1024 are included:

[0058] Step S1021: adjusting the flow rates of atomizing gas and makeup gas to a preset flow rate ratio;

[0059] Step S1022: adjusting the voltage of the extraction lens 1 within a preset range until the signal response reaches a maximum value;

[0060] Step S1023: performing batch self-tuning of the inductively coupled plasma mass spectrometer;

[0061] Step S1024: adjusting the radio frequency matching voltage and the octopole radio frequency voltage.

[0062] According to the implementation scheme of the present invention, by pre-setting the flow ratio of nebulization gas and makeup gas, the stable generation and transmission of sample aerosol is ensured, the difference in droplet size distribution is reduced, and the consistency of plasma load is improved. Makeup gas such as argon can assist in stabilizing the plasma flame and reduce the background noise in high-salt / organic matrix samples. The optimization of the extraction lens 1 voltage can accurately regulate the transmission path of ions from the plasma to the mass analyzer, reduce ion loss, and make the target signal intensity reach the peak value. The auto-tuning function optimizes key parameters such as plasma power, ion lens voltage, and detector gain through a built-in algorithm to achieve the global optimal configuration of instrument performance. The adjustment of the RF matching voltage can optimize the energy coupling efficiency between the plasma and the RF generator, improve the plasma stability, and reduce the matrix effect. The adjustment of the octopole RF voltage can improve the focusing performance of the ion beam, reduce the diffusion of ion clouds, and improve the mass resolution. A personalized tuning mode was adopted. By modifying parameters such as the extraction lens 1 voltage, the ion beam was focused more efficiently, the transmission rate of ions entering the system (especially light-mass element ions) was improved, and a higher level of analytical sensitivity was achieved. Through the full-link optimization of gas path control-ion transmission-system tuning-energy coupling, the sensitivity, stability and anti-interference ability of ICP-MS were significantly improved, providing reliable technical support for ultra-trace element and single-cell analysis.

[0063] In step S1021, the preset flow ratio of the atomizing gas and the makeup gas is 0.25~0.65 L min -1 .

[0064] The preset flow ratio of nebulizer gas and makeup gas can be 0.25 L min -1 、0.3L min -1 , 0.35 L min -1 , 0.4 L min -1 , 0.45 L min -1 , 0.5 L min -1 、0.55 L min -1 , 0.6 L min -1 、0.65 L min -1 , but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0065] In step S1022, the preset adjustment range of the voltage of the extraction lens 1 is -10~-20V.

[0066] The preset adjustment range of the extraction lens 1 voltage can be -10~-15V, -15~-20V; the lens voltage can be -10V, -11 V, -12 V, -13 V, -14 V, -15 V, -16 V, -17 V, -18 V, -19 V, -20 V, but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0067] In step S1024, the adjustment range of the RF matching voltage is 0-1V.

[0068] The RF matching voltage can be 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, or 1 V, but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0069] In step S1024, the octopole radio frequency (RF) voltage is adjusted in the range of 120-140V when detecting light elements; the octopole radio frequency voltage is adjusted in the range of 140-180V when detecting heavy elements.

[0070] The octopole radio frequency voltage can be 120V, 130V, 140V when detecting light elements; and can be 140V, 150V, 160V, 170V, 180V when detecting heavy elements, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0071] Specifically, the octopole RF voltage is adjusted to 120-140 V when detecting light elements such as phosphorus (P), and is adjusted to 180 V when detecting heavy elements such as mercury (Hg), cadmium (Cd), lead (Pb), gold (Au), etc., wherein the real-time monitoring signal response is tuned to a higher range during the process.

[0072] In some specific embodiments of the present invention, the preset parameter adjustment method can be to manually adjust the atomizing gas-compensation gas flow rate ratio to 0.25-0.65 L min after starting the startup item tuning. -1 , then perform the instrument startup item self-tuning (mainly including the matrix tube tuning item); in batch tuning, first manually adjust the extraction lens 1 voltage to the -20V range until the real-time monitoring signal response value is the highest, and then perform the instrument batch self-tuning. After the automatic tuning is completed, adjust individual parameters separately: RF matching voltage is lowered to 0.9V, octopole RF voltage is adjusted to 120-140V when detecting P elements, and adjusted to 180V when detecting heavy mass elements (during the process, the real-time monitoring signal response is tuned to a higher range).

[0073] According to an embodiment of the present invention, in step S1, the internal reference phosphorus element is used to calculate the number of cells in the cell suspension sample; and the transmission efficiency of the cell suspension sample is calculated according to the number of cells in the cell suspension sample.

[0074] According to an embodiment of the present invention, the constituent element P of the cell is selected as the internal reference element "pointer". At the same time, in order to ensure the accuracy of the method application, the measured trace elements and P are kept in the same detection mode, and the P element of the single cell is determined in the "No gas" mode without using a collision reaction cell. It is used to indicate the number of detectable cell signals, calculate the sample TE, and perform TE correction calculations.

[0075] Furthermore, the appropriate injection flow rate, sample density, carrier gas flow ratio, atomization temperature and integration time within a certain test range were adjusted, and the TE calculation results were compared to evaluate and determine the final injection conditions.

[0076] According to an embodiment of the present invention, in step S2, the injection conditions determined include injection flow rate, sample density, injection flux, and atomization temperature.

[0077] Specifically, the injection flow rate is 15~20 µL min -1 The sample density is 1×10 5 ~2×10 5 cells mL -1 , injection throughput is 1500~4000 min -1 , the atomization temperature is 100~120℃.

[0078] According to an embodiment of the present invention, the injection flow rate can be 15 μL min -1 、16µL min -1 、17µL min -1 、18µL min -1 、19µL min -1 、20µL min -1 ; The sample density can be 1×10 5 cells mL -1 , 1.2×10 5 cells mL -1 , 1.4×10 5 cells mL -1 , 1.6×10 5 cells mL -1 , 1.8×10 5 cells mL -1 , 2×10 5 cells mL -1; Injection throughput can be 1500 min -1 , 2000 min -1 , 2500 min -1 、3000 min -1 、3500 min -1 , 4000 min -1 The atomization temperature may be 100°C, 105°C, 110°C, 115°C, or 120°C, but is not limited to the listed values. Other values ​​not listed within the range are also applicable.

[0079] According to an embodiment of the present invention, in step S3, a pre-established element ion standard curve is established based on an ion standard curve establishment method in the related art, and then the content of trace elements is detected, which will not be described in detail here.

[0080] The scheme of the present invention will be further explained below with reference to specific examples. Unless otherwise stated in the following examples, conventional commercial reagents are used.

[0081] Example 1 Construction of a microfluid injection workstation and a fully consumable atomization temperature control platform

[0082] A fully consumable temperature controlled sample injection system for use with Agilent ICP-MS, such as Figure 1 As shown, it includes a microfluidic injection workstation and a fully consumable nebulization temperature control platform.

[0083] Figure 1 Schematic diagram of the sample injection system for the inductively coupled plasma mass spectrometry detection method of trace elements in a single mammalian cell according to an embodiment of the present invention.

[0084] The microfluidic injection workstation is used to provide a stable driving force for the cell suspension sample in the isotropic horizontal direction, including a syringe pump 1 that provides a constant injection flow rate, a 100 µL microinjector 2 for aspirating, storing and pushing the cell suspension sample, a capillary connection tube 3 (inner diameter 0.15 mm) for transmitting a single cell suspension, and a 3D-printed syringe pump support bracket 4. The capillary connection tube 3 is connected to the microinjector 2 through a threaded interface and is connected to the nebulizer 5 at the same time.

[0085] The fully consumable atomization temperature control platform is used to evenly disperse the cell liquid path transmitted to the capillary connecting tube 3 into droplets containing single cell individuals through pneumatic atomization, so as to enter the connecting tube of ICP-MS for subsequent ionization. It includes a standard glass concentric atomizer Micro Mist 5 connected to the atomization gas pipeline, a stackable atomization temperature control device 6 with two compensation gas pipelines connected on both sides, and a magnetic bracket 7 for supporting the temperature control device 6, which provides good coaxiality for sample ion transmission.

[0086] Example 2 Personalized tuning process and optimization of detection conditions for trace element detection in single mammalian cells

[0087] Batch personalized tuning process:

[0088] The purpose of batch processing personalized tuning is to provide higher sensitivity for accurate quantification of trace elements. After starting the startup tuning, first adjust the nebulizer gas-makeup gas flow ratio to 0.20-0.70 L min -1 , then perform self-tuning of the instrument startup items (mainly including the matrix tube tuning items); in batch tuning, first autonomously lower the extraction lens 1 voltage to the range of -10 to -20V, observe until the real-time monitoring signal response value is the highest, then perform batch automatic tuning of the instrument, and adjust individual parameters separately after the automatic tuning is completed: adjust the RF matching voltage to below 1.0V, and observe that the feedback power drops to the daily level; adjust the octopole RF voltage to 120~140V when detecting light mass elements, and adjust it to 180V when detecting heavy mass elements (during the process of tuning, the real-time monitoring signal response is adjusted to a higher range).

[0089] To better explore the applicability of the tuning process for trace elements, a 1 µg L -1 Mercury ion (Hg 2+ ) response, different extraction lens 1 voltages were adjusted for comparison, such as Figure 2 shown.

[0090] Figure 2 1 is a comparison chart of ion signal intensity of standard mercury ion solution under different extraction lens 1 voltages in an embodiment of the present invention.

[0091] according to Figure 2 It can be seen that as the voltage of extraction lens 1 is gradually reduced, 1 µg L -1 Hg 2+ The total quantitative signal gradually increases, indicating that more ions enter the mass spectrometer and the sensitivity is improved. Therefore, the extraction lens 1 voltage in the range of -13V is more suitable for the determination of the heavy element mercury (Hg).

[0092] Methods for optimizing detection conditions:

[0093] THP-1 cells (average diameter 11.6 µm) were used as experimental objects to optimize the measurement conditions. Since the P element is present in large quantities in biological molecules with extremely high content such as nucleic acids and proteins, the internal reference "probe" P was selected as the TE evaluation indicator, and "No gas" was selected as the detection mode, which is consistent with the conventional heavy mass element detection. It can effectively identify unlabeled single mammalian cells. Among them, TE is calculated by the "particle frequency method" proposed in previous studies, which refers to the ratio of the number of cells obtained by detecting P to the total number of cells injected. The specific calculation process is shown in formula (2):

[0094] η n = ×100% (2)

[0095] Where η n That is the sample transmission efficiency TE; n P is the number of cell signals obtained by detecting P (cell); Q sam is the sample injection flow rate (µL min -1 ); c is the sample density (cells mL -1 );t s is the injection time (min), 1min is usually selected as the injection time.

[0096] To avoid cell rupture while maintaining a higher TE, the nebulizer gas flow rate was finally set to 0.3 L min -1 The compensation gas flow rate is optimized to 0.6 L min -1 The sample injection flow rate is stabilized at 15~20 µL min through the 100 µL microinjector 2 and the syringe pump 1. -1 The corresponding cell density is optimized to 1×10 5 ~2×10 5 mL -1 , the injection throughput can be as high as 1500~4000 min -1 .

[0097] Therefore, the rate of cell introduction into plasma can be stabilized within a certain range, which will neither reduce the number of single cells that can be used to study the distribution law nor easily block the injection capillary. At the same time, the integration time is optimized to 0.1 ms, and the TE at 0.1 ms is significantly higher than the TE when the integration time is longer than 0.1 ms. At the same time, the ion intensity is compared based on different nebulization temperatures, such as Figure 3 shown.

[0098] Figure 3 This is a comparison chart of ion signal intensities of standard mercury ion solutions at different atomization temperatures in an embodiment of the present invention.

[0099] according to Figure 3 It can be seen that the atomization temperature is finally optimized to 80℃~120℃. The atomization environment at 120℃ can better promote droplet formation, and the peak TE reaches 27.3%. At 80℃, the TE of THP-1 can reach 18.4%. After testing at room temperature, the TE under the optimized conditions can only reach 1.5%, less than one tenth of that. At the same time, 1 µg L -1 Hg 2+ The signal response (CPS) of the standard solution at 80°C is 16875, which is nearly three times that at room temperature 25°C (6066).

[0100] Example 3 Reliability and authenticity test of the results of the SC-ICP-MS detection method for trace elements in a single mammalian cell based on a temperature-controlled sampling system

[0101] Reliability test process of SC-ICP-MS detection method for trace elements in single mammalian cells:

[0102] The reliability of the single-cell detection method established in the present invention was verified by the cell nucleus iridium (Ir) labeling strategy. The P signal and the labeled Ir signal of the same batch of THP-1 cells were measured using the established and optimized single-cell detection method. The results are as follows: Figure 4 shown.

[0103] Figure 4 The figure is a schematic diagram for comparing the reliability of the verification method based on the Ir marking strategy according to an embodiment of the present invention.

[0104] according to Figure 4 It can be seen that the TE results obtained by the two element detections are almost at the same level, and there is no statistical difference in the raw data of Ir and P detection under the same conditions for the same batch of labeled cells (p>0.05). The highly consistent results effectively show that the optimization conditions of this method and the selection of P element as an internal reference tool are reliable for the evaluation of single cell TE detection.

[0105] Authenticity verification process of SC-ICP-MS detection method for trace elements in single mammalian cells:

[0106] The SC-ICP-MS method established by the present invention and the traditional ICP-MS total amount method were used to detect THP-1 cells exposed to different concentrations of Hg. The results are as follows: Figure 5 shown.

[0107] Figure 5 This is a schematic diagram comparing the results of detecting single-cell mercury content using conventional ICP-MS and SC-ICP-MS methods in an embodiment of the present invention.

[0108] according to Figure 5It can be seen that when the exposure concentrations were 5, 10, 50, and 100 µg L -1 The Hg contents in THP-1 cells obtained by SC-ICP-MS (corresponding to the traditional total ICP-MS method) were 0.013 fg cell -1 (0.035 fg cell -1 )、0.100(0.154 fg cell -1 )、0.170(0.142 fg cell -1 ) and 0.455 fg cell -1 (0.565 fgcell -1 There was no statistically significant difference in the quantitative results of the two methods (p>0.05). In addition, the established SC-ICP-MS method could detect Hg concentrations even when THP-1 cells were exposed to low levels of Hg (≤1 µg L -1 ), the Hg content in a single cell can be quantified successfully. However, the ICP-MS total amount detection method did not detect effective results for the low-level Hg exposure experimental group. This result not only verifies the authenticity of the results of the single-cell method in the present invention using the traditional ICP-MS total amount method, but also demonstrates the superior ability of the established SC-ICP-MS in the determination of trace elements in mammalian cells.

[0109] Example 4 Application of SC-ICP-MS detection method for trace elements in single mammalian cells based on temperature-controlled injection system

[0110] Process for determination of Hg content in single THP-1 cells using SC-ICP-MS detection of trace elements in single mammalian cells:

[0111] Limit of quantification (LOQ) of Hg in a single cell m Related to the background signal, it is calculated from the mean and ten times standard deviation of the Hg signal response of THP-1 at low exposure concentrations. After analysis, the LOQ of the SC-ICP-MS method in a single THP-1 cell is m 0.006 fg cell -1 , which is lower than the value reported in previous studies (~0.01 fg cell -1 ), indicating that the optimized SC-ICP-MS has ultra-high sensitivity under the full-consumption temperature-controlled injection system. Cell density detection limit LOD d As low as 7.18×10 at 80°C 3 cells mL -1 , at 120℃, it is as low as 8.14×10 2 cells mL -1 .LODd The calculation method is shown in formula (3):

[0112] LOD d = (3)

[0113] Among them, η n is the sample transmission efficiency of the cell sample obtained by measuring the internal reference element P; Q sam is the cell sample injection flow rate per minute at the corresponding nebulization temperature; t s LOD is the single injection time, and 60 s, i.e. 1 min, is generally used in the experimental determination process of the present invention. d The results show that the high-temperature atomization environment used in the method of the present invention effectively reduces the cell density detection limit under the same conditions, providing a certain basis and guarantee for the applicability and breadth of the method.

[0114] Considering that there are always some undetected individual cells in a fixed population, their internal Hg content is lower than LOQ m , especially at low exposure concentrations, individuals without detected Hg content may account for a larger proportion of the cell population. Therefore, referring to the concept proposed in the reported study, the single-cell Hg content m (fg cell) in the THP-1 population was calculated using formula (1) -1 ), the quantitative results are as follows Figure 6 shown.

[0115] Figure 6 This is a graph showing changes in mercury content in a single cell of the THP-1 cell line according to an embodiment of the present invention.

[0116] according to Figure 6 It can be seen that the m of THP-1 shows a good exponential growth trend with the increase of exposure concentration. -1 ), the single-cell Hg content remained almost unchanged; when the exposure level was higher than 5 µg L -1 When the concentration of Hg was higher than 1.0477m, the Hg content entered a period of rapid exponential growth until the highest exposure concentration, when the Hg content reached 0.455fg cell. -1 .

[0117] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is 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 in the scope of protection of the present invention.

Claims

1. A SC-ICP-MS detection method for trace elements in a single mammalian cell based on a temperature-controlled sampling system, comprising: Correcting and calculating the transmission efficiency of the cell suspension sample based on the internal reference phosphorus element in a non-reactive gas mode; Determining injection conditions according to the calculated transmission efficiency; The content of trace elements in single cells was detected according to the determined injection conditions and the pre-established element ion standard curve.

2. The detection method according to claim 1, further comprising adjusting the parameters of the inductively coupled plasma mass spectrometer by a preset parameter adjustment method; The preset parameter adjustment method includes: Adjust the flow rates of atomizing gas and make-up gas to the preset flow ratio; Adjust the extraction lens 1 voltage within a preset range until the signal response reaches a maximum value; performing batch auto-tuning of the inductively coupled plasma mass spectrometer; Adjust the RF matching voltage and the octopole RF voltage.

3. The detection method according to claim 2, wherein: The preset adjustment range of the extraction lens 1 voltage is -10~-20V; The adjustment range of the RF matching voltage is 0~1V.

4. The detection method according to claim 2 or 3, wherein: The octapole radio frequency voltage is adjusted in the range of 120-140V when detecting light elements; The octapole radio frequency voltage is adjusted in the range of 140-180V when detecting heavy elements.

5. The detection method according to claim 1, wherein the trace element content in a single cell is corrected based on the internal reference phosphorus element by the calculation formula shown in the following formula (1): m= m det or d + (1st) d ) (1) Where m is the trace element content of a single cell, fg cell -1 ;m det is the median mass of the single-cell trace elements detected in the population, fg cell -1 ; η d is the trace element detection rate, which specifically refers to the ratio of the number of cells in the population where trace elements are detected to the number of cells in P; The detection limit for single cell element quantification.

6. The detection method according to claim 1, wherein: The internal reference phosphorus element is used to calculate the number of cells in the cell suspension sample; The transfer efficiency of the cell suspension sample was calculated according to the number of cells in the cell suspension sample.

7. The detection method according to claim 2, wherein: The preset flow ratio of the nebulizer gas and the makeup gas is 0.25~0.65 L min -1 .

8. The detection method according to claim 1, wherein: It also includes transmitting the cell suspension sample to the inductively coupled plasma mass spectrometer through a microfluidic injection workstation via a fully consumable nebulization temperature control platform; The fully consumable atomization temperature control platform comprises an atomizer, a fully consumable atomization chamber and a temperature control device.

9. The detection method according to claim 1, wherein: The injection conditions include injection flow rate, sample density, injection flux, and atomization temperature.

10. The detection method according to claim 1 or 8, wherein: The determined injection conditions include: The injection flow rate is 15~20 µL min -1 The sample density is 1×10 5 ~2×10 5 cells mL -1 , injection throughput is 1500~4000 min -1 , the atomization temperature is 100~120℃.

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